EA041220B1 - CRYSTAL FORMS - Google Patents

Description

Cross-reference to related applications

This patent application claims priority from U.S. Provisional Applications Serial Numbers 62/570573 filed October 10, 2017; 62/643950, filed March 16, 2018; 62/656668, filed April 12, 2018; 62/669288, filed May 9, 2018; 62/676417, filed May 25, 2018; and 62/712707, filed July 31, 2018; the entire contents of each are incorporated herein by reference.

State of the art

RET is a single-pass transmembrane receptor belonging to the tyrosine kinase superfamily that is required for the normal development, maturation, and maintenance of certain tissues and cell types (Mulligan, L. M., Nature Reviews Cancer (2014) 14:173-186). The extracellular portion of the RET kinase contains four calcium-dependent cadherin-like repeats involved in ligand binding and a cysteine-rich near-membrane region required for proper folding of the RET extracellular domain, while the cytoplasmic portion of this receptor contains two tyrosine kinase subdomains.

RET signaling is mediated by the binding of a group of soluble ligand proteins (GFLs) of the glial cell line neurotrophic factor (GDNF) family, which also includes neuroturin (NTRN), artemin (ARTN), and persephin (PSPN) (Arighi et al., Cytokine Growth Factor Rev. (2005) 16:441-67). Unlike other receptor tyrosine kinases, RET does not directly bind to GFL and requires an additional co-receptor: i.e. one of four members of the GDNF α-receptor (GFRa) family, which are linked to the cell surface by a glycosylphosphatidylinositol bond. GFL and members of the GFRa family form binary complexes, which in turn bind to RET and recruit it into high cholesterol membrane subdomains, which are known lipid rafts where RET signaling occurs.

Upon binding to the ligand-coreceptor complex, RET dimerization and autophosphorylation at intracellular tyrosine residues involve adapter and signaling proteins to activate several subsequent cascades. Binding of the adapter protein to these docking sites results in the activation of the Ras-MAPK and PI3K-Akt/mTOR signaling pathways or recruitment of the CBL family of ubiquitin ligases that act to down-regulate RET in RET-mediated functions.

Aberrant expression and/or activity of RET has been demonstrated in various types of cancer and gastrointestinal disorders such as irritable bowel syndrome (IBS).

The essence of the invention

Compounds of formula I-IV -yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (Formula I); 6-(2-hydroxy-2methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridine-3 yl) pyrazolo[1,5-a]pyridine-3-carbonitrile (formula II); 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1, 5-a]pyridine-3carbonitrile (formula III); and 6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a] pyridine-3-carbonitrile (Formula IV) are RET kinase inhibitors and are useful in the treatment of diseases such as proliferative diseases including cancer.

Accordingly, this document provides compounds of formulas I-IV

- 1 041220

ιν and their pharmaceutically acceptable salts, amorphous and polymorphic forms.

This document also provides a crystalline form of the compound of formula I, said crystalline form being form A and characterized by an X-ray powder diffraction (XRD) diffraction pattern including peaks at °2θ values of 4.4±0.2, 14.6±0.2 and 18.3±0.2.

This document also provides a crystalline form of the compound of formula II, said crystalline form being Form 1 and characterized by an X-ray powder diffraction (XRD) diffraction pattern including peaks at °2θ values of 16.5±0.2, 18.9±0.2 and 26.0±0.2.

This document also provides a crystalline form of the compound of formula III, said crystalline form being form A and characterized by an X-ray powder diffraction (XRD) diffraction pattern including peaks at °2θ values of 17.3±0.2, 19.2±0.2 and 23.9±0.2.

This document also provides a crystalline form of the compound of formula IV, said crystalline form being form A and characterized by an X-ray powder diffraction (XRD) diffraction pattern including peaks at °2θ values of 8.3±0.2, 16.3±0.2 and 21.9±0.2.

This document also provides a solid oral pharmaceutical composition containing a pharmaceutically acceptable carrier and a compound of formulas I-IV, including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

This document also provides a liquid pharmaceutical composition containing a pharmaceutically acceptable carrier and a compound of formulas I-IV, including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

This invention also provides a method of treating cancer in a subject in need thereof, comprising administering a pharmaceutical composition containing a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition obtained using a compound of formula I -IV or its pharmaceutically acceptable salt, amorphous or polymorphic form.

The present invention also provides a method for inhibiting cell proliferation, in vitro or in vivo, comprising contacting a cell with an effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or with a pharmaceutical composition obtained using a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, or with the pharmaceutical composition described in this document.

Also provided herein is a method of treating a RET-associated disease or disorder in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition described in this document.

The present invention provides a method of treating cancer and/or suppressing metastasis associated with a specific type of cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form, or a pharmaceutical composition as described herein.

Also provided herein is a method for treating cancer and/or suppressing metastasis associated with a particular cancer in a subject in need thereof, comprising (a) determining whether the cancer is associated with dysregulation of the RET gene, RET kinase or their expression, or activity, or level; and (b) if the cancer is determined to be associated with dysregulation of the RET gene, RET kinase, or their expression, or activity, or level, administering to said subject a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition obtained using a compound of formula I-IV or its

- 2 041220 pharmaceutically acceptable salt, amorphous or polymorphic form.

Also provided herein is a method of treating RET-associated cancer in a subject, comprising administering to a subject identified or diagnosed with RET-associated cancer a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, or a pharmaceutical composition comprising a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form.

Also provided herein is a method for treating RET-associated cancer in a subject, comprising: determining whether the cancer in the subject is RET-associated cancer; and administering to a subject in whom RET-associated cancer is confirmed, a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, e.g., a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form.

Also provided herein is a method of treating a subject comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, such as a pharmaceutical composition prepared using the compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, to a subject with a medical history indicating that the subject suffers from dysregulation of the RET gene, RET kinase or their expression or activity or level.

Also provided herein is a method for selecting a treatment for a subject, comprising selecting a treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition containing a compound of formula I-IV, e.g., a pharmaceutical composition obtained using a compound of formula I-IV, or a pharmaceutically acceptable salt thereof, in an amorphous or polymorphic form, for a subject identified or diagnosed with RET-associated cancer.

Also provided herein is a method for selecting a treatment for a subject suffering from cancer, comprising: determining whether the subject's cancer is RET-associated cancer; and choosing a treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, for a subject in which RET-associated cancer is confirmed.

This document also provides a method for selecting a subject for treatment, comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition obtained with using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and said method comprising: identifying a subject suffering from RET-associated cancer; and selecting a subject for treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, such as a pharmaceutical composition prepared using a compound of formula I- IV or its pharmaceutically acceptable salt, amorphous or polymorphic form.

Also provided herein is a method for selecting a subject suffering from cancer for treatment, comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form, and said method comprising: determining if the subject's cancer is RET-associated cancer; and selecting a subject in whom RET-associated cancer is confirmed for treatment comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, e.g. a composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form.

This document also provides the use of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, for the manufacture of a medicament for the treatment of RET-associated cancer in a subject.

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Also provided herein is a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, such as a pharmaceutical composition prepared using a compound of formula I-IV, or a pharmaceutically acceptable salt thereof. , amorphous or polymorphic form, for use in the treatment of a subject who has been identified or diagnosed with RET-associated cancer.

Also provided herein is a method for inhibiting RET kinase activity in a mammalian cell, comprising contacting the mammalian cell with a compound of Formula I-VI, or a pharmaceutically acceptable salt thereof, in an amorphous or polymorphic form.

Also provided herein is a method of treating irritable bowel syndrome in a subject, comprising administering to a subject identified or diagnosed with irritable bowel syndrome a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition containing a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form.

Also provided herein is a method for alleviating pain associated with irritable bowel syndrome in a subject in need thereof, comprising administering to a subject identified or diagnosed with irritable bowel syndrome a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or a polymorphic form, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition obtained using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

The present invention also provides a method of providing maintenance therapy to a cancer patient, including preventing or minimizing gastrointestinal disorders such as diarrhea associated with treatment, including chemotherapy treatment, and said method comprises administering to the patient a therapeutically effective amount of a compound of formula I-IV or its a pharmaceutically acceptable salt, amorphous or polymorphic form, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition described herein.

Also provided herein is a method for inhibiting metastasis (e.g., brain metastasis) of a cancer in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form. In some embodiments, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, such as a pharmaceutical composition made using a compound of formula I-IV, or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form is used in combination with another chemotherapeutic agent.

Also provided herein is a method of treating a subject suffering from cancer, comprising: (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a), determining if the cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation that confers increased resistance to treatment with the first RET inhibitor used in step ( A); and (c) administering a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, as monotherapy or in combination with another anti-cancer agent to the specified subject, if the subject has a cancer cell that has at least one RET inhibitor resistance mutation, which gives the cancer cell or tumor increased resistance to treatment with the first RET inhibitor, used in step (a); or (d) administering to the subject additional doses of the first RET inhibitor used in step (a), if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation that renders the cancer cell or tumor more resistant to treatment with the first RET inhibitor used. in step (a).

Also provided herein is a method of treating a subject suffering from cancer, comprising: (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a) - determining if the cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation that confers on the cancer cell

- 4 041220 or tumors increased resistance to treatment with the first RET inhibitor used in step (a); (c) administering to the subject a second RET inhibitor, alone or in combination with another anticancer agent, if said subject has a cancer that has at least one RET inhibitor resistance mutation that renders the cancer cell or tumor more resistant to treatment with the first inhibitor RET used in step (a); or (d) administering to the subject additional doses of the first RET inhibitor used in step (a), if the subject has a cancer cell that does not contain a RET inhibitor resistance mutation that renders the cancer cell or tumor more resistant to treatment with the first RET inhibitor used. at stage (a); wherein said mutation is a substitution at amino acid position 804, eg V804M, V804L or V804E.

This document also provides a method of treating a subject suffering from cancer, comprising: (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously treated with one or more doses of the first RET inhibitor, one or more mutations RET inhibitor resistance, which renders the cancer cell or tumor more resistant to treatment with the first RET inhibitor previously administered to the subject; and (b) administering to said subject a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutical composition thereof. an acceptable salt, amorphous or polymorphic form, as monotherapy or in combination with another anticancer agent, if the subject has a cancer cell that has at least one RET inhibitor resistance mutation that renders the cancer cell or tumor increased resistance to treatment with the first inhibitor RET, which was previously administered to the subject; or (c) administering additional doses of a first RET inhibitor to the subject if the subject has a cancer cell that does not contain a RET inhibitor resistance mutation that renders the cancer cell or tumor more resistant to treatment with the first RET inhibitor previously administered to the subject.

This document also provides a method of treating a subject suffering from cancer, comprising: (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously treated with one or more doses of the first RET inhibitor, one or more mutations RET inhibitor resistance, which renders the cancer cell or tumor more resistant to treatment with the first RET inhibitor previously administered to the subject; and (b) administering to the subject a second RET inhibitor, alone or in combination with another anticancer agent, if the subject has a cancer cell that has at least one RET inhibitor resistance mutation that confers increased resistance to the first treatment to the cancer cell or tumor. an RET inhibitor previously administered to the subject; or (c) administering additional doses of the first RET inhibitor previously administered to the subject if the subject has a cancer cell that does not have a RET inhibitor resistance mutation that confers increased resistance to treatment with the first RET inhibitor previously administered to the subject .

This document also provides a method of treating a subject suffering from cancer, comprising: (a) administering one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition obtained using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, for a certain period of time; (b) after (a) - determining if the cancer cell in the sample obtained from the subject has one or more RET inhibitor resistance mutations that confer increased resistance to treatment with a compound of Formula I-IV or a pharmaceutically acceptable compound thereof to the cancer cell or tumor salt, amorphous or polymorphic form, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition obtained using a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form used in stage (a); and (c) administering a second RET inhibitor or a second compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, to a subject who has a cancer cell that has one or more RET inhibitor resistance mutations that confers increased resistance to treatment with the compound in the cancer cell or tumor formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition obtained using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form, used in step (a); or (d) administering additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing

- 5 041220 compound of formula I-IV, for example, a pharmaceutical composition obtained using a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form used in stage (a), to a subject who has a cancer cell that does not has a RET inhibitor resistance mutation that confers increased resistance to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition containing a compound of formula I-IV, for example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, used in step (a).

Also provided herein is a method of treating a subject suffering from cancer, comprising: (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and who has previously taken one or more doses of a compound of formula I-IV or its pharmaceutical an acceptable salt, amorphous or polymorphic form, one or more RET inhibitor resistance mutations that confer increased resistance to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form previously administered to a subject in a cancer cell or tumor; (b) administering a second RET inhibitor or a second compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, to a subject who has a cancer cell that has one or more resistance mutations to a RET inhibitor that confers increased resistance to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, previously administered to a subject in a cancer cell or tumor; or (c) administering additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, previously administered to a subject who has a cancer cell that does not have a RET inhibitor resistance mutation that confers on the cancer cell or tumor an increased resistance to treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, previously administered to a subject.

This document also provides a pharmaceutical combination for the treatment of cancer (e.g., RET-associated cancer, such as RET-associated cancer having one or more RET inhibitor resistance mutations) in a patient in need thereof, which contains (a) a compound of formula I -IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, (b) an additional therapeutic agent, and (c) optionally at least one a pharmaceutically acceptable carrier, wherein the compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form, and an additional therapeutic agent are in the form of separate compositions or dosage forms for simultaneous, separate or sequential use for the treatment of cancer, wherein the amounts of the compound of formula I-IV or its pharmaceutically an acceptable salt, amorphous or polymorphic form, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and an additional therapeutic agent together are effective for the treatment of cancer. Also provided herein is a pharmaceutical composition containing such a combination. This document also proposes the use of such a combination for the preparation of a medicament for the treatment of cancer. This document also proposes an industrial package or product containing such a combination as a combined preparation for simultaneous, separate or sequential use; and a method for treating cancer in a patient in need thereof.

The present invention also provides a method for reversing or preventing acquired resistance to an anticancer drug, comprising administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, to a patient at risk of developing or having acquired resistance to an anticancer drug. In some embodiments, a dose of an anti-cancer drug is administered to a patient (e.g., administration to a patient occurs at substantially the same time as administration of a dose of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using compounds of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form).

The present invention also provides a method for delaying and/or preventing the development of cancer resistance in an individual to an anticancer drug, comprising administering to said individual an effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition obtained using the compound formulas I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form, before, during or after administration of an effective amount of an anticancer drug.

Also provided herein is a method of treating an individual suffering from cancer who is more likely to develop resistance to an anticancer drug, which comprises administering to said individual (a) an effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, before, during or after administration of (b) an effective amount of an anticancer drug.

The invention also provides methods of treating an individual suffering from a RET-associated cancer that has one or more RET inhibitor resistance mutations that increase the resistance of the cancer to the first RET inhibitor (e.g., one or more amino acid substitutions in the kinase domain (e.g., amino acid positions 700 - 1012 in the wild-type RET protein), in the gatekeeper amino acid (for example, amino acid position 804 in the wild-type RET protein), in the P-loop (for example, amino acid positions 730 - 737 in the wild-type RET protein), in the XDFG residue ( e.g., amino acid position 891 in the wild-type RET protein), in the amino acids of the solvent front of the ATP gap (e.g., amino acid positions 806-811 in the wild-type RET protein), in the activation loop (e.g., amino acid positions 891-916 in the wild-type RET protein) , in the C-helix and in the loop preceding the C-helix (e.g., amino acid positions 768-788 in the wild-type RET protein), and/or in the ATP-binding site (e.g., amino acid positions 730-733, 738, 756, 758, 804, 805, 807, 811, 881, and 892 in the wild-type RET protein) (e.g., a substitution at amino acid position 804, such as V804M, V804L, or V804E, or position 810, for example, G810S, G810R, G810C, G810A, G810V, and G810D, and/or one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4) which comprise administering a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, before, during during or after administration of another anti-cancer drug (eg, a second RET kinase inhibitor). See also J. Kooistra, G. K. Kanev, O. P. J. Van Linden, R. Leurs, I. J. P. De Esch, and C. De Graaf, KLIFS: A structural kinase-ligand interaction database, Nucleic Acids Res., vol. 44, no. D1 , cc. D365-D371, 2016; and O. P. J. Van Linden, A. J. Kooistra, R. Leurs, I. J. P. De Esch, and C. De Graaf, KLIFS: A knowledge-based structural database to navigate kinase-ligand interaction space, J. Med. Chem., Vol. 57, No. 2, pp. 249-277, 2014, both of which are incorporated herein by reference in their entirety. In some embodiments, the wild-type RET protein is an exemplary wild-type RET protein described herein.

Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as is generally understood by a person skilled in the art to which this invention pertains. Methods and materials are described herein in the context of the use of this invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database access numbers and other references mentioned in this document are incorporated herein by reference in their entirety. In case of conflict, this description, including definitions, shall govern.

Other features and advantages of this invention will become apparent from the following detailed description and drawings, as well as from the claims.

Description of graphic materials

Fig. 1A-1E are plots of the solubility of a compound of formula I (free base) in various solvent systems. Fig. 1A is a solubility plot in DCM/EtOH. Fig. 1B is a DMSO/EtOH solubility plot. Fig. 1C is a DMSO/H ₂ O solubility plot. FIG. 1D is a THF/EtOH solubility plot. Fig. 1E is a THF/H2O solubility plot.

Fig. 2A-2G are free base diffraction patterns of the compound of formula I. FIG. 2A is an X-ray powder diffraction pattern of Form A of a compound of formula I (free base). Fig. 2B is an overlay of free base x-ray powder diffraction patterns from different batches before and after EAF analysis. Fig. 2C is a free base differential calorimetry curve. Fig. 2D is an isothermal (25° C.) dynamic sorption curve for free base vapor. Fig. 2E is a free base thermogravimetric curve. Fig. 2F is the Fourier transform IR spectrum of the free base. Fig. 2G is the 1H NMR spectrum of the free base in DMSO-d ⁶ .

In FIG. 3 is a differential scanning calorimetry curve of the maleic acid salt of the compound of Formula I identified in the initial salt screen.

In FIG. 4 is a differential scanning calorimetry curve for the acetic acid salt of the compound of Formula I identified in the initial salt screen.

In FIG. 5 is a differential scanning calorimetry curve of the D-malic acid salt of the compound of Formula I identified in the initial salt screen.

In FIG. 6 is a differential scanning calorimetry curve of the benzoic acid salt of the compound of formula I identified in the initial salt screen.

In FIG. 7 is a differential scanning calorimetry curve of the L-tartaric acid salt of the compound of Formula I identified in the initial salt screen.

In FIG. 8 is a differential scanning calorimetry curve for the citric acid salt of the compound of Formula I identified in the initial salt screen.

In FIG. 9 is a differential scanning calorimetry curve of the propionic acid salt of the compound of formula I identified in the initial salt screen.

In FIG. 10 is a differential scanning calorimetry curve of the D-tartaric acid salt of the compound of formula I identified in the initial salt screen.

In FIG. 11 is a differential scanning calorimetry curve of the L-malic acid salt of the compound of Formula I identified in the initial salt screen.

In FIG. 12 is a differential scanning calorimetry curve for the sulfuric acid salt of the compound of Formula I identified in the initial salt screen.

In FIG. 13 is a differential scanning calorimetry curve of the hydrochloric acid salt of a compound of formula I obtained on a scale of about 0.2 g.

In FIG. 14 is a differential scanning calorimetry curve of the hydrobromic acid salt of the compound of formula I obtained on a scale of about 0.2 g.

Fig. 15 are differential scanning calorimetry curves of the hydrochloric acid salt of the compound of formula I obtained from the optimization process. Fig. 15A is a differential scanning calorimetry curve of hydrochloric acid salt (obtained using DMA as solvent). Fig. 15B is a differential scanning calorimetry curve of hydrochloric acid salt (prepared using DCM/EtOH as solvent).

Fig. 16A-16F is a curve of the hydrochloric acid salt of a compound of formula I, prepared on a 2 g scale. FIG. 16A is a hydrochloric acid salt curve (prepared using DMA as solvent). Fig. 16B is an isothermal (25° C.) dynamic vapor sorption curve of the hydrochloric acid salt (prepared using DMA as solvent). Fig. 16C is a thermogravimetric analysis curve of hydrochloric acid salt (prepared using DMA as solvent). Fig. 16D is an overlay of a differential scanning calorimetry plot and a thermogravimetric analysis plot of the hydrochloric acid salt (prepared using DCM/EtOH as solvent). Fig. 16E is an overlay of X-ray powder diffraction patterns of a hydrochloric acid salt prepared using DMA or DCM/EtOH as solvent at different scales, before and after DSP. Fig. 16F is the 1H NMR spectrum of hydrochloric acid salt in DMSO-d ⁶ .

Fig. 17A-17C are diagrams of the hydrobromic acid salt of a compound of Formula I prepared on a 2 g scale. FIG. 17A is an overlay of a differential scanning calorimetry plot and a thermogravimetric analysis plot of a hydrobromic acid salt. Fig. 17B is an X-ray powder diffraction pattern of a hydrobromic acid salt. Fig. 17C is a Fourier transform IR spectrum of hydrobromic acid salt. Fig. 17D is the 1H NMR spectrum of the salt of hydrobromic acid in DMSO-d ⁶ .

Fig. 18A-18H are diagrams of the L- and D-malic acid salts of the compound of Formula I. FIG. 18A is an overlay of a differential scanning calorimetry curve and an L-malic acid salt thermogravimetric curve. Fig. 18B is an overlay of a DSC curve and a thermogravimetric curve of D-malic acid salt. Fig. 18C is a diagram of isothermal (25°C) dynamic vapor sorption of L-malate salt. Fig. 18D is a graph of isothermal (25° C.) dynamic vapor sorption of D-malic acid salt. Fig. 18E is an overlay of X-ray powder diffraction patterns of L- and D-malic acid salts. Fig. 18F is an overlay of Fourier transform IR spectra of L- and D-malic acid salts. Fig. 18G is the 1H NMR spectrum of malic acid salt in DMSO-d ⁶ . Fig. 18H is the 1H NMR spectrum of a salt of malic acid in DMSO-d ⁶ . Fig. 181 are plots of two batches of the malic acid salt L of the compound of formula I. FIG. 18I is a differential scanning calorimetry curve of L-malic acid salt (lot A). Fig. 18J is a thermogravimetric analysis curve of L-malic acid salt (lot A). Fig. 18K is a differential scanning calorimetry curve of L-malic acid salt (batch B). Fig. 18L is a thermogravimetric analysis curve of L-malic acid salt (batch B). Fig. 18M is an overlay of X-ray powder diffraction patterns of L-malic acid salts (Lot A and Lot B) and the free base of the compound of Formula I.

Fig. 19A-19F are X-ray diffraction patterns of Form 1 polymorph of the compound of formula II.

- 8 041220

Fig. 19A is an X-ray powder diffraction pattern of a completely dried form 1. FIG. 19B is a Form 1 differential scanning calorimetry curve. FIG. 19C is a thermogravimetric/differential thermal analysis curve of Form 1. FIG. 19D is a gravimetric vapor sorption isotherm of Form 1. FIG. 19E is a kinetic gravimetric vapor sorption curve of Form 1. FIG. 19F is the 1H NMR spectrum of form 1 in d ₆ -DMSO.

Fig. 20A-20E are diagrams of the Form 2 polymorph of the compound of formula II. Fig. 20A illustrates the X-ray powder diffraction patterns of Form 2 (suspension on a small scale, suspension on a large scale, and fully dried). Fig. 20B is a Form 2 differential scanning calorimetry curve. FIG. 20C is a thermogravimetric/differential thermal analysis curve of Form 2. FIG. 20D is a gravimetric vapor sorption isotherm of Form 2. FIG. 20E is the kinetic gravimetric vapor sorption curve of Form 2.

Fig. 21A-21F are diagrams of Form 7 polymorph of the compound of formula II. Fig. 21A illustrates the X-ray powder diffraction patterns of Form 7 (suspension on a small scale, suspension on a large scale and fully dried). Fig. 21B is a Form 7 differential scanning calorimetry curve. FIG. 21C is the thermogravimetric/differential thermal analysis curve of Form 7.

Fig. 21D is a gravimetric vapor sorption isotherm of Form 7. FIG. 21E is a kinetic gravimetric vapor sorption curve of form 7. FIG. 21F is the 1H NMR spectrum of form 7 in d ₆ -DMSO.

Fig. 22A-22F are diagrams of Form 8 polymorph of the compound of Formula II. Fig. 22A illustrates the X-ray powder diffraction patterns of Form 8 (suspension on a small scale, suspension on a large scale, and fully dried). Fig. 22B is a Form 8 differential scanning calorimetry curve. FIG. 22C is the thermogravimetric/differential thermal analysis curve of Form 8. FIG. 22D is a gravimetric vapor sorption isotherm of Form 8. FIG. 22E is a kinetic gravimetric vapor sorption curve of Form 8. FIG. 22F is the 1H NMR spectrum of form 8 in d ₆ -DMSO.

Fig. 23A-23F are diagrams of the phosphate salt of the compound of formula II. Fig. 23A is an X-ray powder diffraction pattern of a completely dried phosphate salt. Fig. 23B is a phosphate salt differential scanning calorimetry curve. Fig. 23C is a thermogravimetric/differential thermal analysis curve for a phosphate salt. Fig. 23D is the gravimetric vapor sorption isotherm of the phosphate salt. Fig. 23E is a kinetic gravimetric vapor sorption curve of the phosphate salt. Fig. 23F is the 1H NMR spectrum of form 1 in d ₆ -DMSO.

Fig. 24A-24B are X-ray powder diffraction patterns after screening for hydrochloric acid salts of a compound of formula II. Fig. 24A illustrates plots of the compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4 hour cycles over 24 hours (after cycles were repeated). In FIG. 24B is a plot of a compound of formula II in at least peaks in each solvent tested after samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 25A-25B are X-ray powder diffraction patterns after screening salts of a compound of Formula II with sulfuric acid. Fig. 25A illustrates plots of a Formula II compound in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4-hour cycles over 24 hours (after cycles were repeated). In FIG. 25B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 26A-26B are X-ray powder diffraction patterns after screening salts of a compound of formula II with p-toluenesulfonic acid. Fig. 26A illustrates plots of the compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4 hour cycles over 24 hours (after cycles were repeated). In FIG. 26B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 27A-27B are X-ray powder diffraction patterns after screening salts of a compound of formula II with methanesulfonic acid. Fig. 27A illustrates plots of the compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4-hour cycles for 24 hours (after cycles were repeated). In FIG. 27B is a plot of a compound of formula II in each solvent tested after the samples were left overnight in an oven at 40° C. and relative humidity.

75% (after achieving stability).

Fig. 28A-28B are X-ray powder diffraction patterns after screening salts of a compound of formula II with naphthalene-2-sulfonic acid. Fig. 28A illustrates plots of a compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 28B are plots of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 29A-29B are X-ray powder diffraction patterns after screening for salts of a compound of formula II with benzenesulfonic acid. Fig. 29A illustrates plots of a compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4 hour cycles over 24 hours (after cycles were repeated). In FIG. 29B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 30A-30B are X-ray powder diffraction patterns after screening salts of the compound of formula II with oxalic acid. Fig. 30A illustrates plots of a Formula II compound in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 30B are plots of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 31A-31B are X-ray powder diffraction patterns after screening salts of a compound of formula II with 2-hydroxyethanesulfonic acid. Fig. 31A illustrates plots of a compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4-hour cycles over 24 hours (after cycles were repeated). In FIG. 31B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 32A-32B are X-ray powder diffraction patterns after screening salts of a compound of formula II with L-aspartic acid. Fig. 32A illustrates plots of the Formula II compound in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 32B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 33A-33B are X-ray powder diffraction patterns after screening for maleic acid salts of a compound of formula II. Fig. 33A illustrates plots of a Formula II compound in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 33B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 34A-34B are X-ray powder diffraction patterns after screening salts of a compound of formula II with phosphoric acid. Fig. 34A illustrates plots of the compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4-hour cycles over 24 hours (after cycles were repeated). In FIG. 34B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 35A-35B are X-ray powder diffraction patterns after screening salts of a compound of formula II with ethanesulfonic acid. Fig. 35A illustrates plots of a compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4-hour cycles over 24 hours (after cycles were repeated). In FIG. 35B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 36A-36B are X-ray powder diffraction patterns after screening salts of a compound of formula II with L-glutamic acid. Fig. 36A illustrates diagrams

- 10 041220 compounds of formula II in each tested solvent after processing the samples in temperature cycles from room temperature to 40°C in 4-hour cycles for 24 hours (after repeating the cycles). In FIG. 36B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 37A-37B are X-ray powder diffraction patterns after screening salts of a compound of formula II with L-tartaric acid. Fig. 37A illustrates plots of a compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 37B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 38A-38B are X-ray powder diffraction patterns after screening salts of a compound of formula II with fumaric acid. Fig. 38A illustrates plots of a compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 38B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 39A-39B are X-ray powder diffraction patterns after screening salts of a compound of formula II with citric acid. Fig. 39A illustrates plots of a Formula II compound in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 39B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 40A-40B are X-ray powder diffraction patterns after screening salts of a compound of formula II with D-glucuronic acid. Fig. 40A illustrates plots of the compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 40B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 41A-41B are X-ray powder diffraction patterns after screening salts of the compound of formula II with L-malic acid. Fig. 41A illustrates plots of a compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 41B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 42A-42B are X-ray powder diffraction patterns after screening the hippuric acid salts of the compound of formula II. Fig. 42A illustrates plots of the compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 42B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 43A-43B are X-ray powder diffraction patterns after screening salts of a compound of formula II with D-gluconic acid. Fig. 43A illustrates plots of the compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 43B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 44A-44B are X-ray powder diffraction patterns after screening for salts of a compound of formula II with L-lactic acid. Fig. 44A illustrates plots of the compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 44B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after

- 11 041220 to achieve stability).

Fig. 45A-45B are X-ray powder diffraction patterns after screening salts of a compound of formula II with L-ascorbic acid. Fig. 45A illustrates plots of the compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 45B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 46A-46B are X-ray powder diffraction patterns after screening salts of a compound of formula II with benzoic acid. Fig. 46A illustrates plots of the compound of formula II in each solvent tested after the samples were treated in temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 46B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

Fig. 47A-47B are X-ray powder diffraction patterns after screening salts of the compound of formula II with succinic acid. Fig. 47A illustrates plots of the compound of formula II in each solvent tested after the samples were subjected to temperature cycles from room temperature to 40° C. in 4 hour cycles for 24 hours (after cycles were repeated). In FIG. 47B is a plot of a compound of formula II in each solvent tested after the samples were stored overnight in an oven at 40° C. and 75% relative humidity (after reaching stability).

In FIG. 48 shows thermogravimetric/differential thermal analysis curves for the sulfate salt of the Formula II compound identified in the initial salt screen.

In FIG. 49 shows thermogravimetric/differential thermal analysis curves for the tosylate salt of the compound of formula II identified in the initial salt screen.

In FIG. 50 shows thermogravimetric/differential thermal analysis curves for the naphthalene-2-sulfonate salt of the compound of formula II identified in the initial salt screen.

In FIG. 51 shows thermogravimetric/differential thermal analysis curves for the oxalate salt (1,4-dioxane/10% water) of the Formula II compound identified in the initial salt screen.

In FIG. 52 shows thermogravimetric/differential thermal analysis curves for the oxalate salt (evaporation) of the compound of formula II identified in the initial salt screen.

In FIG. 53 is a thermogravimetric/differential thermal analysis curve of the phosphate salt (acetone/10% water) of the Formula II compound identified in the initial salt screen.

In FIG. 54 is a thermogravimetric/differential thermal analysis curve of the phosphate salt (1PA/10% water) of the Formula II compound identified in the initial salt screen.

In FIG. 55 is a thermogravimetric/differential thermal analysis curve for the tartrate salt of the Formula II compound identified in the initial salt screen.

In FIG. 56 is a thermogravimetric/differential thermal analysis curve for the fumarate salt of the Formula II compound identified in the initial salt screen.

In FIG. 57 shows X-ray powder diffraction patterns of the solids obtained from the solvents tested in the Form I Formula II solvent solubility screen.

Fig. 58A-58D show X-ray powder diffraction patterns of a compound of formula II after temperature cycling experiments in various solvents and after overnight at 40° C. and 75% RH.

In FIG. 59 shows X-ray powder diffraction patterns of a compound of formula II after evaporation experiments using various solvents.

In FIG. 60 shows X-ray powder diffraction patterns of a compound of formula II after quench experiments using various solvents.

In FIG. 61 shows X-ray powder diffraction patterns of a compound of formula II after anti-solvent experiments using various solvents.

Fig. 62A-62F are diagrams of a Formula II compound. Fig. 62A is an X-ray powder diffraction pattern of a compound of formula II. Fig. 62B is a differential scanning calorimetry curve for a compound of formula II. Fig. 62C is a thermogravimetric/differential thermal analysis curve for a compound of formula II. Fig. 62D is a dynamic vapor sorption isotherm of a compound of formula II. Fig. 62E is a kinetic dynamic vapor sorption curve of the compound of formula II. Fig. 62F is the ¹ H NMR spectrum of a compound of formula II in d ₆ -DMSO.

Fig. 63A-63B are diagrams of Form A polymorph of the compound of formula III. Fig. 63A is an X-ray powder diffraction pattern of the Form A polymorph of the compound of formula III. Fig. 63B is a differential scanning calorimetry curve of polymorph form A of the compound of formula III.

Fig. 64A-64B are diagrams of Form A polymorph of the compound of formula IV. Fig. 64A is an X-ray powder diffraction pattern of polymorph Form A of the compound of formula IV. Fig. 64B is a differential scanning calorimetry curve of polymorph form A of the compound of formula IV.

Fig. 65A-64B are diagrams of Form B polymorph of the compound of formula IV. Fig. 65A is an X-ray powder diffraction pattern of Form B polymorph of the compound of formula IV. Fig. 65B is a differential scanning calorimetry curve of polymorph form B of the compound of formula IV.

Fig. 66 is an overlay of X-ray powder diffraction patterns of polymorphs A and B of the compound of formula IV.

Detailed description

1. Definitions.

The term polymorph in this context refers to crystals of one compound having different physical properties, which is due to the order of the molecules in the crystal lattice. Different polymorphs of the same compound differ from one another in one or more chemical, physical, mechanical, electrical, thermodynamic and/or biological properties. Differences in physical properties exhibited by polymorphs can affect pharmaceutical parameters such as storage stability, compressibility, density (an important parameter in formulation and product manufacture), dissolution rate (an important factor in determining bioavailability), solubility, melting point, chemical stability , physical stability, powder flowability, water sorption, caking and particle morphology. Differences in stability can lead to changes in reactivity (eg, differential oxidation, so that the dosage form of one polymorph discolors faster than the dosage form of another polymorph) or mechanical changes (eg, crystalline changes during storage, since the kinetically preferred polymorph is converted to a thermodynamically more stable polymorph), or a change in both of these properties (for example, one polymorph is more hygroscopic than another). Due to solubility/dissolution differences, some changes affect efficacy and/or toxicity. In addition, the physical properties of the crystal may be important for processing; for example, one polymorph may be more likely to form solvates or may be more difficult to filter and wash out free impurities (i.e., the shape and particle size distribution for one polymorph may differ from the shape and particle size distribution of another polymorph). A polymorph in this context does not include amorphous forms of the compound. In this context, amorphous refers to the non-crystalline form of the compound, which may be the solid form of the compound or the solubilized form of the compound. For example, amorphous refers to a compound (eg, the solid form of a compound) without a regularly repeating order of molecules or outer faces.

The term anhydrous as used herein refers to the crystalline form of a compound of formulas I-IV which contains 1% or less water by weight. For example, 0.5% or less, 0.25% or less, or 0.1% or less water by weight.

The term solvate as used herein refers to a crystalline form of a compound of formula IIV, such as a polymorphic form of the compound, in which the crystal lattice contains one or more crystallization solvents.

The term hydrate or hydrated polymorph refers to a crystalline form of a compound of formulas I-IV, such as a polymorph of the compound in which the crystal lattice contains water. Unless otherwise indicated, the term hydrate in this context refers to a stoichiometric hydrate. The stoichiometric hydrate contains water molecules as an integral part of the crystal lattice, and the removal of water molecules leads to instability of the crystal lattice. In comparison, a non-stoichiometric hydrate contains water, but a change in water content does not cause a significant change in the crystal structure. Drying non-stoichiometric hydrates can remove a significant portion of the water without significant disruption of the crystal structure, and the crystals can then be rehydrated to the original non-stoichiometric hydrated crystalline form. Unlike stoichiometric hydrates, dehydration and rehydration of nonstoichiometric hydrates is not accompanied by a phase transition and, therefore, all states of hydration of a nonstoichiometric hydrate represent the same crystalline form.

- 13 041220

Purity in relation to a composition containing a polymorph of a compound of formula I-IV refers to the percentage of one particular polymorph relative to another polymorph or amorph of a compound of formula I-IV in said composition. For example, a composition containing polymorph form 1, having a purity of 90%, contains 90 mass parts of form 1 and 10 mass parts of other polymorphic and/or amorphous forms of the compounds of formula I-IV.

In this context, a compound or composition is substantially free of one or more other components if said compound or composition does not contain a significant amount of such other components. For example, the composition may contain less than 5%, 4%, 3%, 2%, or 1% by weight of other components. Such components may include starting materials, residual solvents, or any other impurities that may occur during the preparation and/or isolation of the compounds and compositions provided herein. In some embodiments, the polymorphic form provided herein is substantially free of other polymorphic forms. In some embodiments, a particular polymorph of a compound of Formula I-IV is substantially free of other polymorphs if that particular polymorph represents at least 95% by weight of the compound of Formula I-IV present. In some embodiments, a particular polymorph of a compound of Formula I-IV is substantially free of other polymorphs if that particular polymorph is at least about 97%, about 98%, about 99%, or about 99.5%, by weight, of the compound of Formula I- IV. In some embodiments, a particular polymorph of a compound of Formula I-IV is substantially free of water if the amount of water is no more than about 2%, about 1%, or about 0.5% by weight of the polymorph.

As used herein, substantially pure with respect to the polymorphic form of a compound of formula I-IV means a sample of the polymorphic form of the compound having a purity greater than 90%, including greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%, and also including exactly or about 100% of the compound relative to the mass of the compound. The remainder of the material contains other form(s) of the compound and/or reactive impurities and/or process impurities arising from its preparation. For example, a polymorphic form of a compound of formulas I-IV may be considered substantially pure if it has a purity greater than 90% of the polymorphic form of a compound of formulas I-IV, measured in a manner currently known and generally accepted in the art, and the remaining less than 10% of the material contains another form(s) of the compound of formula I-IV and/or reaction impurities and/or process impurities. The presence of reactive impurities and/or process impurities can be determined by analytical techniques known in the art, such as, for example, chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry, or infrared spectroscopy.

The term about in front of a DSC, TGA, TG or DTA value that is written in degrees Celsius means a tolerance of ±5°C.

For a more concise description, some quantitative expressions in this context are specified as a range from about an amount of X to about an amount of Y. It should be understood that when a range is indicated, this range is not limited to the specified upper and lower limits, but includes the full range from about an amount of X to about an amount of Y. around the amount of Y, or any range in between.

Room temperature or room temperature t-ra refers to the ambient temperature in a typical laboratory, which is usually around 25°C.

In this context, the terms subject, individual, or patient, used interchangeably, refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the patient is a human. In some embodiments, the subject experiences and/or exhibits at least one symptom of the disease or disorder being treated and/or prevented. In some embodiments, the subject has been identified or diagnosed with cancer with dysregulation of the RET gene, RET protein, or expression or activity, or level of any of them (RET-associated cancer) (e.g., as determined using an assay or kit approved by a government agency). regulations and controls, such as those approved by the FDA). In some embodiments, the subject suffers from a tumor that is positive for dysregulation of the RET gene, RET protein, or expression, or activity, or level of any of them (e.g., as determined using an assay or kit approved by a regulatory agency) . The subject may be a subject with a tumor(s) that is positive for dysregulation of the RET gene, RET protein, or expression, or activity, or level of any of them (e.g., that is positive using an assay or kit approved by a regulatory agency). , for example, approved by the FDA). The subject may be one in which tumors are characterized by dysregulation of the RET gene, RET protein, or expression or activity or level of any of them (e.g., if the tumor is found to be such using a kit or assay approved by a regulatory agency, e.g. FDA). In some embodiments, the subject is predisposed to RET-associated cancer. In some embodiments, the subject has a medical history that indicates that the subject is suffering from a tumor characterized by dysregulation of the RET gene, RET protein, or their expression, or activity, or level (and optionally a medical history that indicates that subject should be treated with any of the compositions provided herein). In some embodiments, the patient is a pediatric patient.

The term pediatric patient in this context refers to a patient under 21 years of age at the time of diagnosis or treatment. The term pediatric can be further classified into various subgroups, including: newborns (from birth to the first month of life); infants (from 1 month to two years); children (from two years to 12 years); and teenagers (from 12 to 21 years old (but not including the twenty-second birthday)). Berhman RE, Kliegman R, Arvin AM, Nelson WE, Textbook of Pediatrics, 15e ed., Philadelphia: W.B. Saunders Company, 1996; Rudolph AM, et al., Rudolph's Pediatrics, 21e ed., New York: McGraw-Hill, 2002; and Avery MD, First LR, Pediatric Medicine, 2e ed., Baltimore: Williams &Wilkins; 1994. In some embodiments, the age of the pediatric patient is from birth to the first 28 days of life, from 29 days to less than two years, from two years to less than 12 years, or from 12 years to 21 years (but not including the twenty-second birthday) . In some embodiments, the age of the pediatric patient is birth to first 28 days of life, 29 days to less than 1 year, one month to less than four months, three months to less than seven months, six months to less than 1 year, 1 less than 2 years, 2 years to less than 3 years, 2 years to less than seven years, 3 years to less than 5 years, 5 years to less than 10 years, 6 years to less than 13 years, 10 years to less than 15 years old or from 15 years old to less than 22 years old.

In this context, the terms treat or treatment refer to therapeutic or palliative measures. Beneficial or desirable clinical results include, but are not limited to, all or partial relief of symptoms associated with the disease or disorder or condition, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease, delaying or slowing the progression of the disease , improvement or amelioration of the disease state (for example, one or more symptoms of the disease) and remission (partial or complete), overt or implicit. Treatment can also mean extending life expectancy compared to life expectancy without treatment.

The term administer or administer refers to a method of administering a dose of a compound or pharmaceutical composition to a vertebrate or invertebrate animal, including mammals, birds, fish or amphibians. The preferred route of administration may vary depending on various factors, such as the components of the pharmaceutical composition, the focus of the disease, and the severity of the disease.

The term pharmaceutically acceptable carrier or pharmaceutically acceptable excipient includes any and all solvents, co-solvents, complexing agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are not biologically or otherwise unwanted. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless the usual medium or agent is incompatible with the active ingredient, their use in the therapeutic compositions of this invention is contemplated. Additional active ingredients may also be incorporated into the compositions. In addition, various adjuvants commonly used in the art can be used. These and other similar compounds are described in the literature, for example, in the publication Merck Index, Merck & Company, Rahway, NJ. The inclusion of various components in pharmaceutical compositions is discussed, for example, in Gilman et al. (ed.) (2010); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th ed., The McGraw-Hill Companies.

A therapeutically effective amount or a pharmaceutically effective amount of a compound provided herein is that amount that is effective to achieve the desired effect, and may vary according to the nature and severity of the disease state and the effectiveness of the compound. The therapeutic effect is the alleviation to some extent of one or more symptoms of the disease and may include the healing of the disease. Healing means eliminating the symptoms of active disease. However, some long-term or permanent disease effect may exist even after healing has been achieved (eg, with extensive tissue damage).

The term RET-associated disease or disorder as used herein refers to diseases or disorders associated with or characterized by dysregulation of the RET gene, RET kinase (also referred to herein as RET kinase protein or RET kinase), or the expression or activity or level of any of them ( eg, one or more) (eg, any type of dysregulation of the RET gene, RET kinase, RET kinase domain, or expression, or activity, or level of any of these, as described herein). Non-limiting examples of a RET associated disease or disorder include, for example, cancer and gastrointestinal disorders such as irritable bowel syndrome (IBS).

The term RET-associated cancer as used herein refers to cancers associated with or characterized by dysregulation of the RET gene, RET kinase (also referred to herein as RET kinase protein or RET kinase), or the expression or activity or level of any of them. Non-limiting examples of RET-associated cancer are described herein.

Expression of dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a translocation of the RET gene that results in the expression of a fusion protein, a deletion in the RET gene that results in the expression of a RET protein that contains a deletion of at least one akino acid compared to the wild-type RET protein, or a mutation in the RET gene that results in the expression of the RET protein with one or more point mutations, or an alternative splice version of RET mRNA that produces a RET protein that results in a deletion at at least one amino acid in the RET protein compared to the wild-type RET protein) or an amplification of the RET gene that results in overexpression of the RET protein or autocrine activity due to overexpression of the RET gene in the cell, resulting in pathogenic upregulation of the kinase domain of the RET protein (e.g., constitutively active kinase domain of the RET protein) in the cell. As another example, dysregulation of the RET gene, the RET protein, or the expression or activity or level of any of these may be a mutation in the RET gene that encodes a RET protein that is constitutively active or has increased activity relative to the protein encoded by the gene. RET that does not contain such a mutation. For example, dysregulation of the RET gene, RET protein, or the expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the RET that contains a functional kinase domain and a second portion of the RET protein. partner (i.e. not RET). In some examples, dysregulation of the RET gene, RET protein, or expression or activity may result from a gene translocation of one RET gene with another non-RET gene. Non-limiting examples of hybrid proteins are described in table. 1. Non-limiting examples of point mutations/inserts/deletions of the RET kinase protein are described in Table 1. 2. Additional examples of RET kinase protein mutations (eg, point mutations) are RET inhibitor resistance mutations. Non-limiting examples of RET inhibitor resistance mutations are described in Table 1. 3 and 4.

The term wild-type describes a nucleic acid (e.g., RET gene or RET mRNA) or protein (e.g., RET protein) that occurs in a subject not suffering from a RET-associated disease, such as RET-associated cancer (and optionally also not at increased risk of developing RET-associated disease and/or not predisposed to RET-associated disease), or which occur in a cell or tissue derived from a subject not suffering from an RET-associated disease, such as RET-associated cancer (and optionally also not at increased risk development of RET-associated disease and/or not predisposed to RET-associated disease).

The term regulatory and control authority refers to the government authority that authorizes the medical use of pharmaceutical agents in a given country. For example, a non-limiting example of a government regulator is the Food and Drug Administration (FDA).

2. Polymorphs and pharmaceutically acceptable salts.

This invention relates to compounds of formula I-IV and their pharmaceutically acceptable salts, which exhibit rearranged kinase inhibition (RET) during transfection. In particular, this document proposes novel crystalline forms of 4-(6-(4-((6methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H- pyrazol-4-yl)pyrazolo[1,5a]pyridine-3-carbonitrile (formula I); 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3 -yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (formula II); 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[ 1,5-a]pyridine-3-carbonitrile (formula III); and 6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5a]pyridine- 3-carbonitrile (formula IV) and their pharmaceutically acceptable salts, pharmaceutical compositions containing said compounds, processes for the preparation of said compounds and the use of said compounds in therapy. More specifically, this invention relates to new crystalline forms of Formulas I-IV and their pharmaceutically acceptable salts, which are useful in the treatment and prevention of diseases that can be treated with a RET kinase inhibitor, including RET-associated diseases and disorders.

- 16 041220

Formula I

This document provides a compound of formula I:

I including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The compound of formula I provided herein can be prepared by methods known and understood by those skilled in the art. For example, synthetic methods can be used, such as those described in US Publication No. 2017/0096425, and said application is incorporated herein by reference in its entirety.

Provided herein are polymorphic forms of a compound of formula I. These forms include, for example, free bases, solvates, hydrates, salts, and unsolvated forms of a compound of formula I, including, for example, polymorphic form A. In some embodiments, a polymorphic form of a compound of formula I is pharmaceutically acceptable salt. In some embodiments, the compound of Formula I is the chloride salt. In some embodiments, the compound of formula I is a bromide salt. In some embodiments, the compound of Formula I is the L-malate salt. In some embodiments, the compound of Formula I is the D-malate salt.

Form A.

One such polymorph is the polymorph known as Form A. Form A is the polymorph of the compound of Formula I. In some embodiments, Form A has a CuKai RPD diffraction pattern that has at least peaks at °2θ 4, 4±0.2, 14.6±0.2 and 18.3±0.2. In some embodiments, Form A has an RPD diffraction pattern with at least peaks at °2θ values of 4.4±0.2, 13.5±0.2, 14.6±0.2, 18.3±0, 2 and 18.8±0.2. In some embodiments, Form A has an RPD diffraction pattern with at least peaks at °2θ values of 4.4±0.2, 13.5±0.2, 14.6±0.2, 17.4±0, 2, 18.3±0.2, 18.8±0.2, 21.0±0.2 and 24.6±0.2. In some embodiments, Form A has an RPD diffraction pattern with at least peaks at °2θ values of 4.4±0.2, 13.5±0.2, 14.6±0.2, 17.4±0, 2, 18.3±0.2, 18.8±0.2, 21.0±0.2 and 24.6±0.2, 22.5±0.2, 24.6±0.2 and 27.7±0.2.

In some embodiments, this document provides a composition containing polymorph Form A. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8% or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I. For example, the composition may contain less than about 15% of the amorphous form.

In some embodiments, provided herein is a Form A polymorph that exhibits an endotherm observed at about 185-195°C, eg, about 192.8°C measured by DSC, associated with adsorbed water. In some embodiments, provided herein is a Form A polymorph that exhibits an endotherm observed at about 220-230°C, eg, about 226.7°C measured by DSC, associated with adsorbed water.

In some embodiments, this document provides a Form A polymorph that exhibits a weight loss of about 1.1% from the start of heating to about 238° C. as measured by TGA.

Provided herein are methods for preparing the Form A polymorph. In some embodiments, the Form A polymorph of a compound of Formula I is prepared by contacting 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazine- 1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3 carbonitrile tetrahydrochloride, sodium triacetoxyborohydride and triethylamine in a polar aprotic solvent. In some embodiments, the polar aprotic solvent is DMSO. In some embodiments, the proposed method further comprises heating a suspension containing 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3yl)pyrazolo[1,5- a]pyridine-3-carbonitrile tetrahydrochloride, sodium triacetoxyborohydride, trimethylamine and DMSO, up to about 30°C. In some embodiments, the proposed method is additionally

- 17 041220 but includes heating the suspension for from about 10 hours to about 15 hours, for example about 13 hours. In some embodiments, the proposed method further includes cooling the suspension to about 19°C after about 13 hours of heating to about 30°C. In some embodiments, the proposed method further comprises adding water to the slurry. For example, the proposed method may further include adding 2 volumes of water to the suspension. In some embodiments, the proposed method includes maturation of the composition containing the specified suspension and water. In some embodiments, the proposed method includes maturation of the composition containing the specified suspension and water, for from about 1 to about 10 hours, such as about 3.5 hours. In some embodiments, the proposed method includes isolating the solid by filtration. In some embodiments, the solid is dried. In some embodiments, the solid is dried under vacuum. In some embodiments, the solid is dried at about 45°C.

Salts of formula I.

In some embodiments, a compound of Formula I is a pharmaceutically acceptable salt. For example, pharmaceutically acceptable salts of a compound of Formula I may include, but are not limited to, the chloride, bromide, sulfate, citrate, L-tartrate, D-tartrate, acetate, L-malate, D-malate, benzoate, propionate, and maleate salts. In some embodiments, the compound of Formula I is the chloride salt. In some embodiments, the chloride salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 1:1 by volume. In some embodiments, the chloride salt is prepared in dimethylacetamide. In some embodiments, the compound of Formula I is the bromide salt. In some embodiments, the bromide salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the bromide salt is prepared in dimethylacetamide. In some embodiments, the compound of Formula I is the sulfate salt. In some embodiments, the sulfate salt is prepared in a mixture of solvents. In some embodiments, the sulfate salt is prepared in a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 4:1 by volume. In some embodiments, the compound of Formula I is the citrate salt. In some embodiments, the citrate salt is prepared in dichloromethane. In some embodiments, the compound of formula I is the L-tartrate salt. In some embodiments, the L-tartrate salt is prepared in dichloromethane. In some embodiments, the compound of formula I is a D-tartrate salt. In some embodiments, the D-tartrate salt is prepared in dichloromethane. In some embodiments, the compound of formula I is an acetate salt. In some embodiments, the acetate salt is prepared in dichloromethane. In some embodiments, the compound of Formula I is the L-malate salt. In some embodiments, the L-malate salt is prepared in dichloromethane. In some embodiments, the L malate salt is prepared in a mixture of solvents. In some embodiments, the L-malate salt is prepared in a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 3.3:1 by volume. In some embodiments, the compound of Formula I is the D-malate salt. In some embodiments, the D-malate salt is prepared in dichloromethane. In some embodiments, the D-malate salt is prepared in a mixture of solvents. In some embodiments, the D-malate salt is prepared in a mixture of dichloromethane and ethanol. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the compound of formula I is a benzoate salt. In some embodiments, the benzoate salt is prepared in dichloromethane. In some embodiments, the compound of formula I is a propionate salt. In some embodiments, the propionate salt is prepared in dichloromethane. In some embodiments, the compound of formula I is a maleate salt. In some embodiments, the maleate salt is prepared in dichloromethane.

Provided herein is a chloride salt of a compound of Formula I. In some embodiments, the chloride salt has an O:free base ratio of about 1.1:1.

In some embodiments, this document provides a composition containing the chloride salt of a compound of Formula I. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some

- 18 041220 In other embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I.

In some embodiments, provided herein is a chloride salt of a compound of Formula I that exhibits an endotherm observed at about 230-245°C, such as about 241°C or 234°C measured by DSC, associated with adsorbed water. In some embodiments, provided herein is a chloride salt of a compound of formula I that exhibits a melting point of about 241° C. as measured by DSC.

In some embodiments, the chloride salt of a compound of formula I undergoes a weight loss of about 7.4% from the start of heating to about 255° C., as measured by TGA.

This document provides methods for preparing the chloride salt of a compound of formula I. In some embodiments, the proposed method includes suspending a composition containing a compound of formula I in a mixture of dichloromethane and ethanol and adding a solution of hydrochloric acid to the resulting mixture to obtain the chloride salt as a residual solid. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 1:1 by volume. In some embodiments, the proposed method includes suspending a composition containing a compound of Formula I in dimethylacetamide and adding a solution of hydrochloric acid to the resulting mixture to form the chloride salt as a residual solid. In some embodiments, hydrochloric acid is added in an aqueous solution. In some embodiments, the solvent is used in an amount of about 46 volumes. In some embodiments, the solvent is used in an amount of about 50 volumes. In some embodiments, the solvent is used in an amount of about 75 volumes. In some embodiments, the temperature of the slurry is periodically changed from about 0° C. to about room temperature. In some embodiments, the temperature of the slurry is periodically changed from about 0°C to about 30°C. In some embodiments, the temperature of the slurry is periodically changed from about 40° C. to about room temperature. In some embodiments, the proposed method includes adding MTBE to the slurry. In some embodiments, the proposed method includes adding about 125 volumes of MTBE. In some embodiments, the proposed method includes the maturation of the suspension. In some embodiments, the proposed method includes aging the slurry for about 3 hours. In some embodiments, the proposed method includes aging the slurry for about 13 hours. In some embodiments, the proposed method includes aging the slurry for about 20 to about 40 hours, for example about 30 hours In some embodiments, the implementation of the proposed method includes stirring the suspension. In some embodiments, the proposed method includes isolating the solid by filtration.

Provided herein is a bromide salt of a compound of Formula I. In some embodiments, the bromide salt has a Br:free base ratio of about 1.1:1.

In some embodiments, provided herein is a composition containing a bromide salt of a compound of Formula I. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I.

In some embodiments, provided herein is a bromide salt of a compound of Formula I that exhibits an endotherm observed at about 235-250°C, eg, about 238°C measured by DSC, associated with sorbed water. In some embodiments, provided herein is a bromide salt of a compound of Formula I that exhibits an endotherm observed at about 220-235°C, eg, about 225°C measured by DSC, associated with sorbed water. In some embodiments, provided herein is a bromide salt of a compound of Formula I that exhibits a melting point of about 225° C. as measured by DSC. In some embodiments, provided herein is a bromide salt of a compound of Formula I that exhibits a melting point of about 238° C. as measured by DSC.

In some embodiments, the bromide salt of a compound of formula I undergoes a weight loss of about 10.3% from the start of heating to about 255° C., as measured by TGA.

This document provides methods for preparing the bromide salt of a compound of formula I. In some embodiments, the proposed method includes suspending a composition containing a compound of formula I in a mixture of dichloromethane and ethanol and adding a solution of hydrobromic acid to the resulting mixture to obtain the bromide salt as a residual solid. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the proposed method includes suspending a composition containing a compound of Formula I in dimethylacetamide and adding a solution of hydrobromic acid to the resulting mixture to form the bromide salt as a residual solid. In some embodiments, hydrobromic acid is added in an aqueous solution. In some embodiments, the solvent is used in the amount of 46 volumes. In some embodiments, the solvent is used in an amount of about 50 volumes. In some embodiments, the temperature of the slurry is periodically changed from about 0° C. to about room temperature. In some embodiments, the temperature of the slurry is periodically changed from about 30° C. to about room temperature. In some embodiments, the temperature of the slurry is periodically changed from about 40° C. to about room temperature. In some embodiments, the proposed method further comprises adding MTBE to the slurry. In some embodiments, the proposed method includes adding about 150 volumes of MTBE. In some embodiments, the proposed method includes the maturation of the suspension. In some embodiments, the proposed method includes aging the slurry for about 1 hour. In some embodiments, the proposed method includes aging the slurry for about 13 hours. In some embodiments, the proposed method includes aging the slurry for about 10 hours to about 30 hours, for example, about 20 hours. In some embodiments, the proposed method includes isolating the solid by filtration.

Provided herein is an L-malate salt of a compound of Formula I. In some embodiments, the L-malate salt has a malate:free base ratio of about 0.97:1.

In some embodiments, provided herein is a composition containing the L malate salt of a compound of Formula I. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8% or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I.

In some embodiments, provided herein is an L-malate salt of a compound of formula I that exhibits an endotherm observed at about 205-220°C, eg, about 208°C measured by DSC, associated with sorbed water. In some embodiments, provided herein is an L-malate salt of a compound of formula I that exhibits a melting point of about 208° C. as measured by DSC.

In some embodiments, the L-malate salt of a compound of Formula I undergoes a weight loss of about 17.7% from the start of heating to about 253° C., as measured by TGA.

Provided herein are methods for preparing the L-malate salt of a compound of formula I. In some embodiments, the proposed method comprises suspending a composition containing a compound of formula I in a mixture of dichloromethane and ethanol and adding a solution of L-malic acid to the resulting mixture to form the L-malate salt as residual solid. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 3.3:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is about 6:1 by volume. In some embodiments, L-malic acid is added in an ethanolic solution. In some embodiments, the solvent is used in an amount of about 46 volumes. In some embodiments, the solvent is used in an amount of about 26 volumes. In some embodiments, the temperature of the slurry is periodically changed from about 0° C. to about room temperature. In some embodiments, the proposed method includes the maturation of the suspension. In some embodiments, the proposed method includes maturing the slurry for about 5 hours to about 24 hours, such as about 13 hours. In some embodiments, the proposed method includes maturing the suspension for about 10 hours to about 30 hours, such as about 20 hours. In some embodiments, the proposed method includes agitation of the slurry. In some embodiments, the proposed method includes isolating the solid by filtration.

Provided herein is a D-malate salt of a compound of Formula I. In some embodiments, the D-malate salt has a malate:free base ratio of about 0.97:1.

In some embodiments, provided herein is a composition containing the D malate salt of a compound of Formula I. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula I. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula I. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula I.

In some embodiments, provided herein is a D-malate salt of a compound of Formula I that exhibits an endotherm observed at about 205-215°C, eg about 208°C measured by DSC, associated with adsorbed water. In some embodiments, provided herein is a D-malate salt of a compound of Formula I that exhibits a melting point of about 209° C. as measured by DSC.

In some embodiments, the D-malate salt of a compound of Formula I undergoes a weight loss of about 18.4% from the start of heating to about 250° C., as measured by TGA.

Provided herein are methods for preparing the D-malate salt of a compound of formula I. In some embodiments, the proposed method comprises suspending a composition containing a compound of formula I in a mixture of dichloromethane and ethanol and adding a solution of D-malic acid to the resulting mixture to form the D-malate salt as residual solid. In some embodiments, the ratio of dichloromethane to ethanol is about 3.6:1 by volume. In some embodiments, the ratio of dichloromethane to ethanol is 3.3:1 by volume. In some embodiments, D-malic acid is added in an ethanolic solution. In some embodiments, the solvent is used in an amount of about 46 volumes. In some embodiments, the temperature of the slurry is periodically changed from about 0° C. to about room temperature. In some embodiments, the proposed method includes the maturation of the suspension. In some embodiments, the proposed method includes maturing the suspension for about 13 hours. In some embodiments, the proposed method includes maturing the suspension for about 20 hours. In some embodiments, the proposed method includes isolating the solid by filtration.

Formula II.

The present invention provides a compound of formula II

including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The compound of formula II provided herein can be prepared by methods known and understood by those skilled in the art. For example, synthetic methods can be used, such as those described in US Provisional Applications Serial Nos. 62/406252; 62/447850; 62/491164; 62/554817; or 62/566093, and these applications are incorporated herein by reference in their entirety.

This document provides polymorphic forms of the compound of formula II. These forms include, for example, free bases, solvates, hydrates, salts, and unsolvated forms of the compound of formula II, including, for example, polymorphs 1, 2, 7, and 8. In some embodiments, the polymorph of the compound of formula II is a pharmaceutically acceptable salt. In some embodiments, the compound of formula II is a phosphate salt.

Form 1.

One such polymorph is the polymorph known as form 1. Form 1 is an anhydrous polymorph of the compound of formula II. In some embodiments, Form 1 has an X-ray powder diffraction (XRD) diffraction pattern obtained with CuKα1 radiation, with at least peaks at °2θ values of 16.5±0.2, 18.9±0.2, and 26, 0±0.2. In some embodiments, Form 1 has an RPD diffraction pattern with at least peaks at °2θ values of 16.5±0.2, 18.9±0.2, 23.8±0.2, 25, 3±0.2 and 26.0±0.2. In some embodiments, Form 1 has an RPD diffraction pattern with at least peaks at °2θ values of 16.5±0.2, 17.8±0.2, 18.9±0.2, 23.8±0, 2, 25.3±0.2, 25.6±0.2, 26.0±0.2 and 28.3±0.2. In some embodiments, Form 1 has an RPD diffraction pattern with at least peaks at °2θ values of 9.8±0.2, 16.5±0.2, 17.8±0.2, 18.9+0, 2, 23.8±0.2, 25.0±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2 and 28.3±0.2.

In some embodiments, this document provides a composition containing polymorph form 1. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula II. For example, in some embodiments, the composition is substantially free of other anhydrous forms of the compound of formula II. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less by weight of one or more other forms of the compound of formula II. For example, the composition may contain less than about 15% Form 2, Form 7, Form 8, or a combination of two or more of these.

In some embodiments, this document provides a Form 1 polymorph that exhibits an endotherm observed at about 185-200°C, eg, about 195°C, as measured by differential scanning calorimetry (DSC) associated with sorbed water. In some embodiments, polymorph form 1 exhibits an endothermic event observed at about 200-210°C, for example, about 207°C. In some embodiments, these endotherms are observed using a scan rate of 10° C. per minute.

In some embodiments, this document provides a Form 1 polymorph that exhibits an endothermic event observed from its onset at about 190° C. as measured by thermogravimetric/differential thermal analysis (TG/DTA). In some embodiments, polymorph form 1 undergoes a weight loss of about 0.4% to about 200°C, for example, from about 190 to about 200°C. In some embodiments, Polymorph Form 1 exhibits an endothermic event from its initiation at about 204°C. In some embodiments, said endothermic event is accompanied by a corresponding weight loss of about 0.2%.

The present invention provides methods for preparing polymorph form 1. In some embodiments, the proposed method includes suspending a composition containing a compound of Formula II in a solvent selected from the group consisting of 1,4-dioxane, 1-butanol, 1-propanol, acetone, anisole, chloroform, cyclohexane, cyclohexanone, dichloromethane, DMSO, ethanol, ethyl acetate, isopropyl alcohol, methyl ethyl ketone, methyl acetate, 2-ethoxyethanol, 2-methyl-THF, methyl isobutyl ketone (MIBK), nitromethane, and THF to give polymorph Form 1 as a residual solid . In some embodiments, the solvent is ethyl acetate. In some embodiments, the solvent is in a mixture with water, for example, the solvent may be a mixture of water and acetone or water and acetonitrile. In some embodiments, water is present in an amount of about 20% by weight. In some embodiments, water is present in an amount of about 50% by weight. In some embodiments, the temperature of the slurry is periodically changed from about 40° C. to about room temperature. In some embodiments, the temperature cycling is carried out for about 60 hours to about 84 hours, such as about 72 hours. In some embodiments, the proposed method further includes collecting residual solids. In some embodiments, the residual solid is collected by filtration. In some embodiments, the proposed method further comprises drying the residual solid, such as under vacuum. In some embodiments, drying is carried out at a temperature of from about 30 to about 50°C, for example, about 40°C.

In some embodiments, a method for preparing a Form 1 polymorph is provided. The proposed method includes providing a composition containing a compound of Formula II in a solvent. In some embodiments, Form 1 polymorph can be prepared by evaporating the solvent from a composition containing a Formula II compound to give Form 1 polymorph as a residual solid, the solvent being selected from the group consisting of dichloromethane, DMSO, methyl acetate, 2-ethoxyethanol, nitromethane and mixtures of acetonitrile and water (20%). In some embodiments, the proposed method includes evaporating the solvent from the composition containing the compound of formula II, obtaining a mixture of polymorph Form 1 and another polymorph form as a residual solid, and the solvent is selected from the group consisting of acetone, chloroform and THF. In some embodiments, the residual solid is a mixture of Form 1 and Form 8.

In some embodiments, polymorph form 1 can be obtained by cooling a solution containing the compound of formula II in acetone, to a temperature of about 5°C, to precipitate polymorph form 1 as a residual solid. In some embodiments, the residual solid is a mixture of Form 1 and Form 8.

In some embodiments, polymorph Form 1 can be obtained by recrystallization of a composition containing a compound of formula II to obtain polymorph Form 1, wherein the recrystallization solvent is selected from the group consisting of a mixture of DMSO and water and a mixture of dichloromethane and heptane.

Form 2.

The present invention also provides a polymorph known as form 2. Form 2 is the hydrated polymorph of the compound of formula II. In some embodiments, form 2 has a CuKα1 RPD diffraction pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, and 24.2±0.2 . In some embodiments, Form 2 has an RPD pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0, 2 and 24.2±0.2. In some embodiments, form 2 has an RPD diffraction pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0, 2, 21.1±0.2, 23.4±0.2, 24.2±0.2 and 24.6±0.2. In some embodiments, form 2 has an RPD diffraction pattern with at least peaks at °2θ values of 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0, 2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2 and 31.2±0.2.

In some embodiments, this document provides a composition containing polymorph Form 2. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8% or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula II. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula II. For example, the composition may contain less than about 15% Form 1, Form 7, Form 8, or a combination of two or more of these.

In some embodiments, provided herein is a form 2 polymorph that exhibits an endotherm observed at about 190-200°C, eg, about 197.5°C measured by DSC, associated with sorbed water. In some embodiments, Form 2 polymorph exhibits an endothermic event observed at about 200-210°C, eg, about 207.5°C. In some embodiments, these endotherms are observed using a scan rate of 10° C. per minute.

In some embodiments, this document provides a Form 2 polymorph that exhibits a weight loss of about 0.7% from the start of heating to about 165° C. as measured by TG/DTA. In some embodiments, polymorph form 2 exhibits an endothermic event observed from its onset at about 194°C. In some embodiments, polymorph form 2 undergoes a weight loss of about 0.2% to about 200°C, for example, from about 194°C to about 200°C. In some embodiments, Polymorph Form 2 exhibits an endothermic event from its initiation at about 205°C.

Provided herein are methods for preparing Form 2 polymorph. In some embodiments, the proposed method comprises suspending a composition containing a compound of formula II in a mixture of ethanol and water to form Form 2 polymorph as a residual solid. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the temperature of the slurry is periodically changed from about 40° C. to about room temperature. In some embodiments, the temperature cycling is carried out for about 60 hours to about 84 hours, such as about 72 hours. In some embodiments, the proposed method further includes collecting residual solids. In some embodiments, the residual solid is collected by filtration. In some embodiments, the residual solid is dried. In some embodiments, the residual solid is dried on the filter bed.

Form 7.

The present invention provides a polymorph known as form 7. Form 7 is the hydrated polymorph of the compound of formula II. In some embodiments, form 7 has a CuKα1 RPD diffraction pattern with at least peaks at

- 23 041220 values of °2θ 16.6±0.2, 18.0±0.2 and 19.9±0.2. In some embodiments, form 7 has an RPD diffraction pattern with at least peaks at °2θ values of 16.6±0.2, 18.0±0.2, 19.3±0.2, 19.9±0, 2 and 23.3±0.2. In some embodiments, form 7 has an RPD diffraction pattern with at least peaks at °2θ values of 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0, 2, 19.3±0.2, 19.9±0.2, 23.3±0.2 and 25.1±0.2. For example, in some embodiments Form 7 has an RPD diffraction pattern with at least peaks at °2θ values of 15.8±0.2, 16.6±0.2, 17.3±0.2, 18.0± 0.2, 19.0±0.2, 19.3±0.2, 19.91±0.2, 21.4±0.2, 23.3±0.2 and 25.1±0, 2.

In some embodiments, this document provides a composition containing polymorph Form 7. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula II. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula II. For example, the composition may contain less than about 15% Form 1, Form 2, Form 8, or a combination of two or more of these.

In some embodiments, provided herein is a Form 7 polymorph that exhibits an endotherm observed at about 145-155°C, eg, about 150°C measured by DSC, associated with sorbed water. In some embodiments, polymorph form 7 exhibits an endotherm observed at about 190-205°C, for example, about 201°C. In some embodiments, polymorph Form 7 exhibits an endothermic event observed at about 205210°C, eg around 207°C. In some embodiments, these endotherms are observed using a scan rate of 10° C. per minute.

In some embodiments, this document provides polymorph Form 7 that exhibits an endothermic event observed from its onset at about 147° C. as measured by TG/DTA. In some embodiments, polymorph form 7 undergoes a weight loss of about 7% to about 150°C, for example, from about 145°C to about 155°C. In some embodiments, the weight loss is a solvent loss. In some embodiments, the weight loss is about two equivalents of solvent compared to the amount of compound present in the sample. In some embodiments, the solvent is water. In some embodiments, polymorph Form 7 exhibits an endothermic event observed from its onset at about 196°C. In some embodiments, Form 7 polymorph dehydrates upon heating to form Form 1 polymorph. In some embodiments, said endothermic event is associated with a transition observed in Form 1. In some embodiments, said transition is associated with a Form 1 endothermic event observed since it began. at about 206°C.

Provided herein are methods for preparing Form 7 polymorph. In some embodiments, the proposed method comprises suspending a composition containing a compound of formula II in a mixture of 1,4-dioxane and water to form Form 7 polymorph as a residual solid. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the temperature of the slurry is periodically changed from about 40° C. to about room temperature. In some embodiments, the temperature cycling is carried out for about 60 to about 84 hours, such as about 72 hours. In some embodiments, the proposed method further includes collecting residual solids. In some embodiments, the residual solid is collected by filtration. In some embodiments, the residual solid is dried. In some embodiments, the residual solid is dried on the filter bed.

Form 8.

The present invention provides a polymorph known as form 8. Form 8 is a solvated polymorph of the compound of formula II. Polymorph Form 8 is the isopropyl alcohol solvate polymorph of the compound of Formula II. In some embodiments, form 8 has a CuKα1 RPD diffraction pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, and 24.2±0.2 . In some embodiments, form 8 has an RPD diffraction pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0, 2 and 24.2±0.2. In some embodiments, form 8 has an RPD diffraction pattern with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0, 2, 21.1±0.2, 23.4±0.2, 24.2±0.2 and 24.6±0.2. In some embodiments, Form 8 has an RPD pattern with,

- 24 041220 at least peaks at °2θ values of 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0, 2, 21.1±0.2,

23.4±0.2, 24.2±0.2, 24.6±0.2 and 31.2±0.2.

In some embodiments, this document provides a composition containing polymorph Form 8. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula II. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula II. For example, the composition may contain less than about 15% Form 1, Form 2, Form 7, or a combination of two or more of these.

In some embodiments, this document provides a Form 8 polymorph that exhibits an endotherm observed at about 165-175°C, eg, about 172°C measured by DSC, associated with sorbed water. In some embodiments, polymorph form 8 shows an endotherm observed at about 185-200°C, for example, about 196°C. In some embodiments, the implementation of the polymorph form 8 shows an endothermic event, observed around 200210°C, for example, around 206°C. In some embodiments, these endotherms are observed using a scan rate of 10° C. per minute.

In some embodiments, this document provides polymorph Form 8 that exhibits an endothermic event observed at about 165° C. as measured by TG/DTA. In some embodiments, polymorph form 8 undergoes a weight loss of about 4% to about 165°C. In some embodiments, the weight loss is a solvent loss. In some embodiments, the weight loss is about 0.5 solvent equivalents. In some embodiments, the solvent is an IPA. In some embodiments, Polymorph Form 8 exhibits an endothermic event observed from its onset at about 191°C. In some embodiments, the endothermic event is associated with the transition observed in Form 1. In some embodiments, the transition is associated with the endothermic event of Form 1, observed from its onset at about 205°C.

Provided herein are methods for preparing Form 8 polymorph. In some embodiments, the proposed method comprises suspending a composition containing a compound of formula II in a solvent selected from the group consisting of IPA and 1-propanol to provide Form 8 polymorph as a residual solid. . In some embodiments, the temperature of the slurry is periodically changed from about 40° C. to about room temperature. In some embodiments, the temperature cycling is carried out for about 60 hours to about 84 hours, such as about 72 hours. In some embodiments, the proposed method further comprises collecting residual solids. In some embodiments, the residual solid is collected by filtration. In some embodiments, the proposed method further comprises drying the residual solid, such as under vacuum. In some embodiments, the drying is carried out at a temperature of from about 30°C to about 50°C, for example, about 40°C.

In some embodiments, a method for preparing a Form 8 polymorph is provided. The proposed method includes providing a composition containing a compound of Formula II in a solvent. In some embodiments, the proposed method includes evaporating the solvent from the composition containing the compound of formula II, including its amorphous and polymorphic forms, to obtain a mixture of polymorphic form 8 and another polymorphic form as a residual solid. In some embodiments, the residual solid is a mixture of Form 8 polymorph and Form 1 polymorph. In some embodiments, the solvent is acetone. In some embodiments, the solvent is chloroform. In some embodiments, the solvent is THF.

Salts of formula II.

In some embodiments, the Formula II compound is a pharmaceutically acceptable salt. For example, pharmaceutically acceptable salts of a compound of formula II may include, but are not limited to, the sulfate, tosylate, naphthalene-2-sulfonate, oxalate, phosphate, tartrate, and fumarate salts. In some embodiments, the compound of formula II is the sulfate salt. In some embodiments, the sulfate salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of IPA and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the compound of formula II is a tosylate salt. In some

- 25 041220 embodiments, the tosylate salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of acetone and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the compound of formula II is the naphthalene-2-sulfonate salt. In some embodiments, the naphthalene-2sulfonate salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of THF and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the compound of formula II is an oxalate salt. In some embodiments, the oxalate salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of 1,4-dioxane and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the oxalate salt is obtained by evaporation from a mixture of solvents. In some embodiments, the solvent is a mixture of THF and water. In some embodiments, the compound of formula II is a tartrate salt. In some embodiments, the tartrate salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of IPA and water. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the compound of formula II is a fumarate salt. In some embodiments, the fumarate salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of THF and water. In some embodiments, the compound of formula II is a phosphate salt. In some embodiments, the phosphate salt is prepared in a mixture of solvents. In some embodiments, the solvent is a mixture of acetone and water. In some embodiments, the solvent is a mixture of IPA and water. In some embodiments, water is present in an amount of about 10% by weight.

This document provides a phosphate salt of a compound of formula II. In some embodiments, the phosphate salt has a PO ₄ :free base ratio of about 1.4:1. In some embodiments, the phosphate salt has a CuKα1 RPD diffraction pattern with at least peaks at °2θ values of 3.6±0.2, 16.7±0.2, and 18.2±0.2 . In some embodiments, the phosphate salt has an RPD diffraction pattern with at least peaks at °2θ values of 3.6±0.2, 15.9±0.2, 16.7±0.2, 17.8±0, 2 and 18.2±0.2. In some embodiments, the phosphate salt has an RPD diffraction pattern with at least peaks at °2θ values of 3.6±0.2, 6.2±0.2, 15.9±0.2, 16.7±0, 2, 17.8±0.2, 18.2±0.2, 20.3±0.2 and 25.5±0.2. For example, in some embodiments, the phosphate salt has an RPD diffraction pattern with at least peaks at °2θ values of 3.6±0.2, 6.2±0.2, 15.9±0.2, 16.7± 0.2,17.8±0.2, 18.2±0.2,19.1±0.2, 20.3±0.2, 20.9±0.2 and 25.5±0, 2.

In some embodiments, this invention provides a composition containing a phosphate salt of a compound of formula II. In some embodiments, the composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula II. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula II. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula II.

In some embodiments, provided herein is a phosphate salt of a compound of formula II that exhibits an endotherm observed at about 165-175°C, eg, about 170°C measured by DSC, associated with sorbed water. In some embodiments, said endotherm is observed using a scan rate of 10° C. per minute.

In some embodiments, provided herein is a phosphate salt of a compound of formula II that exhibits a melting point of about 167° C. as measured by TG/DTA. In some embodiments, the phosphate salt of the compound of formula II undergoes a weight loss of about 1.3% from the start of heating to about 150°C. In some embodiments, the phosphate salt of a compound of Formula II exhibits a second weight loss of about 1.2% starting at about 167°C.

This document provides methods for preparing the phosphate salt of a compound of formula II. In some embodiments, the proposed method includes suspending the composition containing the compound of formula II in a mixture of water and IPA and adding a solution of phosphoric acid to the resulting mixture to obtain a phosphate salt as a residual solid. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the acid is a 1M phosphoric acid solution. In some embodiments, the temperature of the slurry is periodically changed from about 40° C. to about room temperature. In some

- 26 041220 embodiments, the periodic temperature change is carried out for from about 12 to about 48 hours, for example about 24 hours. In some embodiments, the proposed method further includes centrifuging the composition and collecting residual solids. In some embodiments, the residual solid is washed with a solvent. In some embodiments, the solvent is an IPA. In some embodiments, the proposed method further comprises drying the residual solid. In some embodiments, the residual solid is dried under vacuum. In some embodiments, the drying is carried out at a temperature of from about 30°C to about 50°C, for example, about 40°C.

Formula III.

The present invention provides a compound of formula III:

O

III including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The compound of formula III provided herein can be prepared by methods known and understood by those skilled in the art. For example, synthetic methods can be used, such as those described in US Provisional Applications Serial Nos. 62/406252; 62/447850; 62/491164; 62/554817; or 62/566093, and these applications are incorporated herein by reference in their entirety.

This document provides polymorphic forms of the compound of formula III. These forms include, for example, free bases, solvates, hydrates, salts, and unsolvated forms of the compound of formula III, including, for example, polymorph form A. In some embodiments, the polymorph form of the compound of formula III is a pharmaceutically acceptable salt.

Form A.

One such polymorph is the polymorph known as form A. Form A is the polymorph of the compound of formula III. In some embodiments, Form A has a CuKα1 RPD diffraction pattern with at least peaks at °2θ values of 17.3±0.2, 19.2±0.2, and 23.9±0.2 . In some embodiments, Form A has an RPD diffraction pattern with at least peaks at °2θ values of 4.7±0.2, 17.3±0.2, 18.8±0.2, 19.2±0, 2 and 23.9±0.2. In some embodiments, Form A has an RPD diffraction pattern with at least peaks at °2θ values of 4.7±0.2, 6.8±0.2, 15.2±0.2, 17.3±0, 2, 18.8±0.2, 19.2±0.2, 20.2±0.2 and 23.9±0.2. In some embodiments, Form A has an RPD diffraction pattern with at least peaks at °2θ values of 4.7±0.2, 6.8±0.2, 13.4±0.2, 15.2±0, 2, 15.9±0.2, 17.3±0.2, 18.8±0.2, 19.2±0.2, 20.2±0.2 and 23.9±0.2.

In some embodiments, this document provides a composition containing polymorph Form A. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula III. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula III. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula III. For example, the composition may contain less than about 15% amorphous form.

In some embodiments, provided herein is a Form A polymorph that exhibits an endotherm observed at about 135-150°C, such as about 140.5°C or 146.5°C as measured by DSC, associated with adsorbed water.

The present invention provides methods for preparing the Form A polymorph. In some embodiments, the proposed method comprises dissolving a compound of Formula III in acetonitrile and adding water to form the Form A polymorph as a solid. In some embodiments, the proposed method includes heating the composition containing the compound of formula III in acetonitrile to reflux. In some embodiments, the ratio of acetonitrile to water is about 2:3 by volume. In some embodiments, the residual solid is collected by filtration. In some embodiments, the residual solid is dried. In some embodiments, the residual solid is dried under

- 27 041220 high vacuum. In some embodiments, the residual solid is dried at about

40-45°C. In some embodiments, the residual solid is dried overnight.

Formula IV.

This document provides a compound of formula IV

He

IV including its pharmaceutically acceptable salts, amorphous and polymorphic forms.

The compound of formula IV provided herein can be prepared by methods known and understood by those skilled in the art. For example, synthetic methods can be used, such as those described in US Provisional Applications Serial Nos. 62/406275; 62/447849; 62/491180; 62/531690 or 62/566030, and these applications are incorporated herein by reference in their entirety.

This document provides polymorphic forms of the compound of formula IV. These forms include, for example, free bases, solvates, hydrates, salts, and unsolvated forms of the compound of formula IV, including, for example, polymorphs A and B. In some embodiments, the polymorph of the compound of formula IV is a pharmaceutically acceptable salt.

Form A.

One such polymorph is the polymorph known as form A. Form A is the polymorph of the compound of formula IV. In some embodiments, Form A has a CuKα1 RPD diffraction pattern with at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, and 21.9±0.2 . In some embodiments, Form A has an RPD diffraction pattern with at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, 16.6±0.2, 19.4±0, 2 and 21.9±0.2. In some embodiments, Form A has an RPD diffraction pattern with at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, 16.6±0.2, 19.4±0, 2, 20.0±0.2, 20.5±0.2, 21.6±0.2 and 21.9±0.2. For example, in some embodiments Form A has an RPD diffraction pattern with at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, 16.6±0.2, 18.1± 0.2, 18.8±0.2, 19.4±0.2, 20.0±0.2, 20.5±0.2, 21.6±0.2 and 21.9±0, 2.

In some embodiments, this document provides a composition containing polymorph Form A. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula IV. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula IV. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula IV. For example, the composition may contain less than about 15% Form B, amorphous form, or combinations thereof.

In some embodiments, provided herein is a Form A polymorph that exhibits an endotherm observed at about 145-155°C, eg, about 149.9°C measured by DSC, associated with adsorbed water.

The present invention provides methods for preparing polymorph form A. In some embodiments, the proposed method includes suspending a composition containing a compound of Formula IV in a solvent selected from the group consisting of acetone, acetonitrile, 2-butanol, chloroform, ethanol, ethyl acetate, heptane, hexane, isopropanol, MTBE, DMSO, THF, water, and combinations thereof, to give polymorph Form A as a residual solid. In some embodiments, the solvent is acetone, 2-butanol, or acetonitrile. In some embodiments, the solvent is in a mixture with water, for example, the solvent may be a mixture of water and acetone, water and ethanol, or water and DMSO. In some embodiments, water is present in an amount of about 50% by weight. In some embodiments, water is present in an amount of about 40% by weight. In some embodiments, the solvent is in a mixture with heptane, for example, the solvent may be a mixture of chloroform and heptane or heptane and acetone. In some embodiments, heptane is present in an amount of about 50% by weight. In some embodiments, heptane is present in an amount of about

- 28 041220

70% by weight. In some embodiments, Form A is prepared by adding an anti-solvent to a solution of a compound of formula IV in a solvent. In some embodiments, the antisolvent is heptane or water. In some embodiments, the solvent is DMSO and the anti-solvent is water. In some embodiments, the antisolvent vapor diffuses into the solution of the compound of formula IV. In some embodiments, the proposed method further comprises collecting residual solids. In some embodiments, the residual solid is collected by filtration. In some embodiments, the proposed method includes washing the solid. In some embodiments, the proposed method includes washing the solid with water, MTBE, or a combination thereof. In some embodiments, the proposed method further comprises drying the residual solid, such as under vacuum.

Form B.

One such polymorph is the polymorph known as form B. Form B is the polymorph of the compound of formula IV. In some embodiments, form B has a CuKα1 RPD diffraction pattern with at least peaks at °2θ values of 7.5±0.2, 13.7±0.2, and 16.9±0.2 . In some embodiments, form B has an RPD diffraction pattern with at least peaks at °2θ values of 7.5±0.2, 9.7±0.2, 13.7±0.2, 16.9±0, 2 and 19.9±0.2. In some embodiments, form B has an RPD diffraction pattern with at least peaks at °2θ values of 7.5±0.2, 9.7±0.2, 13.7±0.2, 14.5±0, 2, 16.9±0.2, 19.4±0.2, 19.9±0.2 and 21.3±0.2. In some embodiments, form B has an RPD diffraction pattern with at least peaks at °2θ values of 7.5±0.2, 9.7±0.2, 9.9±0.2, 13.7+0, 2, 14.5+0.2, 16.9+0.2, 19.4+0.2, 19.9+0.2, 21.3+0.2 and 27.4+0.2.

In some embodiments, this document provides a composition containing polymorph Form B. In some embodiments, said composition may be substantially pure. For example, the composition may be at least about 90% pure. In some embodiments, the composition may be at least about 95% pure. In some embodiments, the composition may be at least about 98% pure. For example, the composition may have a purity of at least 98.5, 98.6, 98.7, 98.8, 98.9, 99, 99.1, 99.2, 99.3, 99.4, 99.5 , 99.6, 99.7, 99.8 or 99.9%. In some embodiments, the composition is substantially free of other forms of the compound of formula IV. In some embodiments, the composition contains less than about 15% by weight of other forms of the compound of formula IV. For example, the composition may contain less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% by weight of one or more other forms of the compound of formula IV. For example, the composition may contain less than about 15% Form A, an amorphous form, or a combination thereof.

In some embodiments, provided herein is a Form B polymorph that exhibits an endotherm observed at about 160-170°C, eg, about 164.6°C measured by DSC, associated with adsorbed water.

Provided herein are methods for preparing Form B polymorph. In some embodiments, the proposed method comprises suspending a composition containing a compound of formula IV in a mixture of ethanol and water to form Form B polymorph as a residual solid. In some embodiments, water is present in an amount of about 10% by weight. In some embodiments, the slurry is allowed to mature for about 24 to about 72 hours, such as about 36 hours. In some embodiments, the proposed method further comprises collecting residual solids. In some embodiments, the residual solid is collected by filtration. In some embodiments, the residual solid is dried. In some embodiments, the residual solid is dried in a vacuum oven. In some embodiments, the residual solid is dried with a stream of nitrogen. In some embodiments, the residual solid is dried at room temperature. In some embodiments, the residual solid is dried for about 10 to about 20 hours, such as about 18 hours.

It should be understood that the 2-theta values in the RPD diffraction patterns for crystalline forms of the compounds of formulas I-IV, for example, Forms A of Formula I, Forms 1, 2, 7 and 8 of Formula I, Forms A of Formula III, or Forms A and B of Formula IV, as well as their pharmaceutically acceptable salts, e.g., chloride salt, bromide salt, malate salt and phosphate salt, may vary somewhat depending on the instrument, as well as variations in sample preparation and variations from batch to batch, and, therefore, the indicated values \u200b\u200bare not should be understood as absolute. It should be understood that the positions of the peaks in the RPD diffraction pattern are indicated in terms of the angular position (two theta) with an allowable variability of ±0.2° 2θ. The ±0.2° 2θ variability is used when comparing two powder RPD diffraction patterns. In practice, if a diffraction peak in the same diffraction pattern is assigned a range of angular positions (two theta) which is the measured peak position of ±0.2°, and if these peak position ranges overlap, then the two peaks are considered to have the same angular position. For example, if a peak in one diffraction pattern is found to have a position of 11.0° 2θ, then for comparison purposes, the allowable variability allows the peak to be assigned a position in the range of 10.8° - 29 041220

11.2° 2θ. It should also be understood that the relative intensity of the peaks may vary due to orientation effects, so that the intensity shown in the XRD patterns included herein is illustrative and should not be used for absolute comparison. In addition, it should be understood that for the purposes of comparison, some variability in the intensities of the peaks relative to their intensities presented on the RPD diffraction patterns is acceptable. Accordingly, it should be understood that the expression is essentially the same RPD pattern as shown in FIG. 1 means that for comparison purposes, at least 90% of the peaks shown in FIG. 1.

The compounds of this invention may also contain non-natural proportions of atomic isotopes on one or more of the atoms forming these compounds. That is, an atom, in particular when referred to in relation to a compound of formula I-IV, contains all isotopes and isotopic mixtures of that atom, such as naturally occurring isotopes in their natural abundance. For example, when referring to hydrogen, reference is made to 1H, ^2H , ^3H , or mixtures thereof; when mentioning carbon, it is meant to mention ¹² C, ¹³ C, ¹⁴ C, or mixtures thereof; when referring to nitrogen, reference is made to ¹⁴ N, ¹⁵ N or mixtures thereof; and when oxygen is mentioned, ¹⁶ O, ¹⁷ O, ¹⁸ O or mixtures thereof are meant. All isotopic variants of the compounds provided herein are considered to be within the scope of this invention.

For purposes of illustration, Schemes 1-6 present general methods for the preparation of the compounds provided herein as well as major intermediates. A more detailed description of the individual reaction steps is provided, for example, in U.S. Provisional Applications Serial Nos. 62/329895, 62/406252, 62/447850, 62/566093 and 62/566030, the entire contents of which are incorporated herein by reference. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize these compounds. While specific starting materials and reagents are shown and described below in the Schemes, other starting materials and reagents can be readily used in place of them to produce numerous derivatives and/or reaction conditions.

Scheme 1

Scheme 1 provides a general scheme for the synthesis of a compound of formula I (represented as a compound

- 30 041220 and 13a for formula I in scheme 1), where B is 1-methyl-1H-pyrazol-4-yl; X ¹ is N; X ² , X ³ and X ⁴ are CH; and D and E are represented by the structure where the wavy line indicates the point of attachment to the ring containing X ¹ , X ² , X ³ and X ⁴ .

Compound 2 is obtained by treating the MSH reagent with 3-bromo-5-methoxypyridine, which is commercially available. The aminating reagent O-mesitylsulfonylhydroxylamine (MSH) can be prepared as described in Mendiola, J., et al., Org. Process Res. dev. 2009, 13(2), 263-267. Compound 2 can be reacted with ethyl propiolate to give pyrazolo[1,5-a]pyridine, a mixture of compounds 3A and 3B, which are typically prepared in a ratio of about 2:1 to 9:1. A mixture of compounds 3A and 3B can be treated with 48% HBr at elevated temperatures followed by recrystallization or chromatographic purification to isolate compound 4A as the minor isomer and compound 4B as the major isomer.

The isolated compound 4B can be functionalized with a formyl group using POCl ₃ followed by purification to give compound 5. The formyl group of compound 5 can be converted to an oxime group with NH2OH to give compound 6. The oxime group of compound 6 can be converted to a nitrile group using acetic anhydride with obtaining compound 7. The B group can be established by treating compound 7 with the appropriate boron ester having the formula hetAr1-B(OR ^a )(OR ^b ) where hetAr ¹ is 1-methyl-1Hpyrazol-4-yl as described in formula I, using appropriate palladium catalyst cross-coupling conditions, e.g., Suzuki coupling conditions (e.g., a palladium catalyst and optionally a ligand in the presence of an inorganic base, e.g., Pcd ₂ (dba) ₃ , X-Phos and Na ₂ CO ₃ in dioxane at elevated temperatures), to give compound 8, where B is 1-methyl-1H-pyrazol-4-yl , as described in formula I. The methoxy group of compound 8 can be converted to a hydroxy group by treating compound 8 with aluminum trichloride to give compound 9. The free hydroxy group of compound 9 can be converted to a triflate group by treating compound 9 with a triflating reagent, for example, 1 ,1,1-trifluoro-Nphenyl-X-((trifluoromethyl)sulfonyl)methanesulfonamide to give compound 10. Compound 12 can be prepared by condensation of compound 10 with the corresponding boronic ester compound 11, where ring D is where the wavy line represents the point of attachment rings D to the ring containing X ¹ , X ² , X ³ and X ⁴ , and the asterisk denotes the attachment point to P ¹ ; X ¹ , X ² , X ³ and X ⁴ are as described above; P ¹ is an amino protecting group; Z is -B(OR ^x )(OR ^y ), Z is -B(OR ^a )(OR ^b ), and R ^a and R ^b are H or C1-C6 alkyl, or R ^a and R ^b together with the atoms to which they are bonded form a 5-6 membered ring, optionally substituted with one to four C1- _C3 alkyl groups, using appropriate palladium catalyst cross-coupling conditions, e.g., Suzuki coupling conditions (e.g., palladium a catalyst and optionally a ligand in the presence of an inorganic base, eg Pd ₂ (dba) ₃ , X-Phos and Na ₂ CO ₃ in dioxane at elevated temperatures). The protecting group on the D ring of compound 12 can be removed under standard conditions (eg, the Boc protecting group can be removed by treating compound 12 under acidic conditions, eg using HCl). The deprotected ring D can be functionalized (i.e., reacted with or treated with an appropriate reagent) to introduce the E group under standard conditions, such as those described above, to give compound 13, where E is aju

Alternatively, compound 10 can be condensed with compound 14 using appropriate cross-coupling conditions on a palladium catalyst, such as Suzuki coupling reaction conditions (such as a palladium catalyst and optionally a ligand in the presence of an inorganic base, such as Pd(PPh ₃ ) ₄ and Na ₂ CO ₃ ) to give compound 15. Compound 15 can be reacted with compound 16 under appropriate SNAr conditions (e.g., optionally in the presence of a base such as K ₂ CO ₃ and at elevated temperature) to give compound 12a, where the ring D of the compound 16 represents ^N 1 and the wavy line indicates the point of attachment of the ring D to the ring containing X ¹ , X ² , X ³ and X ⁴ and the asterisk indicates the point of attachment to P ¹ ; X ¹ , X ² , X ³ and X ⁴ are as described above;

- 31 041220

P ¹ is an amino protecting group; Z is -B(OR ^x )(OR ^y ), the second nitrogen atom is protected before condensation with a suitable amino protecting group. The protecting group on the D ring of compound 12a, if present, can be removed under standard conditions (eg the Boc group can be removed by treating compound 12a under acidic conditions, eg HCl). The deprotected ring D can be functionalized (i.e., reacted with or treated with an appropriate reagent) to introduce the E group under standard conditions, such as those described above, to give compound 13a, where E is

Scheme 2

Scheme 2 shows an alternative synthesis of compound 13, where B, X ¹ , X ² , X ³ , X ⁴ , D and E are as described in scheme 1. Compound 4A (obtained as in scheme 1) can be functionalized formyl group using POCl ₃ to give compound 17. The formyl group can be converted to an oxime group with NH2OH to give compound 18. The oxime group can be converted to a nitrile group using acetic anhydride to give compound 19. The methoxy group of compound 19 can be converted to a hydroxy group by treating compound 19 with aluminum trichloride to give compound 20. Compound 21 can be prepared by condensation of compound 20 with the corresponding boronic ester compound 11, where ring D is

moreover, the wavy line indicates the point of attachment of the ring D to the ring containing X ¹ , X ² , X ³ and X ⁴ and the asterisk indicates the point of attachment to P ¹ ; X ¹ , X ² , X ³ and X ⁴ are as described above; P ¹ is an amino protecting group; Z is -B(OR ^a )(OR ^b ) and R ^a and R ^b are H or C1-C6 alkyl or R ^a and R ^b together with the atoms to which they are bonded form a 5-6 membered ring , optionally substituted with one to four C1-C3 alkyl groups, using appropriate cross-coupling reactions on a palladium catalyst, e.g., Suzuki coupling reaction conditions (e.g., a palladium catalyst and optionally a ligand in the presence of an inorganic base, e.g., Pd(PPh ₃ ) ₄ and Na ₂ CO ₃ in dioxane at elevated temperatures). The unsubstituted nitrogen atom in ring D is protected before condensation with a suitable amino protecting group. The free hydroxyl group of compound 21 can be converted to a triflate group by treating compound 21 with a triflating reagent, e.g., 1,1,1-trifluoro-N-phenyl-N((trifluoromethyl)sulfonyl)methanesulfonamide, to give compound 22. 22 with the corresponding boronic ester having the formula hetAr ¹ -B(OR ^a )(OR ^b ), where hetAr ¹ is 1-methyl-1H-pyrazol-4-yl as described in formula I, and R ^a and R ^b are H or C1-C6 alkyl, or R ^a and R ^b together with the atoms to which they are bonded form a 5-6 membered ring optionally substituted with one to four C1-C3 alkyl groups using appropriate reaction conditions cross-coupling on a palladium catalyst, e.g., Suzuki coupling reaction conditions (e.g., a palladium catalyst and optionally a ligand in the presence of an inorganic base, such as Pd ₂ (dba) ₃ , X-Phos and Na ₂ CO ₃ in dioxane at elevated temperature), with receiving a connection 12 where B is 1-methyl-1H-32 041220 pyrazol-4-yl as described in formula I. The protecting group on the D ring of compound 12, if present, can be removed under standard conditions (for example, the Boc group can be remove by treating compound 12 under acidic conditions, eg HCl in propan-2-ol). The deprotected ring D can be functionalized (eg, reacted or treated with an appropriate reagent) to introduce the E group under standard conditions such as those described above to give compound 13, where E is

Scheme 3

Scheme 3 shows the general scheme for the synthesis of a compound of formula II or formula III (represented as compound 12 for formula II or III in scheme 3), where B represents -CH ₂ C(CH ₃ ) ₂ OH; X ¹ is N; X ² , X ³ and X ⁴ are CH; and D and E are represented by the structure

or respectively, where the wavy line denotes the point of attachment to the ring containing X ¹ , X ² , X ³ and X ⁴ .

Compound 2 is prepared by treating commercially available 3-bromo-5-methoxypyridine (compound 1) with O-(mesitylsulfonyl)hydroxylamine. O-mesitylsulfonylhydroxylamine can be prepared as described in Mendiola et al., Org. Process Res. dev. (2009) 13(2):263-267. Compound 2 can be reacted with ethyl propiolate to give a mixture of compounds 3A and 3B, which are typically prepared in a ratio of about 2:1 to 9:1, respectively. A mixture of compounds 3A and 3B can be treated with 48% HBr at elevated temperature followed by recrystallization or chromatographic purification to isolate compound 4A as the minor isomer and compound 4B as the major isomer. After isolation, compound 4A can be treated with POCl ₃ to give compound 5. The formyl group can be converted to an oxime group with NH ₂ OH to give compound 6. The oxime group can be converted to a nitrile group with acetic anhydride to give compound 7. The methoxy group of compound 7 can be converted to a hydroxy group by treating compound 7 with aluminum trichloride to give compound 8.

To prepare compound 9, compound 8 can be reacted with a reagent such as ^H0 X?x where X is a leaving atom or group (such as a halide or triflate), in the presence of a suitable base (for example, an alkali metal carbonate such as potassium carbonate ). Compound 11 can then be prepared by condensing compound 9 with the corresponding boronic ester compound 10 (where ring D is

, and the wavy line indicates the point of attachment of the ring D to the ring containing X ¹ , X ² , X ³ and X ⁴ and the asterisk indicates the point of attachment to R ¹ ; X ¹ , X ² , X ³ and X ⁴ are as described above; P ¹ is an amino protecting group; Z is -B(OR ^x )(OR ^y ) and R ^z and R ^y are H or (16C)alkyl, or R ^x and R ^y together with the atoms to which they are attached form a 5-6 membered ring optionally substituted with 1-4 substituents selected from (C ₁ -C ₃ -alkyl)), using appropriate palladium catalyzed cross-coupling reaction conditions, e.g., Suzuki coupling reaction conditions (e.g., a palladium catalyst and optionally a ligand in the presence of bases, for example, Pd(PPh ₃ ) ₄ and Na ₂ CO ₃ in dioxane at elevated temperature). Compound 12 can then be prepared from compound 11 by removing the protecting group P ¹ under standard conditions (e.g., the Boc group can be removed by treating compound 11 under acidic conditions, e.g., HCl), followed by functionalization (i.e., reacting or treating the compound 11 with the appropriate reagent) for the introduction of group E

(for formula II) or (for formula III) under standard conditions.

Alternatively, compound 8 can be condensed with the corresponding boron ester compound 10 to give compound 11a using appropriate palladium catalysed cross-coupling reaction conditions, e.g. Suzuki coupling conditions (e.g. palladium catalyst and optionally ligand in the presence of an inorganic base, e.g. PPh ₃ ) ₄ and Na ₂ CO ₃ in dioxane at elevated temperature). Compound 11a can then be reacted with a reagent such as ^HO X'X where X is a leaving atom or group (such as halide or triflate) under Mitsunobu reaction conditions (e.g. PPh3 and diisopropyl azodicarboxylate) to give compound 11. Then compound 12 can be obtained from compound 11 as described above.

Compound 9 (obtained, for example, as described in scheme 3), in which B is such as

- 34 041220 described above can be condensed with the corresponding boronic ester 13 (where X ¹ , X ² , X ³ and X ⁴ are as described above; L ² is a leaving group such as triflate or halide); Z is -B(OR ^x )(OR ^y ) and R ^z and R ^y are H or (16C)alkyl, or R ^x and R ^y together with the atoms to which they are attached form a 5-6 membered a ring optionally substituted with 1-4 substituents selected from (C1-C3-alkyl)), using appropriate palladium-catalyzed cross-coupling reaction conditions, e.g., Suzuki coupling reaction conditions (e.g., a palladium catalyst and optionally a ligand in the presence of an inorganic base, e.g. Pd(PPh3)4 and Na2CO3 in dioxane at elevated temperature) to give compound 14. Compound 16 can be prepared by condensation of compound 14 with compound 15, where ring D is as defined above and P ¹ is an amino protecting group , under appropriate SNAr conditions (eg optionally in the presence of a base such as K2CO ₃ and at elevated temperature).

The P ¹ protecting group on the D ring of compound 16 can be removed under standard conditions (e.g., the Boc group can be removed by treating compound 16 under acidic conditions, e.g., HCl), to give compound 12 where E is H (i.e., in the ring D unprotected). The deprotected ring D can then be functionalized (i.e., reacted or treated with an appropriate reagent) to introduce the E group under standard conditions such as those described above to give compound 12, where E is as described above for Scheme 3 .

Scheme 5

² SP

4A 4B 5 6

( ^Rb )m

Scheme 5 shows the general scheme for the synthesis of a compound of formula IV (represented as compound 12 for formula IV in scheme 5), where B represents -CH ₂ C(CH ₃ ) ₂ OH; X ¹ is N; X ² , X ³ and X ⁴ are CH; and D, E, (R ^a ) _n and (R ^b )m are represented by the structure

where the wavy line denotes the point of attachment to the ring containing X ¹ , X ² , X ³ and X ⁴ .

Compound 2 is prepared by treating commercially available 3-bromo-5-methoxypyridine (compound

- 35 041220

1) O-(mesitylsulfonyl)hydroxylamine. O-mesitylsulfonylhydroxylamine can be prepared as described in Mendiola, J., et al., Org. Process Res. dev. 2009, 13(2), 263-267. Compound 2 can be reacted with ethyl propiolate to give a mixture of compounds 3A and 3B, which are typically prepared in a ratio of about 2:1 to 9:1, respectively. A mixture of compounds 3A and 3B can be treated with 48% HBr at elevated temperature followed by recrystallization or chromatographic purification to isolate compound 4A as the minor isomer and compound 4B as the major isomer. After isolation, compound 4A can be treated with POCl ₃ to give compound 5. The formyl group can be converted to an oxime group with NH ₂ OH to give compound 6. The oxime group can be converted to a nitrile group with acetic anhydride to give compound 7. The methoxy group of the compound 7 can be converted to hydroxy by treating compound 7 with aluminum trichloride to give compound 8.

Compound 11a can then be reacted with a reagent such as ^H ^ ^χ where X is a leaving atom or group (such as halide or triflate) under Mitsunobu reaction conditions (PPh ₃ and diisopropyl azodicarboxylate) to give compound 11. Compound 12 can then be prepared from compound 11 as described above.

Alternatively, compound 9 can be prepared by reacting compound 8 with a reagent such as ^Ηθ χ- _X where X is a leaving atom or group (such as a halide or triflate) in the presence of a base (for example, an alkali metal carbonate such as carbonate potassium). Compound 11 can then be prepared by condensing compound 9 with the corresponding boron ester compound 10 using appropriate palladium catalyzed cross-coupling reaction conditions, e.g. ₃ ) ₄ and Na ₂ CO ₃ in dioxane at elevated temperature).

Scheme 6

Scheme 6 shows another general scheme for the synthesis of compound 12, where B, X ¹ , X ² , X ³ , X ⁴ , ring D and E are as described above for scheme 5.

Compound 9 (obtained, for example, as described in Scheme 5), in which B is as described for Scheme 5, can be condensed with compound 13 (where X ¹ , X ² , X ³ and X ⁴ are as described for scheme 5, L ² is a leaving group such as triflate or halide); Z is -B(OR ^x )(OR ^y ) and R ^z and R ^y are H or (1-6C)alkyl, or R ^x and R ^y together with the atoms to which they are bonded form 5-6 -membered ring optionally substituted with 1-4 substituents selected from (C1- _C3 -alkyl)), using appropriate palladium-catalyzed cross-coupling reaction conditions, e.g. inorganic base, e.g. Pd(PPh ₃ ) ₄ and Na ₂ CO ₃ in dioxane at elevated temperature) to give compound 14. Compound 12 can be prepared by condensation of compound 14 with compound 15 under appropriate SNAr conditions (e.g., optionally in the presence of a base , such as K ₂ CO ₃ , and at elevated temperature), with compound 15 defined as

- 36 041220

or a salt thereof as described for formula IV.

An amino protecting group in this context refers to a derivative of groups commonly used to block or protect an amino group during reactions on other functional groups of a compound. Examples of suitable protecting groups for use in any of the methods described herein include carbamates, amides, alkyl and aryl groups, imines, as well as many N-heteroatomic derivatives that can be removed to restore the desired amino group. Non-limiting examples of amino protecting groups are acetyl, trifluoroacetyl, t-butyloxycarbonyl (Boc), benzyloxycarbonyl (CBz) and 9fluorenylmethyleneoxycarbonyl (Fmoc).

Additional examples of these groups and other protecting groups are provided in T. W. Greene et al., Greene's Protective Groups in Organic Synthesis. New York: Wiley Interscience, 2006.

The hydroxy groups may be protected with any convenient hydroxy protecting group, for example, as described in T. W. Greene et al., Greene's Protective Groups in Organic Synthesis. New York: Wiley Interscience, 2006. Examples include benzyl, trityl, silyl ethers, and the like.

The nitrogen atoms in the compounds described in any of the above methods may be protected with any convenient nitrogen protecting group, for example, as described in Greene & Wuts, Ed., Protecting Groups in Organic Synthesis, 2nd Ed. NY; John Wiley & Sons, Inc., 1991. Examples of nitrogen protecting groups include acyl and alkoxycarbonyl groups such as t-butoxycarbonyl (BOC), phenoxycarbonyl, and [2-(trimethylsilyl)ethoxy]methyl (SEM).

3. Methods of treatment.

The ability of a compound of formulas I-IV, including its polymorphic forms and pharmaceutically acceptable salts, to act as an inhibitor of RET can be demonstrated using the assays described in examples 8 and 9.

In some embodiments, the compounds provided herein exhibit effective and selective inhibition of RET. For example, the compounds provided herein demonstrate nanomolar efficacy against wild-type RET and RET kinase encoded by a RET gene containing an RET kinase inhibitor activating or resistance mutation, including, for example, the KIF5B-RET fusion, G810R ATP gap front mutations, and G810S, activating mutation M918T and gatekeeper mutations V804M, V804L and V804E, with minimal activity against related kinases.

In some embodiments, the compounds provided herein demonstrate nanomolar efficacy against an altered RET fusion protein encoded by a RET gene that encodes a RET fusion protein (e.g., any of the RET fusion proteins described herein, including, without limitation, CCDC6- RET or KIF5B-RET), and said RET gene contains a RET kinase inhibitor resistance mutation (e.g., any RET mutation described herein, including but not limited to V804M, V804L, or V804E), such that the altered RET protein is a fusion a RET protein that exhibits RET kinase resistance due to the presence of an amino acid substitution or deletion conferring resistance to a RET kinase inhibitor. Non-limiting examples include CCDC6-RET-V804M and KIF5B-RET-V804M. In some embodiments, the compounds provided herein demonstrate nanomolar efficacy against an altered RET protein encoded by the RET gene that contains a RET mutation (e.g., any RET mutation described herein, including, but not limited to, C634W or M918T), and which contains a RET kinase inhibitor resistance mutation (e.g., any RET kinase inhibitor resistance mutation described herein, including but not limited to V804M, V804L, or V804E) such that the altered RET protein exhibits RET kinase resistance due to the presence of an amino acid substitution or deletion responsible for resistance to the RET kinase inhibitor.

In some embodiments, the compounds of Formula I-IV, or their pharmaceutically acceptable salts, amorphous or polymorphic forms, are selectively directed to the RET kinase. For example, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, can selectively and target the RET kinase over other kinases or non-kinase targets.

In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, exhibits at least 30-fold selectivity for RET kinase over another kinase. For example, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, exhibits at least 40-fold selectivity; at least 50-fold selectivity; at least 60-fold selectivity; at least 70-fold selectivity; at least 80-fold selectivity; at least 90-fold selectivity; at least 100-fold selectivity; at least

- 37 041220 measure 200-fold selectivity; at least 300-fold selectivity; at least 400 times selectivity; at least 500-fold selectivity; at least 600-fold selectivity; at least 700-fold selectivity; at least 800-fold selectivity; at least 900-fold selectivity; or at least 1000-fold selectivity for RET kinase over another kinase. In some embodiments, selectivity for a RET kinase over another kinase is measured in a cellular assay (eg, in the cellular assay provided herein).

In some embodiments, the compounds provided herein may exhibit selectivity for RET kinase over KDR kinase (eg, VEGFR2). In some embodiments, selectivity for RET kinase over KDR kinase is observed without loss of potency for a RET kinase encoded by a RET gene that contains an RET kinase inhibitor activating or resistance mutation (eg, a pyloric mutation). In some embodiments, the selectivity to KDR kinase is at least 10-fold (e.g., at least 40-fold selectivity; at least 50-fold selectivity; at least 60-fold selectivity; at least 70-fold selectivity, at least 80-fold selectivity, at least 90-fold selectivity, at least 100-fold selectivity, at least 150-fold selectivity, at least 200-fold selectivity, at least 250-fold selectivity; at least 300-fold selectivity; at least 350-fold selectivity; or at least 400-fold selectivity) compared to inhibition of KIF5B-RET (eg, the proposed compounds are more effective against KIF5B-RET than against KDR). In some embodiments, the selectivity for RET kinase over KDR kinase is about 30-fold. In some embodiments, the selectivity for RET kinase over KDR kinase is at least 100-fold. In some embodiments, the selectivity for RET kinase over KDR kinase is at least 150-fold. In some embodiments, the selectivity for RET kinase over KDR kinase is at least 400-fold. Without wishing to be bound by theory, effective inhibition of KDR kinase is thought to be a common feature for multikinase inhibitors (MKIs) that target RET and may be responsible for the dose-limiting toxicity observed with such compounds.

In some embodiments, the inhibition of V804M is similar to that observed for wild-type RET. For example, inhibition of V804M is within about 2-fold (eg, about 5-fold, about 7-fold, about 10-fold) relative to inhibition of wild-type RET (eg, the proposed compounds are equally effective against wild-type RET and V804M). In some embodiments, selectivity for wild-type RET kinase or V804M over another kinase is measured in an enzyme assay (eg, in an enzyme assay provided herein). In some embodiments, the compounds provided herein exhibit selective cytotoxicity against RET mutant cells.

In some embodiments, inhibition of G810S and/or G810R is similar to that observed for wild-type RET. For example, inhibition of G810S and/or G810R is in the range of about 2-fold (e.g., about 5-fold, about 7-fold, about 10-fold) relative to inhibition of wild-type RET (for example, the proposed compounds are equally effective against wild-type RET and G810S and/ or G810R). In some embodiments, selectivity for wild-type RET kinase or G810S and/or G810R over another kinase is measured in an enzyme assay (eg, in an enzyme assay provided herein). In some embodiments, the compounds provided herein exhibit selective cytotoxicity against RET mutant cells.

In some embodiments, the compounds provided herein exhibit penetration into the brain and/or the central nervous system (CNS). Such compounds can cross the blood-brain barrier and inhibit RET kinase in the brain and/or other CNS structures. In some embodiments, the compounds provided herein may cross the blood-brain barrier in a therapeutically effective amount. For example, treating a patient for cancer (eg, RET-associated cancer, such as RET-associated brain or CNS cancer) may include administering (eg, orally) the subject compound to the patient. In certain such embodiments, the compounds provided herein are useful in the treatment of a primary brain tumor or a metastatic brain tumor. For example, RET-associated primary brain cancer or metastatic brain cancer.

In some embodiments, the compounds of Formula I-IV or their pharmaceutically acceptable salts, amorphous or polymorphic forms exhibit one or more of high FA absorption, low clearance, and low potential for drug-drug interactions.

The compounds of formula I-IV or their pharmaceutically acceptable salts, amorphous or polymorphic forms are useful in the treatment of diseases and disorders that can be treated with a kinase inhibitor

- 38 041220

RET, such as RET-associated diseases and disorders, e.g., proliferative disorders such as cancers, including hematological cancers and solid tumors (e.g., advanced solid tumors and/or RET-positive solid tumors) and gastrointestinal disorders, such as IBS.

In some embodiments, compounds of Formula I-IV, or pharmaceutically acceptable salts, amorphous or polymorphic forms thereof, are useful in preventing the diseases and disorders described herein (eg, autoimmune diseases, inflammatory diseases, and cancer). The term prevention in this context means the total or partial prevention of the occurrence, recurrence or spread of the disease or pathological condition described in this document, or its symptom.

The term RET-associated disease or disorder as used herein refers to diseases or disorders associated with or characterized by dysregulation of the RET gene, the RET kinase (also referred to herein as the RET kinase protein), or the expression or activity or level of any of them (e.g., one or more) (eg, any type of dysregulation of the RET gene, RET kinase, RET kinase domain, or expression or activity or level of any of these, as described herein). Non-limiting examples of a RET-associated disease or disorder include, for example, cancer and gastrointestinal disorders such as irritable bowel syndrome (IBS).

The term RET-associated cancer as used herein refers to cancers associated with or characterized by dysregulation of the RET gene, the RET kinase (also referred to herein as the RET kinase protein), or the expression or activity or level of any of these. Non-limiting examples of RET-associated cancer are described herein.

The expression of a dysregulation of a RET gene, a RET kinase, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing a RET kinase domain and a fusion partner, a mutation in the RET gene that results in the expression of a RET protein that contains a deletion of at least one amino acid compared to the wild-type RET protein, a mutation in the RET gene that results in the expression of the RET protein with one or more point mutations compared to the wild-type RET protein, mutations in the gene RET that results in the expression of a RET protein with at least one inserted amino acid compared to a wild-type RET protein, a gene duplication that results in an increased level of the RET protein in the cell, or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) , which leads to an increased level of the RET protein in the cell), an alternative splice version of the RET mRNA, which leads to images a RET protein having a deletion of at least one amino acid in the RET protein compared to the wild-type RET protein), or overexpression (e.g., elevated levels) of wild-type RET kinase in a mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/ paracrine signaling (eg, compared to a control non-cancerous cell). As another example, dysregulation of the RET gene, the RET protein, or the expression or activity or level of any of these may be a mutation in the RET gene that encodes a RET protein that is constitutively active or has increased activity relative to the protein encoded by the gene. RET that does not contain such a mutation. For example, dysregulation of the RET gene, RET protein, or the expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the RET that contains a functional kinase domain and a second portion of the RET protein. partner (i.e. not RET). In some examples, dysregulation of the RET gene, RET protein, or expression or activity or level of any of these may be the result of a gene translocation of one RET gene with another non-RET gene. Non-limiting examples of hybrid proteins are presented in table. 1. Non-limiting examples of point mutations/insertions/deletions of the RET kinase protein are presented in table. 2 and 2a. Additional examples of RET kinase protein mutations (eg, point mutations) are RET inhibitor resistance mutations. Non-limiting examples of RET inhibitor resistance mutations are described in Table 1. 3 and 4.

In some embodiments, dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them may be caused by an activating mutation in the RET gene (see, for example, chromosomal translocations that result in the expression of any of the fusion proteins listed in Table 1). In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them may be caused by a genetic mutation that results in the expression of a RET kinase having increased resistance to inhibition by a RET kinase inhibitor and/or a multikinase inhibitor (MKI ), for example, compared to wild-type RET kinase (see, for example, amino acid substitutions in Tables 3 and 4). In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity, or level of any of them may be caused by a mutation in a nucleic acid encoding an altered RET protein (e.g., a RET fusion protein or a RET protein having a mutation (e.g., primary mutation), which leads to

- 39 041220 expression of an altered RET protein that has increased resistance to inhibition by a RET kinase inhibitor and/or a multikinase inhibitor (MKI), for example, compared to wild-type RET kinase (see, for example, amino acid substitutions in Tables 3 and 4) . Illustrative point mutations, insertions and deletions of RET kinase, presented in table. 2 and 2a may be caused by an activating mutation and/or may result in the expression of a RET kinase that has increased resistance to inhibition by a RET kinase inhibitor and/or a multikinase inhibitor (MKI).

The term activating mutation describes a mutation in the RET kinase gene that results in the expression of a RET kinase having increased kinase activity, eg compared to wild-type RET kinase, eg when assayed under the same conditions. For example, an activating mutation may result in the expression of a fusion protein that contains a RET kinase domain and a fusion partner. In another example, an activating mutation may be a mutation in the RET kinase gene that results in the expression of a RET kinase having one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid substitutions (e.g., , any combination of any of the amino acid substitutions described herein) that has increased kinase activity, eg, compared to wild-type RET kinase, eg, when assayed under the same conditions. In another example, an activating mutation may be a mutation in the RET kinase gene that results in the expression of a RET kinase having one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid deletions, e.g. , compared to wild-type RET kinase, for example, when assayed under the same conditions. In another example, an activating mutation may be a mutation in the RET kinase gene that results in the expression of a RET kinase containing at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18 or at least 20) inserted amino acids compared to a wild-type RET kinase, eg, the exemplary wild-type RET kinase described herein, eg, when assayed under the same conditions. Additional examples of activating mutations are known in the art.

The term wild-type describes a nucleic acid (e.g., RET gene or RET mRNA) or protein (e.g., RET protein) that occurs in a subject not suffering from a RET-associated disease, such as RET-associated cancer (and optionally also not at increased risk of developing RET-associated disease and/or not predisposed to RET-associated disease), or which occur in a cell or tissue derived from a subject not suffering from an RET-associated disease, such as RET-associated cancer (and optionally also not at increased risk development of RET-associated disease and/or not predisposed to RET-associated disease).

The term regulatory and control authority refers to the government authority that authorizes the medical use of pharmaceutical agents in a given country. For example, a non-limiting example of a government regulator is the Food and Drug Administration (FDA).

Provided herein is a method of treating cancer (e.g., RET-associated cancer) in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof. . For example, provided herein are methods for treating RET-associated cancer in a patient in need of such treatment, comprising a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the patient; and b) administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes one or more fusion proteins. Non-limiting examples of RET gene fusion proteins are described in Table 1. 1. In some embodiments, the fusion protein is KIF5B-RET. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more point mutations/inserts in the RET kinase protein. Non-limiting examples of point mutations/inserts/deletions in the RET kinase protein are described in Table 1. 2 and 2a. In some embodiments, point mutations/inserts/deletions in the RET kinase protein are selected from the group consisting of M918T, M918V, C634W, V804L, V804M, G810S, and G810R. In some embodiments, the point mutations/insertions/deletions in the RET kinase protein are in the RET fusion protein (eg, in any of the RET gene fusion proteins described in Table 1). In some embodiments, the compound of formula IIV is a polymorph. In some embodiments, the compound is the Form A polymorph of the Formula I compound. In some embodiments, the compound is the Form 1 polymorph of the Formula II compound. In some embodiments, the compound is the Form 2 polymorph of the compound of Formula II. In some variations

- 40 041220 ant implementation of the connection is a polymorphic form 7 of the compounds of formula II. In some embodiments, the compound is the Form 8 polymorph of the Formula II compound. In some embodiments, the compound is Form A polymorph of the compound of formula III. In some embodiments, the compound is Form A polymorph of the compound of formula IV. In some embodiments, the compound is the Form B polymorph of the compound of formula IV.

In some embodiments, a compound of Formula I-IV is a pharmaceutically acceptable salt. In some embodiments, the compound is the chloride salt of a compound of formula I. In some embodiments, the compound is the bromide salt of a compound of formula I. In some embodiments, the compound is the L malate salt of a compound of formula I. In some embodiments, the compound is the D-malate salt a compound of formula I. In some embodiments, the compound is the phosphate salt of a compound of formula II. In some embodiments, the phosphate salt is a sesqui phosphate salt (eg, PO ₄ :free base 1.4:1).

In some embodiments of any method or use described herein, the cancer (eg, RET-associated cancer) is a hematologic cancer. In some embodiments of any method or use described herein, the cancer (eg, RET-associated cancer) is a solid tumor (eg, advanced solid tumor and/or RET fusion positive solid tumor). In some embodiments of any method or use described herein, the cancer (e.g., RET-associated cancer) is lung cancer (e.g., small cell lung carcinoma or non-small cell lung carcinoma), thyroid cancer (e.g., papillary thyroid cancer, medullary cancer thyroid cancer (eg, sporadic medullary thyroid cancer or hereditary medullary thyroid cancer), differentiated thyroid cancer, recurrent thyroid cancer, or refractory differentiated thyroid cancer), thyroid adenoma, endocrine neoplasms, lung adenocarcinoma, bronchiolar lung cell carcinoma , multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, breast cancer, breast carcinoma, breast neoplasm, colon and rectal cancer (eg, metastatic colon and rectal cancer) , papillary renal cell carcinoma, ganglioneuromatosis of the gastrointestinal mucosa, inflammatory myofibroblastic tumor or cervical cancer. In some embodiments of any method or use described herein, the cancer (e.g., RET-associated cancer) is selected from the group: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult cancer, adrenocortical carcinoma, anal cancer , appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, unknown primary carcinomas, cardiac tumors, cervical cancer, childhood cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, focal neoplasms, neoplasms, colon cancer, colon and rectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, cutaneous angiosarcoma, bile duct cancer, ductal carcinoma in situ u, embryonic tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial embryonic cell tumor, extragonadal embryonic cell tumor, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrous histiocytoma of bone, gallbladder cancer, gastric cancer , gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), embryonic cell tumors, gestational trophoblastic disease, gliomas, hairy cell tumors, hairy cell leukemia, head and neck cancer, thoracic neoplasms, head and neck neoplasms, tumors CNS, primary CNS tumor, cardiac cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip cancer, and oral cavity, liver cancer lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone, osteocarcinoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck, midline carcinoma, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome , myelodysplastic/myeloproliferative neoplasms, focal neoplasms, neoplasms, myelogenous leukemia, myeloid leukemia, multiple myeloma, myloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, cancer of the nasopharynx, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, neoplasm of the lung, cancer of the respiratory tract, neoplasm lung, respiratory tract neoplasm, bronchogenic carcinoma, bronchial neoplasm, oral cavity cancer, lip cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pa

- 41 041220 pillomatosis, paraganglioma, cancer of the paranasal sinuses and nasal cavity, cancer of the parathyroid gland, penile cancer, cancer of the larynx, pheochromositoma, cancer of the pituitary gland, plasma cell neoplasm, pleuropulmonary blastoma, breast cancer during pregnancy, primary lymphoma of the central nervous system, primary peritoneal cancer, prostate cancer, rectal cancer, colon cancer, colon neoplasms, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezari syndrome, skin cancer, Spitz tumors, small cell lung cancer, small intestine cancer, soft sarcoma tissue, squamous cell carcinoma, squamous cell carcinoma of the neck, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, mimoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, unknown primary carcinoma, urethral cancer, uterine cancer, sarcoma uterus, vaginal cancer, vulvar cancer, and Wilms' tumor .

In some embodiments, the hematologic cancer (e.g., hematologic cancers that are RET-associated cancers) is selected from the group consisting of leukemias, lymphomas (non-Hodgkin's lymphoma), Hodgkin's disease (also referred to as Hodgkin's lymphoma), and myeloma, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocytic leukemia (JMML), adult T-cell ALL, AML with trilinear myelodysplasia (AML/TMDS), mixed linear leukemia (MLL), myelodysplastic syndromes ( MDS), myeloproliferative disorders (MPD) and multiple myeloma (MM). Additional examples of hematologic cancers include myeloproliferative disorders (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic primary myelofibrosis (IMF/IPF/PMF). In one embodiment, the hematologic cancer (eg, a hematologic cancer that is RET-associated cancer) is AML or CMML.

In some embodiments, the cancer (eg, RET-associated cancer) is a solid tumor. Examples of solid tumors (eg, solid tumors that are RET-associated cancers) include, for example, thyroid cancer (eg, papillary thyroid carcinoma, medullary thyroid carcinoma), lung cancer (eg, lung adenocarcinoma, small cell lung carcinoma) , pancreatic cancer, pancreatic ductal carcinoma, breast cancer, colon cancer, colon and rectal cancer, prostate cancer, renal cell carcinoma, head and neck tumors, neuroblastoma, and melanoma. See, for example, Nature Reviews Cancer, 2014, 14, 173-186.

In some embodiments, the cancer is selected from the group consisting of lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colon and rectal cancer, papillary renal cell carcinoma, gastrointestinal ganglioneuromatosis, and cervical cancer.

In some embodiments, the patient is a human.

The compounds of formula I-IV or their pharmaceutically acceptable salts, amorphous or polymorphic forms are also suitable for the treatment of RET-associated cancer.

Accordingly, this document also provides a method of treating a patient diagnosed or identified with RET-associated cancer, for example, any of the exemplary RET-associated cancers described herein, comprising administering to said patient a therapeutically effective amount of a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, or its pharmaceutical composition, as described in this document.

Deregulation of the RET kinase, the RET gene, or the expression or activity or level of any (eg, one or more) of these may contribute to tumorigenesis. For example, dysregulation of a RET kinase, a RET gene, or the expression or activity or level of any of them may be a translocation, overexpression, activation, amplification, or mutation of a RET kinase, a RET gene, or a RET kinase domain. The translocation may include gene translocation resulting in the expression of a fusion protein that contains a RET kinase domain and a fusion partner. For example, the fusion protein may have increased kinase activity compared to the wild-type RET protein. In some embodiments, a mutation in the RET gene may include mutations in the RET ligand-binding site, extracellular domains, the kinase domain, and in regions involved in protein:protein interactions and downstream signaling. In some embodiments, a mutation (e.g., an activating mutation) in the RET gene may result in the expression of a RET kinase having one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid substitutions (e.g., , one

- 42 041220 or more amino acid substitutions in the kinase domain (for example, at amino acid positions 7231012 in the wild-type RET protein), the gatekeeper amino acid (for example, amino acid position 804 in the wild-type RET protein), in the P-loop (for example, at amino acid positions 730-737 in the wild-type RET protein), in the DFG motif (for example, at amino acid positions 892-894 in the wild-type RET protein), in the amino acids of the solvent front of the ATP gap (for example, at amino acid positions 758, 811 and 892 in the wild-type RET protein type), in the activation loop (for example, at amino acid positions 891-916 in the wild-type RET protein), in the C-helix in the loop preceding the C-helix (for example, at amino acid positions 768-788 in the wild-type RET protein), and/ or at an ATP binding site (eg, at amino acid positions 730-733, 738, 756, 758, 804, 805, 807, 811, 881, and 892 in the wild-type RET protein). In some embodiments, the mutation may be a gene amplification of the RET gene. In some embodiments, a mutation (e.g., an activating mutation) in the RET gene may result in the expression of a RET kinase or RET receptor that lacks at least one amino acid (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids) compared to the wild-type RET protein. In some embodiments, the RET kinase dysregulation may be increased expression (e.g., increased levels) of wild-type RET kinase in a mammalian cell due to aberrant cellular signaling and/or deregulated autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). In some embodiments, a mutation (e.g., an activating mutation) in the RET gene may result in the expression of a RET kinase or RET receptor that has at least one inserted amino acid (e.g., at least 2, at least 3, at least 4 , at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18 , at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids) compared to the wild-type RET protein. In some embodiments, the dysregulation of RET kinase may be increased expression (e.g., increased levels) of wild-type RET kinase in a mammalian cell (e.g., compared to a control non-cancerous cell), e.g., due to aberrant cellular signaling and/or deregulated autocrine/paracrine signal transmission. Other deregulations may include splice variants of the RET mRNA. In some embodiments, the wild-type RET protein is an exemplary wild-type RET protein described herein.

In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes overexpression of the wild-type RET kinase (eg, resulting in autocrine activation). In some embodiments, dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes overexpression, activation, amplification, or mutation in a chromosomal segment containing the RET gene or a portion thereof, including, for example, a kinase domain portion or a portion of capable of exhibiting kinase activity.

In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes one or more chromosomal translocations or inversions resulting in RET gene fusion. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity, or level of any of them is the result of genetic translocations in which the expressed protein is a fusion protein containing residues from a non-RET partner protein and contains a minimum of functional kinase domain RET.

Non-limiting examples of RET fusion proteins are shown in Table 1. 1.

- 43 041220

Table 1

Illustrative RET Merger Partners and Cancer

Merger partner Non-limiting examples of RET-associated cancer(s) BCR Chronic myelomonocytic leukemia (CM ML) CLIP1 Adenocarcinoma KIF5B NSCLC, ovarian cancer, Spitz neoplasms; lung adenocarcinoma ^{3 4 14 28} ; adenosquamous carcinomas ¹⁵ CCDC6 (also called PTC1, D10S170 or H4) NSCLC, colon cancer, papillary thyroid cancer; adenocarcinomas; lung adenocarcinoma; metastatic cancer of the colon and rectum ⁵ ; adenosquamous carcinomas ¹⁵ , breast cancer ³⁰ PTC1ex9 (new permutation CCDC6) Metastatic papillary thyroid cancer ² NCOA4 (also called RTSP, ELE1 and RFG) Papillary thyroid cancer ²¹ , NSCLC, colon cancer, salivary gland cancer, metastatic colorectal cancer ⁵ ; lung adenocarcinoma ¹⁵ ; adenosquamous carcinomas ¹⁵ ; diffuse sclerosing variant of papillary thyroid cancer ¹⁰ , breast cancer ³⁰ , acinar- cellular carcinoma ³² , the mammary analogue of secretory carcinoma ³³ TRIM33 (also called RTS7, RFG7HTIF1G) NSCLC, papillary thyroid cancer, lung adenocarcinoma ⁴⁰ , miscellaneous ²² ERC1 (also called ELKS and RAB61P2) Papillary thyroid cancer, breast cancer FGFR1OP CMML, primary myelofibrosis with secondary acute myeloid leukemia MBD1 (also called PCM1) papillary thyroid cancer PRKAR1A (also called RTS2) papillary thyroid cancer TRIM24 (also called RTS6) papillary thyroid cancer KTN1 (also called RTS8) papillary thyroid cancer GOLGA5 (also called RTS5) Papillary thyroid cancer, spiceoid neoplasms NOOC papillary thyroid cancer KIAA1468 (also called RTS9 and RFG9) Papillary thyroid cancer, lung adenocarcinoma ^{8 12} TRIM27 (also called RFP) papillary thyroid cancer ACAR13 papillary thyroid cancer FKBP15 Papillary thyroid cancer, acute myeloid leukemia ⁴⁰ SPECC1L papillary thyroid cancer; thyroid carcinoma TBL1XR1 papillary thyroid cancer; thyroid carcinoma

- 44 041220

CEP55 Diffuse gastric cancer ⁷ CUX1 lung adenocarcinoma ACBD5 papillary thyroid carcinoma MYH13 Medullary thyroid carcinoma ¹ Not described Inflammatory myofibroblastic Tumor® PIBF1 Lung cell carcinoma bronchioles ⁹ KIAA1217 (also called SKT) Papillary thyroid cancer ^{10 13} Lung adenocarcinoma ¹⁴ NSCLC ¹⁴ MPRIP NSCLC ¹¹ HRH4-RET Thyroid cancer and/or papillary thyroid carcinoma ¹⁷ Ria-RET Thyroid cancer and/or papillary thyroid carcinoma ¹⁷ RFG8 Papillary thyroid carcinoma ¹⁸ FOXP4 Lung adenocarcinoma ¹⁹ MYH10 Childhood myofibromatosis ²⁰ HTIF1 Miscellaneous ²² H4L Miscellaneous ²² RTS4 (new permutation of NCO4/ELE1) Papillary thyroid cancer ²³ FRMD4A NSCLC ²⁴ SQSTM1 Papillary thyroid carcinoma ²⁵ AFAP1L2 Papillary thyroid carcinoma ²⁵ AFAP1 NSCLC ³¹ PPFIBP2 Papillary thyroid carcinoma ²⁵ EML4 NSCLC PARD3 NSCLC ²⁷ RASGEF1A breast cancer ³⁰ TEL (also called ETV6) In vitro ³⁴ , secretory carcinoma ⁵¹ RUFY1 Cancer of the colon and rectum ³⁵ OLFM4 Cancer of the small intestine ³⁸ UEVLD Papillary thyroid carcinoma ²⁹ DLG5 Non-anaplastic thyroid cancer (NAT) ³⁷ RRBP1 Colon cancer ³⁸ AN KZ Papillary thyroid carcinoma ³⁹ PICALM NSCLC ⁴⁰ MYO5C NSCLC ⁴¹ ERHA5 NSCLC ⁴⁰ RUFY2 Lung cancer ⁴² KIF13A Adenocarcinoma of the lung ⁴³ , NSCLC ⁴⁵ TNIP1 Cancer of the colon and rectum ⁴⁴ SNRNP70 Cancer of the colon and rectum ⁴⁴

- 45 041220

MRLN Thyroid carcinoma ⁴⁸ LMNA Spitzoid melanoma ⁴⁷ RUFY3 papillary thyroid carcinoma TFG MYO5A Pigmented spindle cell nevus (PSCN) Reed ⁴⁸ ADD3 Lung adenocarcinoma ⁴⁹ JMJD1C NSCLC ⁵⁰ RBPMS DOCK1 TAF3 NCOA1 NSCLC ⁵²

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10.1097/PAS.0000000000001074 ⁴⁹ Cao, et al. Onco. Targets. Ther. 2018(11):2637-2646, 2018. doi: 10.2147/OTT.S155995 ⁵⁰ Luo, et al. Int. J. Cancer, 2018. Email. publ. to print, doi: 10.1002/ijc.31542 ⁵¹ Guilmette, et al. Hum Pathol. pii: S0046-8177(18)30316-2, 2018. doi:

10.1016/j.humpath.2018.08.011 ⁵² Zhao, et al. Journal of Clinical Oncology vol. 36, no. 15, add. [S], MA e21139.

In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes one or more deletions (e.g., deletion of amino acid at position 4), insertions, or point mutation(s) in the RET kinase. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes deletion of one or more residues in the RET kinase, resulting in constitutive activity of the RET kinase domain.

In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes at least one point mutation in the RET gene that results in the production of a RET kinase having one or more amino acid substitutions, insertions, or deletions. compared to wild-type RET kinase (see, for example, point mutations listed in Table 2).

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table 2

Amino acid substitutions/insertions/deletions in the RETA kinase protein

Amino acid position 2

Amino acid position 3

Amino acid position 4

Amino acid position 5

Amino acid position 6

Amino acid position 7

Amino acid position 8

Amino acid position 11

Amino acid position 12

Amino acid position 13

Amino acid position 20

Amino acid position 32 (ex. S32L)

Amino acid position 34 (e.g. D34S)

Amino acid position 40 (ex. L40P)

Amino acid position 45 (e.g. A45A) ³⁹

Amino acid position 56 (e.g. L56M) ³⁰

Amino acid position 64 (ex. P64L)

Amino acid position 67 (e.g. R67H)

Amino acid position 77 (e.g. R77C)® ⁵

Amino acid position 114 (e.g. R114H)

Amino acid position 136 (e.g. glutamic acid for the stop codon)

Amino acid position 145 (ex. V145G)

Amino acid position 177 (e.g. R177L)® ⁷

Amino acid position 180 (e.g. arginine for stop codon)

Amino acid position 200

Amino acid position 270 (e.g. P270L)® ⁵

Amino acid position 278 (e.g. T278N) ⁵⁷

Amino acid position 292 (for example,

V292M)

Amino acid position 294

Amino acid position 321 (for example,

G321R)

Amino acid position 330 (e.g. R330Q)

Amino acid position 338 (ex. T338I)

Amino acid position 360 (for example,

R360W)

Amino acid position 373 (e.g. alanine for frameshift)

Δ Amino acid positions 378 - 385 with one amino acid insertion (e.g. D378 G385>E)

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Amino acid position 393 (ex. F393L)

Amino acid position 423 (e.g. G423R) ²⁷

Amino acid position 428 (for example, E428K) ⁵⁷

Amino acid position 432 (for example, A432A ³⁹ )

Amino acid position 446 (e.g. G446R) ²⁸

Δ Amino acid positions 505-506 (deletion of 6 base pairs within the germline reading frame in exon 7) ³

Amino acid position 510 (e.g. A510V)

Amino acid position 511 (for example, E511K)

Amino acid position 513 (e.g. G513D) ⁷ *

Amino acid position 515 (eg C515S, C515W ⁴ )

Amino acid position 525 (ex. R525W) ⁷ *

Amino acid position 531 (e.g. C531R or 9 bp duplication ² )

Amino acid position 532 (e.g. duplication) ²

Amino acid position 533 (e.g. G533C, G533S)

Amino acid position 534 (e.g. L534L)®

Amino acid position 550 (e.g. G550E)

Amino acid position 591 (e.g. V591I)

Amino acid position 593 (e.g. G593E)

Amino acid position 595 (e.g. E595D and E595A) ¹⁸

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Amino acid position 600 (for example,

R600Q)

Amino acid position 602 (e.g. I602V) ⁶

Amino acid position 603 (for example, K603Q, K603E ² )

Amino acid position 606 (e.g. Y606C)

Amino acid position 609 (e.g. C609Y, C609S, C609G, C609R, C609F, C609W, C609C ³² )

Amino acid position 611 (e.g. C611R, C611S, C611G, C611Y, C611F, C611W)

Amino acid position 616 (e.g. E616Q) ²³

Δ Amino acid position 616 ^s4

Amino acid position 618 (C618S, C618Y, C618R, C618G, C618F, C618W, stop ⁵⁶ )

Amino acid position 619 (ex. F619F)

Amino acid position 620 (e.g. C620S, C620W, C620R, C620G, C620L, C620Y, C620F, C620A ⁴⁷ )

Δ Amino acid positions 612-620 ⁷⁴

Amino acid position 622 (e.g. P622L) ⁶⁸

Amino acid position 623 (for example, E623K)

Amino acid position 624 (e.g. D624N)

Amino acid position 628 (e.g. P628N) ⁷³

Amino acid positions 629-631 (e.g. L629-D631delinsH) ⁸⁰

Amino acid position 630 (e.g. C630A, C630R, C630S, C630Y, C630F, C630W)

Δ Amino acid position 630 ⁵⁶

Amino acid position 631 (e.g. D631N,

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D631Y, D631A, D631G, D631V, D631E)

Δ Amino acid position 631 ^s9

Amino acid positions 631-633>V (i.e.

residues 631-633 are replaced by one valine residue)

Amino acid positions 631-633>A (i.e.

residues 631-633 are replaced by one alanine residue)

Amino acid positions 631-633>E (i.e.

residues 631-633 are replaced by one glutamic acid residue)

Δ Amino acid positions 631-633 (e.g. D631 - L633)

Δ Amino acid positions 631-634 (e.g. D631-C634)

Amino acid position 632 (e.g. E632K, E632G ^{5 11} , E632V® ² , 632 for frameshift ⁴⁷ )

Amino acid positions 632-633>V (i.e.

residues 632 and 633 are replaced by one valine residue) ⁷⁴

Δ Amino acid positions 632-633 (e.g., E632 - L633 in somatic cells or a 6 bp deletion within the germline reading frame in exon 11 ⁹ )

Amino acid positions 632-639>HR (i.e. residues 632-639 are replaced by two residues, histidine and arginine)

Amino acid position 633 (e.g. L633R® ² , 9 bp duplication ² , L633delinsLCR ⁷¹ )

Amino acid position 634 (e.g., C634W, C634Y, C634S, C634R, C634F, C634G, C634L, C634A, or C634T, 9 bp ² deletion, 9 bp ⁵ ® duplication, or 12 bp 2 duplication ⁾ (eg, causing MTS)

Δ Amino acid position 634 ⁵ ® Amino acid position 632/633/634

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(E632V/L633R/634, 9 bp deletion) ⁶² Amino acid position 635 (e.g. R635G or ELCR ² insert) Amino acid position 636 T636R ² , T636M ⁴ ) (For example, Amino acid positions 636-637 T636-V637insCRT) ⁸⁰ (For example, Amino acid position 638 isoleucine for frameshift ⁴⁷ ) (For example, Amino acid position 640 (e.g. A640G) Amino acid position 634/640 O634R/A640G) ⁵⁶ (For example, Amino acid position 641 (eg A641S, A641T ⁸ ) Amino acid position 634/641 O634S/A641S) ⁵⁶ (For example, Amino acid position 639/641 A639G/A641R) ⁵⁶ (For example, Amino acid position 644 T644M) ⁵⁹ (For example, Amino acid position 648 (e.g. V648I) Amino acid positions 634/648 O634R/V648I) ⁷⁷ (For example, Amino acid position 649 S649L) ²⁸ (For example, Amino acid position 661 H661H) ⁶ (For example, Amino acid position 664 (e.g. A664D) Amino acid position 665 H665Q) (For example, Amino acid position 666 (e.g. K666E, K666M, K666N, K666R) Amino acid position 675 (T675T, silent

- 53 041220

nucleotide substitution) ¹⁸ Amino acid Р679Р) ⁶ position 679 (For example, Amino acid position 680 (e.g. A680T, alanine for frameshift) ⁶ Amino acid position 686 (e.g. S686N) Amino acid S689T) ¹⁸ position 689 (For example, Amino acid position 691 (e.g. G691S) Amino acid R694Q) position 694 (For example, Amino acid position 700 (ex. M700L) Amino acid position 706 (e.g. V706M, V706A) Amino acid position 713, splice variant (eg, E713K (eg, splice variant)) ⁶ Amino acid D714Y) ⁵⁷ position 714 (For example, Amino acid G727E) ⁶ position 727 (For example, Amino acid E732K) ²⁰ position 732 (For example, Amino acid E734K) ⁴⁸ position 734 (For example, Amino acid G736R) ⁶ position 736 (For example, Amino acid V738V) ⁶ position 738 (For example, Amino acid T742M) ⁵¹ position 742 (For example, Amino acid G748C) position 748 (For example, Amino acid position 749 (For example,

- 54 041220

^R749T36 )

Amino acid position 750 (e.g. A750P, A750G®)

Amino acid position 752 (for example,

Y752Y)®

Amino acid position 751 (e.g. G751G)®

Amino acid position 762 (for example,

^E762Q3® )

Amino acid position 765 (e.g. S765P, S765F)

Amino acid position 766 (eg P766S, P766M®)

Amino acid position 768 (e.g. E768Q, E768D, E768N ⁴⁶ , E768G ⁷² )

Amino acid position 769 (e.g. L769L®)

Amino acid position 770 (for example,

R770Q)

Amino acid position 771 (e.g. D771N)

Amino acid position 777 (e.g. N777S)

Amino acid position 778 (e.g. V778I)

Amino acid position 781 (for example,

Q781R)

Amino acid position 788 (e.g. I788I ³² , I788N ⁷⁸ )

Amino acid position 790 (e.g. L790F)

Amino acid position 768/790 (e.g. E768D/L790T) ⁴⁰

Amino acid position 791 (eg Y791F, Y791N ²⁴ )

Amino acid position 634/791 (e.g. C634Y/Y791F) ⁵⁵

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Amino acid position 790/791 (e.g. L790F/Y791F) ⁵⁵

Amino acid position 802

Amino acid position 804 (eg V804L ^{15 16} , V804M ^{15 16} , V804E ¹² ) (eg causing MTS)

Amino acid position 778/804 ⁵⁰ (e.g. V778I/V804M ⁵⁴ )

Amino acid position 781/804 (e.g. Q781R/V804M) ⁴¹

Amino acid position 805 (e.g. E805K)

Amino acid position 804/805 (e.g. V804M/E805K) ¹⁷

Amino acid position 806 (e.g. Y806F, Y806S ¹² , Y806G, ^{Y806C 2 12 14} , Y806E ¹⁴ , Y806H ¹² , Y806N ¹² -Y806Y ³² )

Amino acid position 804/806 (e.g. V804M/Y806C) ³⁸

Amino acid position 810 (e.g. G810R ¹² , G810S ¹² , G810A ¹³ , G810C, G810V and G810D)

Amino acid position 818 (for example, E818K)

Amino acid position 819 (e.g. S819I)

Amino acid position 820 (e.g. R820L) ⁵⁷

Amino acid position 823 (e.g. G823E)

Amino acid position 826 (e.g. Y826M, Y826S) ¹⁰

Amino acid position 828 (e.g. G828R) ⁵⁷

Amino acid position 833 (e.g. R833C)

Amino acid position 836 (e.g. S836S) ¹⁹

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Amino acid position 841 (e.g. P841L, P841P)

Amino acid position 843 (e.g. E843D)

Amino acid position 844 (e.g. R844W, R844Q, R844L)

Amino acid position 804/844 (e.g. V804M/R844L) ⁷⁶

Amino acid position 845 (for example,

A845A)® ³

Amino acid position 848 (e.g. M848T)

Amino acid position 852 (eg I852M)

Amino acid position 853 (e.g. S853T) ⁵⁷

Amino acid position 865 (e.g. L865V) ¹²

Amino acid position 866 (e.g. A866W) ³³

Amino acid position 867 (for example, E867K) ³⁷

Amino acid position 870 (e.g. L870F) ¹²

Amino acid position 873 (e.g. R873W, R873Q ⁴² )

Amino acid position 876 (e.g. A876V)

Amino acid position 881 (e.g. L881V)

Amino acid position 882

Amino acid position (e.g. A883F, A883S, A883T, A883Y ⁵³ , A883V)

Amino acid position 884 (e.g. E884K, E884V ³⁵ )

Amino acid position 886 (for example,

R886W)

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Amino acid position 891 (e.g. S891A, S891S ³² , S891L ³⁵ )

Amino acid position 893 (e.g. F893L) ⁴²

Amino acid position 894 (e.g. G894S) ⁴³

Amino acid position 897 (e.g. R897Q, R897P)

Amino acid position 898 (e.g. D898V, D898Y ⁶⁶ )

Δ Amino acid position 898

Δ Amino acid positions 898-902 ⁵⁸

Δ Amino acid positions 899-902 ⁴⁷

Δ Amino acid positions 898-901 ⁴⁷

Δ Amino acid positions 632-633/Δ

Amino acid provisions 898-901 ⁴⁷

Amino acid position 900 (e.g. Y900F) ²²

Amino acid position 901 (for example, E901K)

Amino acid position 904 (ex. S904F, S904S, S904C ² , S904T ⁵⁷ )

Amino acid position 691/904 (e.g. G691S/S904S) ⁴⁹

Amino acid position 804/904 (e.g. V804M/S904C) ³⁸

Amino acid position 905 (e.g. Y905F) ²²

Amino acid position 907 (e.g. K907E, K907M)

Amino acid position 908 (e.g. R908K)

Amino acid position 911 (e.g. G911D, G911G (e.g. splice variant) ⁸ )

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Amino acid position 912 (e.g. R912P, R912Q)

Amino acid position 918 (e.g., M918T ² , M918V, M918L ⁶ ) (e.g. causing MTS)

Amino acid position 591/918 (e.g. V5911/M918T) ⁶¹

Amino acid position 620/918 (e.g. C620F/M918T) ⁴⁷

Amino acid position 891/918 (e.g. S891A/M918T) ⁴⁷

Δ Amino acid positions 898-901/M918T ⁴⁷

Amino acid position 919 (e.g. A919V, A919P ⁵² )

Amino acid position 768/919 ⁵⁴

Amino acid position 921 (e.g. E921K, E921D)

Amino acid position 911/918/921 (e.g. G911E/M918T/E921K) ⁶¹

Amino acid position 922 (e.g. S922P, S922Y)

Amino acid position 924 (e.g. F924S)®

Amino acid position 930 (ex. T930M)

Amino acid position 961 (ex. F961L)

Amino acid position 972 (e.g. R972G)

Amino acid position 973 (e.g. P973T) ⁵⁷

Amino acid position 977 (e.g. S977R) ³⁷

Amino acid position 981 (e.g. Y981F) ²²

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Amino acid R982C) ⁷⁰ position 982 (For example, Amino acid position (e.g. C634R/G691S/R982C) ⁴⁵ 634/691/982 Amino acid position 292/67/982 (e.g. V292M/ R67H/R982O) ⁷⁵ Amino acid position 634/292/67/982 (e.g. C634R/ V292M/ R67H/R982C) ⁷⁵ Amino acid M1009V) position 1009 (For example, Amino acid Y1015F) ²² position 1015 (For example, Amino acid D1017N) position 1017 (For example, Amino acid S1024F) ⁷⁹ position 1024 (For example, Amino acid V1041G) position 1041 (For example, Amino acid P1047S) ⁶⁵ position 1047 (For example, Amino acid A1051T) ⁵⁷ position 1051 (For example, Lamino acid position 1059 ⁵⁷ Amino acid M1064T) position 1064 (For example, Amino acid Y1096F) ²¹ position 1096 (For example, Amino acid A1105V) ⁵⁷ position 1105 (For example, Amino acid M1109T) ³⁴ position 1109 (For example, RET+3 ¹ (In frame deletion in exons 6 and 11) ²⁵ (3 bp deletion in reading frame in exon 15) ²⁶ Nucleotide ι 2136+2T>G) ²⁹ position 2136+2 (For example, (del632-636 ins6) ³¹ Amino acids Y791F + I852M) ³¹ regulations 791 and 852 (for example, Amino acid positions 634 C634R + I852M) ³¹ I and 852 (for example, c.1893_1895del ⁴⁴

^{A The} RET kinase mutations shown may be activating mutations and/or may confer increased resistance of the RET kinase to a RET kinase inhibitor and/or a multikinase inhibitor (MKI), for example, compared to wild-type RET kinase.

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7006.1999.tb00701.x ⁵⁹ Krampitz. Cancer, 120(13), 1920-1931, 2014: 10.1002/cncr.28661 ⁸⁰ Jhiang et al, Thyroid 6(2), 1996. doi: 10.1089/thy.1996.6.115 ⁶¹ Dvofakova, et al, Thyroid, 16( 3), 311-316, 2006. doi: 10.1089/thy.2006.16.311 ⁶² Severskaya et al, Genomics Transcriptomics Proteomics, 40(3) 425-435.

⁶³ Elisei, et al, Journal of Genetic Syndromes & Gene Therapy, 5(1), 1, 2014. doi: 10.4172/21577412.1000214 ⁶⁴ Ahmed et al, The Journal of Molecular Diagnostics, 7(2), 283-288, 2005. doi: 10.1016/S15251578(10)60556-9 ⁶⁵ Oliveira, et al. J. Exp. Clin. Cancer Res. 37(84), 2018. doi: 10.1186/s13046-018-0746-y ⁶⁶ Yi, et al. Case Rep. Endocrinol. 2018:8657314, 2018. doi: 10.1155/2018/8657914 ⁶⁷ Huang, et al. cell. 173(2): 355-370, 2018. doi: 10.1016/j.cell.2018.03.039 ⁶⁸ Bosic, et al. pathology. 50(3):327-332, 2018. doi: 10.1016/j.pathol.2017.10.011 ⁶⁹ Yao, et al. Zhonghua Yi Xue Za Zhi. 87(28):1962-1965, 2007. PMID: 17923033 ⁷⁰ Quintela-Fandino, et al. Mol. oncol. 8(8):1719-1728, 2014. doi: 10.1016/j.molonc.2014.07.005 ⁷¹ Urbini, et al. Int J Genomics 2018: 6582014. doi: 10.1155/2018/6582014 ⁷² Yu, et al. Clin Lung Cancer, pii: S1525-7304(18)30204-3, 2018. doi:

10.1016/j.cllc.2O18.08.010 ⁷³ Soca-Chafre, et al. Oncotarget 9(55):30499-30512, 2018. doi: 10.18632/oncotarget.25369 ⁷⁴ Kim, et al. BMC Urol 18(1):68, 2018. doi: 10.1186/s 12894-018-0380-1 ⁷⁵ Qi, et al. PLoS One 6(5):e20353, 2011. doi: 10.1371/journal.pone.0020353

- 62 041220 ⁷⁶ Bartsch, et al Exp Clin Endocrinol Diabetes 108(2):128-132, 2000. doi: 10.1055/S-2000-5806 ⁷⁷ Nunes, et al. J Clin Endocrinol Metab. 87(12):5658-5661,2002. doi: 10.1210/jc.2002-020345 ⁷⁸ Plenkeret al., Sci. Transl. Med., 9(394), doi: 10.1126/scitranslmed.aah6144, 2017 ⁷⁹ Romei, et al., European Thyroid Journal, vol. 7, add. 1, cc. 63. Abstract number: P1-07-69. Event info: 41st Annual Meeting of the European Thyroid Association, ETA 2018. 15 Sept. 2018-18 Sept. 2018. doi: 10.1159/000491542 ⁸⁰ Ciampi, et al., European Thyroid Journal, vol. 7, add. 1, cc. 63. Abstract number: OP-09-66. Event info: 41st Annual Meeting of the European Thyroid Association, ETA 2018. 15 Sept. 2018-18 Sept. 2018. doi: 10.1159/000491542

In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes at least one point mutation in the RET gene that results in the production of a RET kinase having one or more amino acid substitutions, insertions, or deletions. compared to wild-type RET kinase (see, for example, point mutations listed in Table 2a).

Table 2a

Amino acid substitutions/insertions/deletions in the RET ^A kinase protein

Amino acid position 20

Amino acid position 32 (ex. S32L)

Amino acid position 34 (e.g. D34S)

Amino acid position 40 (ex. L40P)

Amino acid position 64 (ex. P64L)

Amino acid position 67 (e.g. R67H)

Amino acid position 114 (e.g. R114H)

Amino acid position 145 (ex. V145G)

Amino acid position 200

Amino acid position 292 (for example,

V292M)

Amino acid position 294

Amino acid position 321 (for example,

G321R)

Amino acid position 330 (for example,

R330Q)

Amino acid position 338 (ex. T338I)

Amino acid position 360 (for example,

- 63 041220

R360W)

Amino acid position 393 (ex. F393L)

Amino acid position 432

Δ Amino acid residues 505-506 (deletion of 6 base pairs within the germline reading frame in exon 7)

Amino acid position 510 (e.g. A510V)

Amino acid position 511 (for example, E511K)

Amino acid position 513 (e.g. G513D)

Amino acid position 515 (eg C515S, C515W ⁴ )

Amino acid position 525 (e.g. R525W)

Amino acid position 531 (e.g. C531R or 9 bp duplication)

Amino acid position 532 (e.g. duplication)

Amino acid position 533 (e.g. G533C, G533S)

Amino acid position 550 (e.g. G550E)

Amino acid position 591 (e.g. V591I)

Amino acid position 593 (e.g. G593E)

Amino acid position 595 (e.g. E595D

- 64 041220 and E595A)

Amino acid position 600 (e.g. R600Q)

Amino acid position 602 (e.g. I602V)

Amino acid position 603 (e.g. K603Q, K603E)

Amino acid position 606 (e.g. Y606C)

Amino acid position 609 (e.g. C609Y, C609S, C609G, C609R, C609F, C609W)

Amino acid position 611 (e.g. C611R, C611S, C611G, C611Y, C611F, C611W)

Amino acid position 616 (e.g. E616Q)

Amino acid position 618 (C618S, C618Y, C618R, C618G, C618F, C618W)

Amino acid position 620 (e.g. C620S, C620W, C620R, C620G, C620L, C620Y, C620F)

Amino acid position 623 (for example, E623K)

Amino acid position 624 (e.g. D624N)

Amino acid position 630 (e.g. C630A, C630R, C630S, C630Y, C630F, C630W)

Amino acid position 631 (e.g. D631N, D631Y, D631A, D631G, D631V, D631E)

Amino acid position 632 (for example, E632K,

E632G)

- 65 041220

Δ Amino acid residues 632-633 (deletion of 6 base pairs within the germline reading frame in exon 11)

Amino acid position 633 (e.g. 9 bp duplication)

Amino acid position 634 (eg, C634W, C634Y, C634S, C634R, C634F, C634G, C634L, C634A, or C634T, or ELCR insert, or 12 bp duplication) (eg, causing MTC)

Amino acid position 635 (e.g. R635G)

Amino acid position 636 (e.g. T636P, T636M)

Amino acid position 640 (e.g. A640G)

Amino acid position 641 (e.g. A641S, A641T)

Amino acid position 648 (e.g. V648I)

Amino acid position 649 (e.g. S649L)

Amino acid position 664 (e.g. A664D)

Amino acid position 665 (ex. H665Q)

Amino acid position 666 (e.g. K666E, K666M, K666N, K666R)

Amino acid position 686 (e.g. S686N)

- 66 041220

Amino acid position 689 (e.g. S689T)

Amino acid position 691 (e.g. G691S)

Amino acid position 694 (e.g. R694Q)

Amino acid position 700 (ex. M700L)

Amino acid position 706 (for example, V706M,

V706A)

Amino acid position 713, splice variant (e.g., E713K)

Amino acid position 732 (for example, E732K)

Amino acid position 736 (for example,

G736R)

Amino acid position 748 (for example,

G748C)

Amino acid position 750 (e.g. A750P)

Amino acid position 765 (e.g. S765P)

Amino acid position 766 (e.g. P766M)

Amino acid position 768 (e.g. E768Q, E768D)

Amino acid position 769 (e.g. L769L)

Amino acid position 770 (e.g. R770Q)

- 67 041220

Amino acid position 771 (e.g. D771N)

Amino acid position 777 (e.g. N777S)

Amino acid position 778 (e.g. V778I)

Amino acid position 781 (e.g. Q781R)

Amino acid position 790 (e.g. L790F)

Amino acid position 791 (e.g. Y791F, Y791N)

Amino acid position 802

Amino acid position 804 (for example, V804L,

V804M, V804E) (e.g. MTC callers)

Amino acid position 805 (e.g. E805K)

Amino acid position 804/805 (e.g. V804M/E805K)

Amino acid position 806 (e.g. Y806F, Y806S, Y806G, Y806C, Y806E, Y806H, Y806N)

Amino acid position 810 (for example, G810R, G810S, G810A, G810C, G810V, and G810D)

Amino acid position 818 (for example, E818K)

Amino acid position 819 (e.g. S819I)

Amino acid position 823 (e.g. G823E)

Amino acid position 826 (e.g. Y826M, Y826S)

- 68 041220

Amino acid position 833 (e.g. R833C)

Amino acid position 836 (e.g. S836S)

Amino acid position 841 (e.g. P841L, P841P)

Amino acid position 843 (e.g. E843D)

Amino acid position 844 (e.g. R844W, R844Q, R844L)

Amino acid position 848 (e.g. M848T)

Amino acid position 852 (eg I852M)

Amino acid position 865 (e.g. L865V)

Amino acid position 870 (e.g. L870F)

Amino acid position 873 (e.g. R873W)

Amino acid position 876 (e.g. A876V)

Amino acid position 881 (e.g. L881V)

Amino acid position 882

Amino acid position (e.g. A883F, A883S, A883T)

Amino acid position 884 (for example, E884K)

Amino acid position 886 (e.g. R886W)

Amino acid position 891 (e.g. S891A)

- 69 041220

Amino acid position 897 (e.g. R897Q)

Amino acid position 898 (e.g. D898V)

Amino acid position 900 (e.g. Y900F)

Amino acid position 901 (for example, E901K)

Amino acid position 904 (e.g. S904F, S904S, S904C)

Amino acid position 907 (e.g. K907E, K907M)

Amino acid position 908 (e.g. R908K)

Amino acid position 911 (for example,

G911D)

Amino acid position 912 (e.g. R912P, R912Q)

Amino acid position 918 (eg, M918T, M918V, M918L) (eg, causing MTS)

Amino acid position 919 (e.g. A919V)

Amino acid position 921 (for example, E921K)

Amino acid position 922 (e.g. S922P, S922Y)

Amino acid position 930 (e.g. T930M)

Amino acid position 961 (ex. F961L)

Amino acid position 972 (for example,

- 70 041220

R972G)

Amino acid position 982 (e.g. R982C)

Amino acid position 1009 (for example,

M1009V)

Amino acid position 1015 (for example,

Y1015F)

Amino acid position 1017 (for example,

D1017N)

Amino acid position 1041 (for example,

V1041G)

Amino acid position 1064 (for example,

M1064T)

Amino acid position 1096 (for example,

Y1096F)

RET+3 (In frame deletion in exons 6 and 11) (3 bp deletion in frame in exon 15) ^A The above RET kinase mutations may be activating mutations and/or may confer increased RET kinase resistance to a RET inhibitor and/or a multikinase inhibitor (MKI), for example, compared to wild-type RET kinase.

In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes a splice change in RET mRNA that results in the expression of a protein that is an alternative RET splice variant having at least one deleted residue (compared to wild-type RET kinase), resulting in constitutive activity of the RET kinase domain.

The RET kinase inhibitor described above includes any compound that exhibits RET inhibitory activity. In some embodiments, the RET kinase inhibitor is selective for RET kinase. Exemplary RET kinase inhibitors may exhibit an inhibitory activity (IC ₅₀ ) against RET kinase of less than about 1000 nM, less than about 500 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM less than about 10 nM, or less than about 1 nM, as measured in the assay described herein. In some embodiments, the RET kinase inhibitor may exhibit an inhibitory activity (IC50) against RET kinase of less than about 25 nM, less than about 10 nM, less than about 5 nM, or less than about 1 nM as measured in the assay provided herein.

In this context, the first RET kinase inhibitor or the first RET inhibitor is the RET kinase inhibitor described herein, but which does not contain a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, described herein. In this context, the second RET kinase inhibitor or the second RET inhibitor is the RET kinase inhibitor described herein, but which does not contain the compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form described herein. If the method provided herein includes both the first and second RET inhibitor, then the first and second RET kinase inhibitors are different.

In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises at least one point mutation in the RET gene that results in the production of a RET kinase having one or more amino acid substitutions or insertions, or deletions in the RET gene, which results in the production of a RET kinase having one or more amino acids inserted or deleted compared to wild-type RET kinase. In some instances, the resulting RET kinase is more resistant to inhibition of its phosphotransferase activity by one or more of the first RET inhibitor(s) than wild-type RET kinase or RET kinase lacking such a mutation. Optionally, such mutations do not reduce the sensitivity of a cancer cell or tumor containing the RET kinase to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form (for example, compared to a cancer cell

- 71 041220 coy or tumor that does not contain a specific RET inhibitor resistance mutation). In such embodiments, the RET inhibitor resistance mutation can result in a RET kinase that has one or more of increased Vmax, decreased Km for ATP, and increased KD for the first RET kinase inhibitor in the presence of the first RET kinase inhibitor, compared to the wild-type RET kinase. type or RET kinase not containing such a mutation in the presence of the same first RET kinase inhibitor.

In other embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises at least one point mutation in the RET gene that results in the production of a RET kinase that has one or more amino acid substitutions compared to a wild-type RET kinase, and which has increased resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, compared to a wild-type RET kinase or a RET kinase lacking such a mutation. In such embodiments, a RET inhibitor resistance mutation can result in a RET kinase that has one or more of increased Vmax, reduced Km, and reduced KD in the presence of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, an amorphous or polymorphic form compared to wild-type RET kinase or RET kinase not containing such a mutation in the presence of the same compound of formulas I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

Examples of RET inhibitor resistance mutations may, for example, include point mutations, insertions or deletions within and near the ATP binding site in the RET kinase tertiary structure (e.g., amino acid positions 730-733, 738, 756, 758, 804, 805, 807, 810, 811, 881, and 892 wild-type RET kinases, e.g., the exemplary wild-type RET kinase described herein) including, but not limited to, pylorus residue (e.g., amino acid position 804 in wild-type RET kinase), P- loops (e.g., amino acid positions 730-737 in wild-type RET kinase), residues within or near the DFG motif (e.g., amino acid positions 888-898 in wild-type RET kinase), and amino acid residues at the ATP gap solvent front (e.g., amino acid positions 758, 811 and 892 in the wild-type RET kinase). Additional examples of these types of mutations include changes in a residue that can affect enzyme activity and/or drug binding, including, but not limited to, residues in the activating loop (e.g., amino acid positions 891-916 of wild-type RET kinase), residues near or interacting with the activating loop, residues involved in active or inactive conformations of the enzyme, changes including mutations, deletions and insertions in the loop preceding the C-helix and within the C-helix (e.g., amino acid positions 768-788 in the wild-type RET protein). In some embodiments, the wild-type RET protein is an exemplary wild-type RET kinase described herein. Specific residues or regions of residues that can be altered (and are RET inhibitor resistance mutations) include, but are not limited to, those listed in Table 1. 3, where the numbering is based on the sequence of the wild-type human RET protein (eg, SEQ ID NO: 1). As will be appreciated by those skilled in the art, an amino acid position in a reference protein sequence that corresponds to a particular amino acid position in SEQ ID NO: 1 can be determined by aligning the sequence of a reference protein with SEQ ID NO: 1 (e.g., using software such as ClustalW2 ). Additional examples of RET inhibitor resistance mutation positions are shown in Table 1. 4. Changes to these residues may include single or multiple amino acid substitutions, insertions within or at the ends of sequences, and deletions within or at the ends of sequences. See also J. Kooistra, G. K. Kanev, O. P. J. Van Linden, R. Leurs, I. J. P. De Esch, and C. De Graaf, KLIFS: A structural kinase-ligand interaction database, Nucleic Acids Res., vol. 44, no. D1 , c. D365-D371, 2016), the entire contents of which are incorporated herein by reference.

Иллюстративная последовательность зрелого белка RET человека (SEQ ID NO: 1) MAKATSGAAG LRLLLLLLLP LLGKVALGLY FSRDAYWEKL YVDQAAGTPL LYVHALRDAP EEVPSFRLGQ HLYGTYRTRL HENNWICIQE DTGLLYLNRS LDHSSWEKLS VRNRGFPLLT VYLKVFLSPT SLREGECQWP GCARVYFSFF NTSFPACSSL KPRELCFPET RPSFRIRENR PPGTFHQFRL LPVQFLCPNI SVAYRLLEGE GLPFRCAPDS LEVSTRWALD REQREKYELV

AVCTVHAGAR EEVVMVPFPV TVYDEDDSAP TFPAGVDTAS AVVEFKRKED TWATLRVFD ADWPASGEL VRRYTSTLLP GDTWAQQTFR VEHWPNETSV QANGSFVRAT VHDYRLVLNR NLSISENRTM QLAVLVNDSD FQGPGAGVLL LHFNVSVLPV SLHLPSTYSL SVSRRARRFA QIGKVCVENC QAFSGINVQY KLHSSGANCS TLGWTSAED TSGILFVNDT KALRRPKCAE LHYMVVATDQ QTSRQAQAQL LVTVEGSYVA EEAGCPLSCA VSKRRLECEE CGGLGSPTGR CEWRQGDGKG ITRNFSTCSP STKTCPDGHC DWETQDINI CPQDCLRGSI VGGHEPGEPR GIKAGYGTCN CFPEEEKCFC EPEDIQDPLC DELCRTVIAA AVLFSFIVSV LLSAFCIHCY HKFAHKPPIS SAEMTFRRPA QAFPVSYSSS GARRPSLDSM ENQVSVDAFK ILEDPKWEFP RKNLVLGKTL GEGEFGKWK ATAFHLKGRA GYTTVAVKML KENASPSELR DLLSEFNVLK QVNHPHVIKL YGACSQDGPL LLIVEYAKYG SLRGFLRESR KVGPGYLGSG GSRNSSSLDH PDERALTMGD LISFAWQISQ GMQYLAEMKL VHRDLAARNI LVAEGRKMKI SDFGLSRDVY EEDSYVKRSQ GRIPVKWMAI ESLFDHIYTT QSDVWSFGVL LWEIVTLGGN PYPGIPPERL FNLLKTGHRM ERPDNCSEEM YRLMLQCWKQ EPDKRPVFAD ISKDLEKMMV KRRDYLDLAA STPSDSLIYD DGLSEEETPL VDCNNAPLPR ALPSTWIENK LYGMSDPNWP GESPVPLTRA DGTNTGFPRY PNDSVYANWM LSPSAAKLMD TFDS

In some embodiments, the RET inhibitor resistance mutation may include

- 72 041220 deregulation of the MET gene, MET kinase, or expression, or activity, or the level of any of them.

The expression of a dysregulation of a MET gene, a MET kinase, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a MET gene translocation that results in the expression of a fusion protein, a deletion in the MET gene that results in the expression of a MET protein that contains a deletion of at least one amino acid compared to the wild-type MET protein, or a mutation in the MET gene that results in the expression of the MET protein with one or more point mutations, or an alternative splice version of the MET mRNA that produces the MET protein, resulting in a deletion of at least one amino acid in the MET protein compared to the wild-type MET protein) or amplification of the MET gene that results in overexpression of the MET protein or autocrine activity due to overexpression of the MET gene in the cell, resulting in pathogenic upregulation of the kinase domain of the MET protein (e.g. , the constitutively active kinase domain of the MET protein) in the cell. As another example, dysregulation of the MET gene, the MET protein, or the expression or activity or level of any of these may be a mutation in the MET gene that encodes a MET protein that is constitutively active or has increased activity relative to the protein encoded by the gene. MET that does not contain such a mutation. For example, deregulation of the MET gene, MET protein, or expression or activity or level of any of them may be the result of a gene or chromosome translocation that results in the expression of a fusion protein containing a first portion of the MET that contains a functional kinase domain and a second portion of the MET protein. partner (i.e. not MET). In some examples, dysregulation of a MET gene, MET protein, or expression or activity may result from a gene translocation of one MET gene with another non-MET gene.

The term wild-type MET describes a nucleic acid (e.g., MET gene or MET mRNA) or protein (e.g., MET protein) that occurs in a subject not suffering from MET-associated cancer (and optionally also not at increased risk of developing MET-associated cancer and/or not predisposed to MET-associated cancer), or that occur in a cell or tissue derived from a subject not suffering from MET-associated cancer (and optionally also not at increased risk of developing MET-associated cancer and/or not predisposed to MET-associated cancer ). The term MET-associated cancer as used herein refers to cancers associated with or characterized by dysregulation of the MET gene, MET kinase, or expression, or activity, or level of any of them.

In some embodiments, compounds of Formula I-IV, or pharmaceutically acceptable salts, amorphous or polymorphic forms thereof, are useful in the treatment of patients who develop cancers with RET inhibitor resistance mutations (e.g., which result in increased resistance to a first RET inhibitor, e.g., with a substitution at amino acid position 804, such as V804M, V804L, or V804E, with a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D, and/or with one or more of the RET inhibitor resistance mutations listed in tables 3 and 4) through the introduction of a combination or sequential, or additional (for example, subsequent) therapy to existing medical treatment (for example, other RET kinase inhibitors; for example, the first and/or second RET kinase inhibitor). Exemplary first and second RET kinase inhibitors are described herein. In some embodiments, the first or second RET kinase inhibitor may be selected from the group consisting of cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667, and BLU6864.

In some embodiments, the compounds of Formula I-IV, or pharmaceutically acceptable salts, amorphous or polymorphic forms thereof, are useful in the treatment of cancer that is identified as having one or more RET inhibitor resistance mutations (that result in increased resistance to a first or second RET inhibitor, e.g. , a substitution at amino acid position 804, such as V804M, V804L, or V804E, or, for example, a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D). In some embodiments, one or more RET inhibitor resistance mutations are found in a nucleic acid sequence encoding a RET fusion protein (e.g., any of the RET gene fusion proteins described in Table 1) to form a RET fusion protein that exhibits resistance to the inhibitor. RET kinases. In some embodiments, one or more RET inhibitor resistance mutations are found in a nucleic acid sequence encoding a mutant RET protein (e.g., a mutant RET protein having one of the mutations described in Table 2), producing a mutant RET protein that exhibits resistance. RET kinases. Non-limiting examples of RET inhibitor resistance mutations are listed in Table 1. 3 and 4.

- 73 041220

Table 3

RET inhibitor resistance mutations

Illustrative RET resistance mutations

Amino acid position 634 (e.g. C634W) ¹⁰

Amino acid position 732 (for example, E732K) ⁷

Amino acid position 788 (for example, I788N) ⁸

Amino acid position 790 (e.g. L790F) ⁹

Amino acid position 804 (e.g. V804M ¹² , V804L ¹ ' ² , V804E ⁶ )

Amino acid position 778/804 ¹³

Amino acid position 804/805 (e.g. V804M/E805K) ³

Amino acid position 806 (e.g. Y806C ⁴ ' ⁶ , Y806E ⁴ , Y806S ⁶ , Y806H ⁶ , Y806N ⁶ )

Amino acid position 804/806 (e.g. V804M/Y806C) ¹¹

Amino acid position 810 (for example, G810A ⁵ , G810R ⁶ , G810S ⁶ , G810C, G810V and G810D)

Amino acid position 865 (e.g. L865V ^S )

Amino acid position 870 (eg L870F ⁶ )

Amino acid position 891 (e.g. S891A) ¹⁰

Amino acid position 904 (e.g. S904F) ¹²

Amino acid position 804/904 (e.g. V804M/S904C) ¹¹

Amino acid position 918 (eg M918T) ^{10 1} Yoon et al., J. Med. Chem. 59(1):358-73, 2016.

² US Patent No. 8629135.

³ Cranston, et al., Cancer Res. 66(20):10179-87, 2006.

⁴ Carlomagno, et al., Endocr. Rei. Cancer 16(1):233-41,2009.

⁵ Huang et aL, Mol. Cancer Then, 2016 Aug 5. pii: molcanther.0258.2016. [Email] publ. before publication].

⁶ PCT Patent Application Publication No. WO 2016/127074.

⁷ Mamedova et al., Summer Undergraduate Research Programs (SURP) Student Abstracts, University of Oklahoma Health Sciences Center, 2016.

⁸ Plenker et al., Sc. Transl. Med., 9(394), doi: 10.1126/scitranslmed.aah6144, 2017.

⁹ Kraft et al, Cancer Research, 2017, vol. 77, no. 13, add. Appendix 1. Abstract number: 4882; American Association for Cancer Research Annual Meeting 2017. Washington DC, USA. 01 Apr. 2017-05 Apr. 2017.

¹⁰ U.S. Patent Application Publication No. 2018/0022732.

¹¹ Roskoski and Sadeghi-Nejad, Pharmacol. Res., 128, 1-17. doi: 10.1016/j.phrs.2017.12.021, 2018.

¹² Nakaoku, et al. NatCommun, 9(1), 625. doi: 10.1038/S41467-018-02994-7, 2018.

¹³ Roy et al. Oncologist, 18(10): 1093-1100. doi: 10.1634/theoncologist.2013-0053, 2013.

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Table 4 Additional Illustrative Amino Acid Positions of RET Inhibitor Resistance Mutations

Amino acid RET and position Illustrative mutation Mechanistic substantiation resistance L730 R Steric hindrance and/or active conformational effect G731 V Steric hindrance and/or active conformational effect E732 TO Steric hindrance and/or active conformational effect G733 V Steric hindrance and/or active conformational effect E734 To Steric hindrance and/or active conformational effect L760 m Active conformational effect K761 E Active conformational effect E762 TO Active conformational effect N763 D Active conformational effect A764 V Active conformational effect S765 N Active conformational effect R766 A Active conformational effect S767 WITH Active conformational effect E768 TO Active conformational effect L779 M Steric hindrance and/or active conformational effect I788 M Steric hindrance and/or active conformational effect M868 R Steric hindrance and/or active conformational effect K869 E Steric hindrance and/or active conformational effect L870 Q Steric hindrance and/or active conformational effect V871 M Steric hindrance and/or active conformational effect H872 R Steric hindrance and/or active conformational effect R873 R Steric hindrance and/or active conformational effect D874 Y Steric hindrance and/or active conformational effect L881 R Steric hindrance and/or active conformational effect L895 M Active conformational effect S896 N Active conformational effect

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R897 C Active conformational effect D898 Y Active conformational effect V899 G Active conformational effect Y900 D Active conformational effect E901 TO Active conformational effect E902 TO Active conformational effect D903 Y Active conformational effect S904 WITH Active conformational effect Y905 D Active conformational effect V906 M Active conformational effect K907 Ε Active conformational effect R908 P Active conformational effect S909 Ο Active conformational effect Q910 R Active conformational effect G911 Ο Active conformational effect R912 P Active conformational effect

The oncogenic role of RET was first described in papillary thyroid carcinoma (PTC) (Grieco et al., Cell, 1990, 60, 557-63), which is derived from thyroid follicular cells and is the most common thyroid malignancy. About 2030% of RTSs have somatic chromosomal rearrangements (translocations or inversions) linking the promoter and 5' fragments of constitutively expressed, unrelated genes with the RET tyrosine kinase domain (Greco et al., Q. J. Nucl. Med. Mol. Imaging, 2009, 53, 440 -54), controlling its ectopic expression in thyroid cells. The fusion proteins resulting from such rearrangements are referred to as RET/PTC proteins. For example, RET/PTC 1 is a fusion between CCDD6 and RET that is commonly found in papillary thyroid carcinomas. Similarly, RET/PTC3 and RET/PTC4 are ELE1 and RET fusions that are common in papillary thyroid carcinomas, although the fusion events leading to the formation of RET/PTC3 and RET/PTC4 result in different proteins with different molecular weights ( see, for example, Fugazzola et al., Oncogene, 13(5):1093-7, 1996). Some RET fusions associated with PTC are not referred to as RET/PTC, but instead are referred to as the fusion protein itself. For example, a fusion between RET and both ELKS and PCM1 occurs in PTC, but the fusion proteins are called ELKS-RET and PCM1-RET (see, e.g., Romei and Elisei, Front. Endocrinol. (Lausanne), 3:54, doi: 10.3389 /fendo.2012.00054, 2012). The role of RET-PTC rearrangements in the pathogenesis of RTS has been confirmed in transgenic mice (Santoro et al., Oncogene, 1996, 12, 1821-6). To date, various fusion partners have been identified from PTC and other types of cancer, all of which provide a protein/protein interaction domain that induces ligand-independent RET dimerization and constitutive kinase activity (see, for example, Table 1). A 10.6 Mb pericentric inversion on chromosome 10, where the RET gene map is located, has recently been identified in about 2% of lung adenocarcinoma patients, creating different variants of the KIF5B-RET chimeric gene (Ju et al., Genome Res., 2012, 22, 436-45; Kohno et al., 2012, Nature Med., 18, 375-7; Takeuchi et al., Nature Med., 2012, 18, 378-81; Lipson et al., 2012, Nature Med., 18 , 382-4). Fusion transcripts are highly expressed and all generated chimeric proteins contain the N-terminal portion and supercoil region of KIF5B that mediates homodimerization and the complete RET kinase domain. None of the RET-positive patients have other known oncogenic changes (such as EGFR or K-Ras mutation, ALK translocation), supporting the possibility that KIF5B-RET fusion may be the leading mutation in lung adenocarcinoma. The oncogenic potential of KIF5B-RET was confirmed by transfection of the fusion gene into cultured cell lines: similar to what was observed with RET-PTC fusion proteins, KIF5B-RET is constitutively phosphorylated and causes NIH-3T3 transformation and IL-3-independent growth of BA-F3 cells. However, other RET fusion proteins have been identified in lung adenocarcinoma patients, such as the CCDC6RET fusion protein, which has been found to play a key role in the proliferation of the human lung adenocarcinoma cell line LC-2/ad (Journal of Thoracic Oncology, 2012, 7(12 ): 1872-1876). RET inhibitors have been shown to be useful in the treatment of lung cancer involving RET rearrangements (Drilon, A.E. et al. J Clin Oncol 33, 2015 (added; ref. 8007)). RET fusion proteins have also been identified in patients with colon and rectal cancer (Song Eun-Kee, et al. International Journal of Cancer, 2015, 136:

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1967-1975).

In addition to rearrangements in the RET sequence, point mutations to acquire the function of the RET proto-oncogene are also leading oncogenic events, as demonstrated in medullary thyroid carcinoma (MTC), which is derived from calcitonin-producing parafollicular cells (de Groot, et al., Endocrine Rev. ., 2006, 27, 535-60; Wells and Santoro, Clin. Cancer Res., 2009, 15, 7119-7122). Approximately 25% of MTS cases are associated with multiple endocrine neoplasia type 2 (MEN2), a group of hereditary cancer syndromes affecting the neuroendocrine organs that are caused by RET germline activating point mutations. In the MEN2 subtypes (MEN2A, MEN2B, and familial MTC/FMTC), mutations in the RET gene have a strong phenotype-genotype correlation, which determines different MTS aggressiveness and clinical manifestations of the disease. Mutations in the MEN2A syndrome include one of six cysteine residues (mainly C634) located in the extracellular region with high cysteine content, resulting in ligand-independent homodimerization and constitutive RET activation. Patients at an early age (onset at 5–25 years) develop MTS and may also develop pheochromocytoma (50%) and hyperparathyroidism. MEN2B is caused primarily by the M918T mutation located in the kinase domain. This mutation constitutively activates RET in its monomeric state and alters kinase recognition of the substrate. The MEN2B syndrome is characterized by early onset (<1 year) and is a highly aggressive form of MTS, pheochromocytoma (50% of patients), and ganglioneuroma. In FMTC, the only manifestation of the disease is MTS, which usually occurs in adulthood. Many different mutations have been found spanning the entire RET gene. The remaining 75% of MTS cases are sporadic, and about 50% of them carry somatic RET mutations: the most common mutation is M918T, which, as in the case of MEN2B, is associated with the most aggressive phenotype. Somatic RET point mutations have also been described in other tumors such as colon and rectal cancer (Wood et al., Science, 2007, 318, 1108-13) and small cell lung carcinoma (Jpn. J. Cancer Res., 1995, 86, 1127-30). In some embodiments, the MTC is a RET fusion positive MTC.

RET signaling components have been shown to be expressed in primary breast tumors and functionally interact with the estrogen receptor-cc cascade in breast tumor cell lines (Boulay et al., Cancer Res. 2008, 68, 3743-51; Plaza-Menacho et al ., Oncogene, 2010, 29, 4648-57), and RET expression and activation by GDNF family ligands may play an important role in perineural invasion of various types of cancer cells (Ito et al., Surgery, 2005, 138, 78894; Gil et al. , J. Natl Cancer Inst., 2010, 102, 107-18; Iwahashi et al., Cancer, 2002, 94, 167-74).

RET is also expressed in 30-70% of invasive breast cancers, with expression relatively more common in estrogen receptor positive tumors (Plaza-Menacho, I., et al., Oncogene, 2010, 29, 4648-4657; Esseghir, S., et al., Cancer Res., 2007, 67, 11732-11741; Morandi, A., et al., Cancer Res., 2013, 73, 3783-3795; Gattelli, A., EMBO Mol. Med. , 2013, 5, 1335-1350).

The identification of RET rearrangements is described in a subset of PDX (patient-derived xenografts) derived from colon and rectal cancer. Although the frequency of these events in patients with colon and rectal cancer has not yet been established, the data obtained suggest a role for RET as a target in this indication (Gozgit et al., AACR Annual Meeting 2014). Various studies have shown that the RET promoter is frequently methylated in colon and rectal cancer, and heterozygous missense mutations that putatively reduce RET expression have been identified in 5-10% of cases, suggesting that RET may have some features of a tumor suppressor. in sporadic colon cancers (Luo, Y., et al., Oncogene, 2013, 32, 2037-2047; Sjoblom, T., et al., Science, 2006, 268-274; Cancer Genome Atlas Network, Nature, 2012, 487, 330-337).

An increasing number of tumor types have now been shown to express significant amounts of wild-type RET kinase, which may influence tumor development and spread. RET is expressed in 50-65% of pancreatic ductal carcinomas, and expression is more common in metastatic and higher grade tumors (Ito, Y, et al., Surgery, 2005, 138, 788-794; Zeng, Q., et al., J. Int. Med. Res. 2008, 36, 656-664).

In neoplasms of hematopoietic lineages, RET is expressed in acute myeloid leukemia (AML) with monocytic differentiation, as well as in CMML (Gattei, V. et al., Blood, 1997, 89, 29252937; Gattei, V., et al., Ann. Hematol , 1998, 77, 207-210; Camos, M., Cancer Res. 2006, 66, 6947-6954). Recent studies have identified rare chromosomal rearrangements affecting RET in patients with chronic myelomonocytic leukemia (CMML). CMML is often associated with rearrangements of several tyrosine kinases that lead to the expression of chimeric cytosolic oncoproteins that lead to activation of the RAS cascades (Kohlmann, A., et al., J. Clin. Oncol. 2010, 28, 2858-2865). In the case of RET, fusion proteins that bind RET to BCR (BCR-RET) or to the oncogenic partner, fibroblast growth factor receptor 1 (FGFR1OP-RET), have been transformed in early hematopoietic progenitor cells, and they can shift the maturation of these cells towards monocytic pathways, probably due to initiation of RET-mediated signaling

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RAS (Ballerini, P., et al., Leukemia, 2012, 26, 2384-2389).

RET expression has also been demonstrated in several other tumor types, including prostate cancer, small cell lung cancer, melanoma, renal cell carcinoma, and head and neck tumors (Narita, N., et al., Oncogene, 2009, 28, 3058-3068; Mulligan, L. M., et al., Genes Chromosomes Cancer, 1998, 21, 326-332; Flavin, R., et al., Urol. Oncol., 2012, 30, 900-905; Dawson, D. M., J Natl Cancer Inst, 1998 , 90, 519-523).

In neuroblastoma, RET expression and its activation by GFL plays a role in tumor cell differentiation, probably with the participation of other neurotrophic factor receptors, to down-regulate N-Myc, the expression of which is a marker of poor prognosis (Hofstra, R. M., W., et al., Hum Genet 1996, 97, 362-364; Petersen, S. and Bogenmann, E., Oncogene, 2004, 23, 213-225; Brodeur, G. M., Nature Ref. Cancer, 2003, 3, 203-216).

Multi-target inhibitors are known that cross-react with RET (Borrello, M.G., et al., Expert Opin. Ther. Targets, 2013, 17(4), 403-419; international patent applications No. WO 2014/141187, WO 2014/184069 and WO 2015/079251). Such multitarget inhibitors (or multikinase inhibitors or MKIs) may also be associated with the development of RET inhibitor resistance mutations. See, for example, Q. Huang et al., Preclinical Modeling of KIF5B-RET Fusion Lung Adenocarcinoma., Mol. Cancer Ther, No. 18, cc. 2521-2529, 2016; Yasuyuki Kaneta et al., abstract B173: Preclinical characterization and antitumor efficacy of DS-5010, a highly potent and selective RET inhibitor, Mol Cancer Ther, January 1, 2018 (17) (1 addition) B173; DOE10.1158/1535-7163.TARG-17-B173, the entire contents of which are incorporated herein by reference.

Accordingly, provided herein are methods of treating a patient diagnosed with (or determined to have) cancer which comprises administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Also provided herein are methods of treating a patient identified or diagnosed with RET-associated cancer, which comprises administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof. In some embodiments, RET-associated cancer is identified or diagnosed in a patient through the use of a government-approved, e.g., FDA-approved test or assay to identify dysregulation of the RET gene, RET kinase, or expression or activity, or level of any of them, in the patient. or in a biopsy sample obtained from a patient, or by performing any of the non-limiting examples of assays described herein. In some embodiments, the test or assay is presented as a kit. In some embodiments, the cancer is a RET-associated cancer. For example, a RET-associated cancer may be a cancer that includes one or more RET inhibitor resistance mutations.

Also provided herein are methods for treating cancer in a patient in need thereof, comprising: (a) detecting RET-associated cancer in the patient; and (b) administering to said patient a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof. Some embodiments of the proposed methods further comprise administering to the subject another anti-cancer agent (eg, a second RET inhibitor, a second compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form, or immunotherapy thereof). In some embodiments, the subject has previously been treated with a first RET inhibitor or has previously been treated with another anticancer agent, such as through at least partial tumor resection or radiation therapy. In some embodiments, a RET-associated cancer has been identified in a patient through the use of a government-approved, e.g., FDA-approved test or assay to identify dysregulation of the RET gene, RET kinase, or expression or activity, or level of any of these, in the patient. or in a biopsy sample obtained from a patient, or by performing any of the non-limiting examples of assays described herein. In some embodiments, the test or assay is presented as a kit. In some embodiments, the cancer is a RET-associated cancer. For example, a RET-associated cancer may be a cancer that includes one or more RET inhibitor resistance mutations.

Also provided are methods of treating a patient, which include performing analysis on a sample obtained from the patient to determine if the patient has dysregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them, and administration (for example, specific or selective administration) of a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof, to a patient who has been found to have dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them. Some embodiments of the proposed methods further comprise administering to the subject another anti-cancer agent (e.g., a second inhibitor

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RET, the second compound of formula I-IV, or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or immunotherapy). In some embodiments of the proposed methods, the subject has previously been treated with a first RET inhibitor or has previously been treated with another anticancer agent, such as through at least partial tumor resection or radiation therapy. In some embodiments, the patient is a patient who is suspected of having RET associated cancer, a patient exhibiting one or more symptoms of RET associated cancer, or a patient at increased risk of developing RET associated cancer. In some embodiments, the assay uses next generation sequencing, pyrosequencing, immunohistochemistry, or FISH analysis with a Break Apart probe. In some embodiments, the assay is a regulatory approved assay, such as an FDA approved kit. In some embodiments, the assay is a liquid biopsy. Additional, non-limiting assays that can be used in these methods are described in this document. Additional assays are also known in the art. In some embodiments, the dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations.

Also provided is a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or a pharmaceutical composition thereof for use in the treatment of RET-associated cancer in a patient who has been diagnosed or found to have RET-associated cancer at the stage of analysis (for example, in vitro analysis) on a sample obtained from a patient to determine whether the patient has dysregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them, and the presence of dysregulation of the RET gene, RET kinase, or expression, or activity, or level any of them is determined by the fact that the patient is suffering from RET-associated cancer. Also proposed is the use of a compound of formula IIV or its pharmaceutically acceptable salt, amorphous or polymorphic form for the manufacture of a medicament for the treatment of RET-associated cancer in a patient who has been identified or diagnosed as having RET-associated cancer at the stage of analysis on a sample obtained from the patient, to determine whether a given patient has dysregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them, and the presence of dysregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them determines the fact that the patient is suffering from RET-associated cancer. Some embodiments of any of the methods or uses described herein further include recording in a patient's medical history (e.g., on a computer-readable medium) that the patient is found to have a dysregulation of the RET gene, RET kinase, or expression or activity, or level of any of these, by analysis, and he needs to enter the compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, or its pharmaceutical composition. In some embodiments, the assay uses next generation sequencing, pyrosequencing, immunohistochemistry, or FISH analysis with a Break Apart probe. In some embodiments, the assay is a regulatory approved assay, such as an FDA approved kit. In some embodiments, the assay is a liquid biopsy. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations.

Also provided is a compound of formula I-IV, or a pharmaceutically acceptable salt thereof, in an amorphous or polymorphic form, for use in the treatment of cancer in a patient in need thereof, or in a patient who has been identified or diagnosed as having RET-associated cancer. This document also provides the use of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, for the manufacture of a medicament for the treatment of cancer in a patient diagnosed or diagnosed with RET-associated cancer. In some embodiments, the cancer is RET-associated cancer, eg, RET-associated cancer, having one or more RET inhibitor resistance mutations. In some embodiments, RET-associated cancer is identified or diagnosed in a patient through the use of a government-approved, e.g., FDA-approved kit to identify dysregulation of the RET gene, RET kinase, or expression or activity, or level of any of these, in the patient. or in a biopsy sample from this sample. As defined herein, RET-associated cancer includes cancers described herein and known in the art.

In some embodiments of any of the methods or uses described herein, a patient is identified or diagnosed as having a cancer with dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them. In some embodiments of any of the methods or uses described herein, a tumor is identified in a patient that is positive for dysregulation of the RET gene, RET kinase, or

- 79 041220 expression, or activity, or the level of any of them. In some embodiments of any of the methods or uses, said patient may be a patient with a tumor(s) that is positive for dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them. In some embodiments of any of the methods or uses described herein, said patient may be a patient whose tumors have dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them. In some embodiments of any of the methods or uses described herein, the patient is predisposed to RET-associated cancer (eg, cancer having one or more RET inhibitor resistance mutations). In some embodiments, provided herein are methods of treating RET-associated cancer in a patient in need of such treatment, comprising a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the patient; and b) administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them includes one or more fusion proteins. Non-limiting examples of RET gene fusion proteins are described in Table 1. 1. In some embodiments, the fusion protein is KIF5B-RET. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more point mutations/insertions/deletions in the RET kinase protein. Non-limiting examples of point mutations/inserts/deletions in the RET kinase protein are described in Table 1. 2 and 2a. In some embodiments, point mutations/inserts/deletions in the RET kinase protein are selected from the group consisting of M918T, M918V, C634W, V804L, V804M, G810S, and G810R. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations. Non-limiting examples of RET inhibitor resistance mutations are described in Table 1. 3 and 4. In some embodiments, the RET inhibitor resistance mutation is V804M. In some embodiments, the RET inhibitor resistance mutation is G810S. In some embodiments, the RET inhibitor resistance mutation is G810R. In some embodiments, a cancer with dysregulation of the RET gene, RET protein, or expression or activity or level of any of these is determined using a regulatory approved assay or kit, such as an FDA approved one. In some embodiments, a tumor that is positive for dysregulation of the RET gene, RET kinase, or expression or activity or level of any of these is a tumor that is positive for one or more RET inhibitor resistance mutations. In some embodiments, a tumor with dysregulation of the RET gene, RET protein, or expression or activity or level of any of these is determined using a regulatory approved assay or kit, such as an FDA approved one.

In some embodiments of any of the methods or uses described herein, the patient has a medical history that indicates that the patient has a tumor that has dysregulation of the RET gene, RET kinase, or expression, or activity, or level of any of these (eg, a tumor having one or more RET inhibitor resistance mutations). In some embodiments, the medical history indicates that the patient is to be treated with one or more compounds of formula I-IV or their pharmaceutically acceptable salts, amorphous or polymorphic forms, or compositions provided herein. In some embodiments, a cancer with dysregulation of the RET gene, RET kinase, or expression or activity or level of any of these is a cancer having one or more RET inhibitor resistance mutations. In some embodiments, cancer with dysregulation of the RET gene, RET protein, or expression or activity or level of any of these is determined using a regulatory approved assay or kit, such as an FDA approved one. In some embodiments, a tumor that is positive for dysregulation of the RET gene, RET kinase, or expression or activity or level of any of these is a tumor that is positive for one or more RET inhibitor resistance mutations. In some embodiments, a tumor with dysregulation of the RET gene, RET protein, or expression or activity or level of any of these is determined using a regulatory approved assay or kit, such as an FDA approved one.

Also provided are methods of treating a patient which comprise administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, in an amorphous or polymorphic form, to a patient with a medical history indicating that the patient has dysregulation of the RET gene, RET kinase, or expression , or activity, or the level of any of them. Also provided is the use of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, for the manufacture of a medicament for the treatment of RET-associated cancer in a patient who has a medical history indicating that the patient has dysregulation of the RET gene, RET kinase, or expression, or activity, or level of any

- 80 041220 of them. Some embodiments of these methods and uses may further include: the step of performing an assay (e.g., an in vitro assay) on a sample obtained from a patient to determine if the patient has a deregulation of the RET gene, RET kinase, or expression or activity, or the level of any of them, and recording the received information in the patient's clinical file (for example, on a computer-readable medium) that the presence of RET gene dysregulation, RET kinase, or expression, or activity, or the level of any of them is identified in this patient. In some embodiments, said assay is an in vitro assay. For example, an assay that uses next generation sequencing, immunohistochemistry, or a FISH assay with a Break Apart probe. In some embodiments, the assay is approved by a regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy. In some embodiments, the dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations.

This document also provides a method of treating a subject. In some embodiments, the proposed method includes performing an analysis on a sample obtained from the subject to determine if the subject has dysregulation of the RET gene, RET protein, or expression or level of any of them. In some such embodiments, the proposed method also includes administering to a subject found to have dysregulation of the RET gene, RET protein, or expression or activity, or level of any of them, a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form. In some embodiments, the proposed method includes determining if the subject has dysregulation of the RET gene, RET protein, or expression or level of any of them by performing an assay that is performed on a sample obtained from the subject. In such embodiments, the proposed method also includes administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the dysregulation of the RET gene, RET kinase protein, or their expression or activity is a gene or chromosomal translocation that results in the expression of a RET fusion protein (eg, any of the RET fusion proteins described herein). In some embodiments, the RET fusion may be selected from a KIF5B-RET fusion and a CCDC6-RET fusion. In some embodiments, the dysregulation in the RET gene, the RET kinase protein, or the expression or activity or level of any of them is one or more point mutations in the RET gene (e.g., any one or more of the RET point mutations described herein) . One or more point mutations in the RET gene may result in, for example, translation of a RET protein having one or more of the following amino acid substitutions: M918T, M918V, C634W, V804L, V804M, G810S, and G810R. In some embodiments, the dysregulation in the RET gene, the RET kinase protein, or the expression or activity or level of any of them is one or more RET inhibitor resistance mutations (e.g., any combination of one or more RET inhibitor resistance mutations described in this document). Some embodiments of the proposed methods further comprise administering to the subject another anti-cancer agent (eg, a second RET inhibitor, a second compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form, or immunotherapy thereof).

In some embodiments, the compounds provided herein exhibit penetration into the brain and/or the central nervous system (CNS). Such compounds can cross the blood-brain barrier and inhibit RET kinase in the brain and/or other CNS structures. In some embodiments, the compounds provided herein may cross the blood-brain barrier in a therapeutically effective amount. For example, treating a patient for cancer (eg, RET-associated cancer, such as RET-associated brain or CNS cancer) may include administering (eg, orally) the subject compound to the patient. In certain such embodiments, the compounds provided herein are useful in the treatment of a primary brain tumor or a metastatic brain tumor. For example, the subject compounds can be used in the treatment of one or more gliomas such as glioblastoma (also known as glioblastoma multiforme), astrocytomas, oligodendrogliomas, ependymas and mixed gliomas, meningiomas, medulloblastomas, gangliogliomas, schwannomas (neurilemmomas) and craniopharyngiomas (see e.g. , tumors listed in Louis, D.N. et al Acta Neuropathol 131(6), 803-820 (June 2016)). In some embodiments, the brain tumor is a primary brain tumor. In some embodiments, the patient has previously been treated with another anti-cancer agent, such as another RET inhibitor (eg, a compound that is not a compound of general formula I) or a multikinase inhibitor. In some embodiments, the brain tumor is a metastatic brain tumor. In some embodiments, the patient has previously been treated with another anti-cancer agent, such as another RET inhibitor (eg, a compound that is not a compound of general formula I) or a multikinase inhibitor.

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Also provided are methods (eg, in vitro methods) for selecting treatment for a patient who has been identified or diagnosed as having RET-associated cancer. Some embodiments may further include administering the selected treatment to a patient who has been identified or diagnosed as having RET-associated cancer. For example, the treatment of choice may include administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Some embodiments may further include the step of performing analysis on a sample obtained from the patient to determine if the patient has dysregulation of the RET gene, RET kinase, or expression or activity, or level of any of them, and identifying and diagnosing in the patient, which is determined to have a deregulation of the RET gene, RET kinase, or the expression or activity, or the level of any of them, the presence of RET-associated cancer. In some embodiments, the cancer is a RET-associated cancer having one or more RET inhibitor resistance mutations. In some embodiments, RET-associated cancer is identified or diagnosed in a patient through the use of a government-approved, e.g., FDA-approved kit to identify dysregulation of the RET gene, RET kinase, or expression or activity, or level of any of these, in the patient. or in a biopsy sample from a given patient. In some embodiments, the RET-associated cancer is a cancer described herein or known in the art. In some embodiments, the assay is an in vitro assay. For example, an assay that uses next generation sequencing, immunohistochemistry, or a FISH assay with a Break Apart probe. In some embodiments, the assay is approved by a regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy.

Also provided herein are methods for selecting a treatment for a patient, the methods comprising the step of assaying a sample obtained from the patient to determine if the patient has dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of of them (e.g., one or more RET inhibitor resistance mutations), and identifying or diagnosing, in a patient, that RET gene, RET kinase dysregulation, or expression, or activity, or level of any of them, has RET-associated cancer. Some embodiments further include administering the selected treatment to a patient who has been identified or diagnosed as having RET-associated cancer. For example, the treatment of choice may include administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, to a patient who has been identified or diagnosed as having RET-associated cancer. In some embodiments, said assay is an in vitro assay. For example, an assay that uses next generation sequencing, immunohistochemistry, or a FISH assay with a Break Apart probe. In some embodiments, the assay is approved by a regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy.

Also provided are methods for selecting a patient for treatment, the methods comprising selecting, identifying or diagnosing a patient suffering from RET-associated cancer and selecting a patient for treatment comprising administering a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form. In some embodiments, identifying or diagnosing a patient as suffering from a RET-associated cancer may include the step of performing an analysis on a sample obtained from the patient to determine if the patient has dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them, and identifying and diagnosing in a patient who is found to have dysregulation of the RET gene, RET kinase, or the expression, or activity, or level of any of them, the presence of RET-associated cancer. In some embodiments, the method for selecting a patient for treatment may be used as part of a clinical trial that includes the administration of various treatments for RET-associated cancer. In some embodiments, the RET-associated cancer is a cancer having one or more RET inhibitor resistance mutations. In some embodiments, said assay is an in vitro assay. For example, an assay that uses next generation sequencing, immunohistochemistry, or a FISH assay with a Break Apart probe. In some embodiments, the assay is approved by a regulatory agency, such as an FDA-approved kit. In some embodiments, the assay is a liquid biopsy. In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations.

In some embodiments of any of the methods or uses described herein, an assay used to determine if a patient has dysregulation of the RET gene or RET kinase, or the expression or activity or level of any of them, using a sample obtained from a patient may include, for example, next generation sequencing,

- 82 041220 immunohistochemistry, fluorescence microscopy, FISH analysis with Break Apart probe, Southern blot, Western blot, FACS analysis, Northern blot, and PCR-based amplification (eg, RT-PCR and real-time quantitative RT-PCR). As is known in the art, these assays are typically performed, for example, using at least one labeled nucleic acid probe or at least one labeled antibody or antigen-binding fragment thereof. The assays can use other detection methods known in the art to detect dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of these (see, for example, the references cited herein). In some embodiments, the dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them comprises one or more RET inhibitor resistance mutations. In some embodiments, the sample is a biological sample or a biopsy sample (eg, a biopsy sample embedded in paraffin) obtained from a patient. In some embodiments, the patient is a patient suspected of having RET associated cancer, a patient having one or more symptoms of RET associated cancer, and/or a patient at increased risk of developing RET associated cancer.

In some embodiments, dysregulation of the RET gene, RET kinase, or expression or activity or level of any of these can be identified using a liquid biopsy (otherwise referred to as a liquid biopsy or liquid phase biopsy). See, for example, Karachialiou et al., Realtime liquid biopsies become a reality in cancer treatment, Ann. Transl. Med, 3(3):36, 2016. Liquid biopsy techniques can be used to detect overall tumor burden and/or dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them. Liquid biopsy can be performed on biological samples relatively easily obtained from a subject (eg, through routine blood sampling) and is usually less invasive than conventional methods used to detect tumor burden and/or dysregulation of the RET gene, RET kinase, or expression , or activity, or the level of any of them. In some embodiments, a liquid biopsy can be used to detect the presence of dysregulation of the RET gene, RET kinase, or expression or activity or level of any of these at an earlier stage than conventional methods. In some embodiments, the biological specimen to be used for a liquid biopsy may include, for example, blood, plasma, urine, cerebrospinal fluid, saliva, sputum, bronchoalveolar lavage, bile, lymphatic fluid, cystic fluid, feces, peritoneal effusion, and their combinations. In some embodiments, a liquid biopsy can be used to detect circulating tumor cells (TCCs). In some embodiments, a liquid biopsy can be used to detect cell-free DNA. In some embodiments, cell-free DNA detected by liquid biopsy is circulating tumor DNA (cDNA) derived from tumor cells. ctDNA analysis (eg, using sensitive detection technologies such as, but not limited to, next generation sequencing (NGS), conventional PCR, digital PCR, or microarray analysis) can be used to identify dysregulation of the RET gene, RET kinase, or expression or activity , or the level of any of them.

In some embodiments, liquid biopsy can detect ctDNA derived from a single gene. In some embodiments, liquid biopsy can detect ctDNA formed from multiple genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more, or any number of genes in between). In some embodiments, coDNA derived from multiple genes can be detected using any of a variety of commercially available test panels (e.g., commercially available test panels configured to detect RET gene dysregulation, RET kinase, or expression or activity, or the level of any of them). Liquid biopsies can be used to detect dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them, including, but not limited to, point mutations or single nucleotide variants (SNVs), copy number variants (CNVs), genetic fusions (e.g. , translocations or rearrangements), insertions, deletions, or any combination thereof. In some embodiments, a liquid biopsy can be used to detect a gamete mutation. In some embodiments, a liquid biopsy can be used to detect a somatic mutation. In some embodiments, a liquid biopsy can be used to detect a primary genetic mutation (eg, primary mutation or primary fusion associated with the initial development of a disease, such as cancer). In some embodiments, a liquid biopsy can be used to detect a genetic mutation that develops after the development of a primary genetic mutation (eg, a resistance mutation that occurs in response to a drug that is administered to a subject). In some embodiments, the dysregulation of the RET gene, RET kinase, or expression or activity, or the level of any of them, as determined by liquid biopsy, is also present in a cancer cell that is in the subject's body (eg, a tumor). In some embodiments, liquid biopsy can detect any type of gene dysregulation

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RET, RET kinase, or expression, or activity, or the level of any of them, as described in this document. In some embodiments, a genetic mutation identified by a liquid biopsy can be used to identify a subject as potentially suitable for a particular treatment. For example, detection of dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a subject may indicate that the subject will be responsive to treatment that includes administering a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

A liquid biopsy may be performed at various points in time during the course of diagnosis, course of observation, and/or course of treatment to determine one or more clinically relevant parameters, including, without limitation, disease progression, treatment efficacy, or the development of resistance mutations following administration of a drug to a subject. For example, the first liquid biopsy may be performed at a first time point and the second liquid biopsy may be performed at a second time point during the course of diagnosis, course of observation, and/or course of treatment. In some embodiments, the first time point may be a point in time before the subject is diagnosed with a disease (e.g., when the subject is healthy), and the second point in time may be a point in time after the subject has developed a disease (e.g., the second point in time can be used to make the subject of the disease diagnosis). In some embodiments, the first time point may be a point in time before the subject is diagnosed with a disease (eg, when the subject is healthy), after which the subject is observed, and the second point in time may be a point in time after the subject is observed. In some embodiments, the first time point may be the time point after the subject is diagnosed with the disease, after which the subject is treated, and the second time point may be the time point after the treatment is administered; in such cases, the second time point can be used to assess the effectiveness of the treatment (for example, if the genetic mutation(s) found at the first time point is reduced in number or not detected), or to determine the presence of a resistance mutation resulting from the treatment. In some embodiments, the treatment to be administered to a subject may include a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

In some embodiments, the efficacy of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, can be determined by analyzing the allelic frequency of RET gene dysregulation in scDNA obtained from a patient at different time points, for example, in scDNA obtained from a patient at the first time point, and scDNA obtained from the patient at a second time point, wherein at least one dose of a compound of formula I-IV is administered to the patient between the first and second time points. Some embodiments of such methods may further include administering to the patient at least one dose of a compound of formula IIV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, between the first and second time points. For example, reduction (e.g., reduction from 1% to about 99%, reduction from 1% to about 95% reduction, reduction from 1% to about 90%, reduction from 1% to about 85%, reduction from 1% to about 80 %, decrease from 1% to about 75%, decrease from 1% to about 70%, decrease from 1% to about 65%, decrease from 1% to about 60%, decrease from 1% to about 55%, decrease from 1 % to about 50%, decrease from 1% to about 45%, decrease from 1% to about 40%, decrease from 1% to about 35%, decrease from 1% to about 30%, decrease from 1% to about 25% , decrease from 1% to about 20%, decrease from 1% to about 15%, decrease from 1% to about 10%, decrease from 1% to about 5%, from about 5% to about 99%, from about 10% to about 99%, from about 15% to about 99%, from about 20% to about 99%, from about 25% to about 99%, from about 30% to about 99%, from about 35% to about 99%, about 40% to about 99%, about 45% to about 99%, about 50% to about 99%, about 55% to about 99%, about 60% to about 99%, about 65% toabout 99%, about 70% to about 99%, about 75% to about 95%, about 80% to about 99%, about 90% to about 99%, about 95% to about 99%, from about 5% to about 10%, about 5% to about 25%, about 10% to about 30%, about 20% to about 40%, about 25% to about 50%, about 35% to about 55%, about 40% to about 60%, about 50% to about 75%, about 60% to about 80%, or about 65% to about 85% reduction in scDNA RET gene dysregulation allelic frequency (AF) obtained from the patient at the second time point, compared with the allelic frequency (AF) of RET gene dysregulation in scDNA obtained from the patient at the first time point, indicates that the compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic the form is effective in that subject. In some embodiments, AF is reduced such that the level falls below the detection limit of the instrument. Alternatively, an increase in the allelic frequency (AF) of the RET gene dysregulation in the patient-derived scDNA at the second time point compared to the allelic frequency (AF) of the RET gene dysregulation in the patient-derived scDNA at the first time point indicates that that a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is not effective in the subject (e.g., the subject has developed a resistance mutation to

- 84 041220 to the compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form). Some embodiments of these methods may further include administering additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof to a patient who has been found to be efficacious of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Some embodiments of these methods may further include administering another treatment (e.g., a treatment that does not include administering a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy) to a patient who has been found to be ineffective with a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form.

In some examples of such methods, the time difference between the first and second time points may be from about 1 day to about 1 year, from about 1 day to about 11 months, from about 1 day to about 10 months, from about 1 day to about 9 months. , from about 1 day to about 8 months, from about 1 day to about 7 months, from about 1 day to about 6 months, from about 1 day to about 5 months, from about 1 day to about 4 months, from about 1 day up to about 3 months, from about 1 day to about 10 weeks, from about 1 day to about 2 months, from about 1 day to about 6 weeks, from about 1 day to about 1 month, from about 1 day to about 25 days, from about 1 day to about 20 days, from about 1 day to about 15 days, from about 1 day to about 10 days, from about 1 day to about 5 days, from about 2 days to about 1 year, from about 5 days to about 1 year, about 25 days to about 1 year, about 1 month to about 1 year, about 6 weeks to about 1 year, about 2 months to about 1 year, about 3 months to about 1 g about 4 months to about 1 year, about 5 months to about 1 year, about 3 months to about 1 year, about 4 months to about 1 year, about 5 months to about 1 year, from about 6 about 7 months to about 1 year, about 8 months to about 1 year, about 9 months to about 1 year, about 10 months to about 1 year, about 11 months to about 1 year , from about 1 day to about 7 days, from about 1 day to about 14 days, from about 5 days to about 10 days, from about 5 days to about 20 days, from about 10 days to about 20 days, from about 15 days up to about 1 month, from about 15 days to about 2 months, from about 1 week to about 1 month, from about 2 weeks to about 1 month, from about 1 month to about 3 months, from about 3 months to about 6 months, from about 4 months to about 6 months, from about 5 months to about 8 months, or from about 7 months to about 9 months. In some embodiments of such methods, the patient may have previously been identified with a cancer having a misregulated RET gene (eg, any of the examples of a misregulated RET gene described herein). In some embodiments of such methods, the patient may have previously been diagnosed with any type of cancer described herein. In some embodiments of such methods, the patient may have one or more metastases (eg, one or more brain metastases).

In some of the above embodiments, the scDNA includes tsDNA, such as RET-associated tsDNA. For example, scDNA is tsDNA, such as RET-associated tsDNA. In some embodiments, at least a portion of the scDNA is determined to be RET-associated ctDNA, for example, a sequenced and/or quantified amount of total scDNA is found to have a RET fusion mutation and/or a RET resistance mutation.

In the field of medical oncology, it is standard practice to use a combination of different forms of treatment to treat each patient with cancer. In the field of medical oncology, the other component(s) of such co-treatment or therapy, in addition to the compositions provided herein, may be, for example, surgery, radiation therapy, and chemotherapeutic agents such as other kinase inhibitors, signal transduction inhibitors, and/or monoclonal antibodies . For example, the operation may be an open operation or a minimally invasive operation. Thus, the compounds of formulas I-IV or their pharmaceutically acceptable salts, amorphous or polymorphic forms may also be useful as adjuvants for the treatment of cancer, i. they can be used in combination with one or more additional therapeutic agents or therapeutic agents, for example, with a chemotherapeutic agent that acts by the same or a different mechanism of action. In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphic form thereof, may be used prior to the administration of an additional therapeutic agent or additional therapy. For example, a patient in need thereof may be administered one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, over a period of time and then directed to at least partial tumor resection. In some embodiments, treatment with one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, may reduce tumor size (e.g., tumor burden) prior to at least partial resection of the tumor. In some embodiments, a patient in need thereof may be administered one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, over a period of time and then placed on one or more courses of radiation therapy. In some embodiments, treatment with one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof may result in a reduction in tumor size (e.g., tumor burden) prior to one or more courses of radiation therapy.

In some embodiments, the patient is suffering from a cancer (e.g., a locally advanced or metastatic tumor) that is resistant or intolerant to standard therapy (e.g., administration of a chemotherapeutic agent such as a first RET inhibitor or a multikinase inhibitor, immunotherapy, or radiation (e.g., radioactive iodine). In some embodiments, the patient is suffering from a cancer (e.g., a locally advanced or metastatic tumor) that is resistant or intolerant to prior therapy (e.g., administration of a chemotherapeutic agent such as a first RET inhibitor or a multikinase inhibitor, immunotherapy, or radiation (e.g., to radioactive iodine). In some embodiments, the patient is suffering from a cancer (eg, locally advanced or metastatic tumor) for which there is no standard therapy. In some embodiments, the patient has not previously been treated with RET kinase inhibitors. For example, the patient has not previously been treated with a selective RET kinase inhibitor. In some embodiments, the patient has previously been treated with RET kinase inhibitors.

In some embodiments, the patient has previously received treatment. In some embodiments, the patient suffering from NSCLC (e.g., NSCLC positive for RET fusion) was previously treated with platinum-based chemotherapy, PD-1/PDL1 immunotherapy, or both prior to treatment with a compound of Formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form. In some embodiments, a patient suffering from thyroid cancer (e.g., RET fusion positive thyroid cancer) was previously treated with one or more of sorafenib, lenvatinib, and radioactive iodine prior to treatment with a compound of Formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form. In some embodiments, a patient suffering from colon and rectal cancer (e.g., RET fusion positive colon and rectal cancer) has previously been treated with fluoropyrimidine-based chemotherapy, targeted anti-VEGF therapy, or targeted anti-EGFR therapy. or without it, prior to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a patient suffering from pancreatic cancer (e.g., RET fusion positive pancreatic cancer) was previously treated with one or more of fluoropyrimidine-based chemotherapy, gemcitabine-based chemotherapy, and S-1 chemotherapy prior to treatment. using a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a patient suffering from breast cancer (e.g., RET fusion positive breast cancer) was previously treated with one or more of an anthracycline, a taxane, HER2-targeted therapy, and hormone therapy prior to treatment with a compound of Formula I -IV or its pharmaceutically acceptable salt, amorphous or polymorphic form. In some embodiments, a patient suffering from MTS (e.g., RET fusion positive MTS cancer) was previously treated with one or more of caboxantinib and vandetanib prior to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form.

In some embodiments of any of the methods described herein, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered in combination with a therapeutically effective amount of at least one additional therapeutic agent selected from one or more additional therapeutic agents. or therapeutic (eg, chemotherapeutic) agents.

Non-limiting examples of additional therapeutic agents include: other RET-targeting therapeutic agents (i.e., a first or second RET kinase inhibitor), other kinase inhibitors (i.e., receptor tyrosine kinase-targeting therapeutic agents (i.e., Trk inhibitors or EGFR inhibitors). )) signal transduction pathway inhibitors, checkpoint inhibitors, apoptotic pathway modulators (eg obataclax); cytotoxic chemotherapeutic agents, angiogenesis-targeting therapeutics, immune system-targeting agents, including immunotherapy, and radiation therapy.

In some embodiments, the other RET-targeting therapeutic is a multikinase inhibitor that exhibits RET inhibition activity. In some embodiments, another RET-targeting therapeutic inhibitor is selective for RET kinase. Exemplary RET kinase inhibitors may exhibit an inhibitory activity (IC ₅₀ ) against RET kinase of less than about 1000 nM, less than about 500 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM less than about 10 nM, or less than about 1 nM, as measured in the assay described herein. In some embodiments, RET kinase inhibitors may exhibit inhibitory

- 86 041220 activity (IC ₅₀ ) against RET kinase is less than about 25 nM, less than about 10 nM, less than about 5 nM, or less than about 1 nM as measured in the assay provided herein.

Non-limiting examples of therapeutic agents targeting RET (e.g., a first RET inhibitor or a second RET inhibitor) include alectinib (9-ethyl-6,6-dimethyl-8-[4-(morpholin4-yl)piperidin-1-yl]- 11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile); amuvatinib (MP470, HPK56) (N-(1,3-benzodioxol-5-ylmethyl)-4-([1]benzofuro[3,2-d]pyrimidin-4-yl)piperazin-1carbothioamide); apatinib (YN968D1) (M-[4-(1-cyanocyclopentyl)phenyl-2-(4-picolyl)amino-3-nicotinamide methanesulfonate); cabozantinib (Cometriq XL-184) (N-(4-((6,7-dimethoxyquinolin-4yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); dovitinib (TKI258; GFKI-258; CHIR-258) ((3Z)-4-amino-5-fluoro-3-[5-(4-methylpiperazin-1-yl)-1,3-dihydrobenzimidazole-2ylidene]quinoline- 2-on); famitinib (5-[2-(diethylamino)ethyl]-2-[(Z)-(5-fluoro-2-oxo-1H-indole-3ylidene)methyl]-3-methyl-6,7-dihydro-1H- pyrrolo[3,2-c]pyridin-4-one); fedratinib (SAR302503, TG101348) (N-(2-methyl-2-propanyl)-3-{[5-methyl-2-({4-[2-(1-pyrrolidinyl)ethoxy]phenyl}amino)-4pyrimidinyl] amino}benzenesulfonamide); foretinib (XL880, EXEL-2880, GSK1363089, GSK089) (N'[3-fluoro-4-[[6-methoxy-7-(3-morpholinopropoxy)-4-quinolyl]oxy]phenyl]-N1-(4- fluorophenyl)cyclopropane-1,1-dicarboxamide); fostamantinib (R788) (2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one, 6-[[5-fluoro2-[(3,4,5-trimethoxyphenyl)amino]- 4-pyrimidinyl]amino]-2,2-dimethyl-4-[(phosphonooxy)methyl]-, sodium salt (1:2)); ilorasertib (AVT-348) (1-(4-(4-amino-7-(1-(2-hydroxyethyl)-1H-pyrazol-4yl)thieno[3,2-c]pyridin-3-yl)phenyl) -3-(3-fluorophenyl)urea); lenvatinib (E7080, Lenvima) (4-[3chloro-4-(cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide); motesanib (AMG 706) (N-(3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-[(pyridin-4-ylmethyl)amino]pyridine-3carboxamide); nintedanib (3-Z-[1-(4-(N-((4-methylpiperazin-1-yl)methylcarbonyl)-N-methylamino)anilino)-1-phenylmethylene]-6-methyloxycarbonyl-2-indolinone); ponatinib (AP24534) (3-(2-imidazo[1,2b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl) phenyl]benzamide); PP242 (torkinib) (2-[4-amino-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]-1Nindol-5-ol); quisartinib (1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-(7-(2-morpholinoethoxy)benzo[d]imidazo[2,1-b]thiazol-2-yl) phenyl)urea;regorfenib (BAY 73-4506, Stivarga) (4-[4-({[4-chloro-3(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide hydrate ); RXDX-105 (CEP-32496, agerafenib) (1-(3-((6,7-dimethoxyquinazolin-4-yl)oxy)phenyl)-3-(5-(1,1,1trifluoro-2-methylpropane -2-yl)isoxazol-3-yl)urea); semaxanib (SU5416) ((3Z)-3-[(3,5-dimethyl-1Hpyrrol-2-yl)methylidene]-1,3-dihydro-2H- indol-2-one); sitravatinib (MGCD516, MG516) (M-(3-fluoro-4{[2-(5-{[(2-methoxyethyl)amino]methyl}-2-pyridinyl)thieno[3.2 -b]pyridin-7-yl]oxy}phenyl)-N-(4fluorophenyl)-1,1-cyclopropanedicarboxamide);sorafenib (BAY 43-9006) (4-[4-[[[[4-chloro-3( trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-2-pyridinecarboxamide); yl)methoxy]quinazolin-4-amine);vatalanib (PTK787, PTK/ZK, ZK222584) (N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine);AD-57 ( N-[4[4-amino-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]phenyl]-N-[3-(trifluoromethyl)phenyl]urea); AD-80 (1-[4-(4-amino-1-propan-2-ylpyrazolo[3,4-d]pyrimidin-3-yl)phenyl]-3-[2-fluoro-5(trifluoromethyl)phenyl] urea); AD-81 (1-(4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3yl)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea ); ALW-II-41-27 (N-(5-((4-((4-ethylpiperazin-1yl)methyl)-3-(trifluoromethyl)phenyl)carbamoyl)-2-methylphenyl)-5-(thiophene-2- yl) nicotinamide); BPR1K871 (1-(3-chlorophenyl)-3-(5-(2-((7-(3-(dimethylamino)propoxy)quinazolin-4-yl)amino)ethyl)thiazol-2yl)urea); CLM3 (1-phenethyl-N-(1-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine); EBI-907 (N-(2chloro-3-(1-cyclopropyl-8-methoxy-3H-pyrazolo[3,4-c]isoquinolin-7-yl)-4-fluorophenyl)-3-fluoropropane-1sulfonamide); NVP-AST-487 (N-[4-[(4-ethyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl]-N'-[4-[[6(methylamino)-4-pyrimidinyl]oxy ]phenyl]urea); NVP-BBT594 (ВВТ594) (5-((6acetamidopyrimidin-4-yl)oxy)-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)indoline-1-carboxamide ); PD173955 (6-(2,6-dichlorophenyl)-8-methyl-2-(3-methylsulfanylanilino)pyrido[2,3-d]pyrimidin-7-one); PP2 (4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine); PZ1 (N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1yl )phenyl)acetamide); RPI-1 (1,3-dihydro-5,6-dimethoxy-3-[(4-hydroxyphenyl)methylene]-H-indol-2-one; (3E)-3-[(4-hydroxyphenyl)methylidene]- 5,6-dimethoxy-1H-indol-2-one); SGI-7079 (3-[2-[[3-Fluoro-4-(4-methyl1-piperazinyl)phenyl]amino]-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]benzolacetonitrile ); SPP86 (1isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine); SU4984 (4-[4-[(E)-(2-oxo-1H-indol3-ylidene)methyl]phenyl]piperazine-1-carbaldehyde); sunitinib (SU11248) (N-(2-diethylaminoethyl)-5-[(Z)(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3 -carboxamide); TG101209 (N-tert-butyl-3-(5-methyl-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-ylamino)benzenesulfonamide); withaferin A ((4β,5β,6β,22R)-4,27-dihydroxy-5,6:22,26-diepoxyergosta-2,24-diene-1,26-dione); XL-999 ((Z)-5-((1-ethylpiperidin-4-yl)amino)-3-((3-fluorophenyl)(5-methyl-1H-imidazol-2-yl)methylene)indolin-2one) ; BPR1J373 (5-phenylthiazol-2-ylamine-pyrimidine derivative); CG-806 (CG'806); DCC-2157; GTX-186; HG-6-63-01 ((E)-3-(2-(4-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)vinyl)-N-(4-((4- ethylpiperazin1-yl)methyl)-3-(trifluoromethyl)phenyl)-4-methylbenzamide); SW-01 (cyclobenzaprine hydrochloride);

- 87 041220

XMD15-44 (N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-4-methyl-3-(pyridin-3ylethynyl)benzamide (derived from structure)); Y078-DM1 (an antibody-drug conjugate consisting of the antibody RET (Y078) linked to a derivative of the cytotoxic agent maytansine); Y078-DM4 (an antibody-drug conjugate consisting of the antibody RET (Y078) linked to a derivative of the cytotoxic agent maytansine); ITRI-305 (D0N5TB, DIB003599); BLU-667 (((1 S,4R)-N-((S)-1 -(6-(4-fluoro-1 H-pyrazol-1 -yl)pyridin-3-yl)ethyl)-1-methoxy -4-(4-methyl-6-((5-methyl-1Hpyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide); BLU6864; DS-5010; GSK3179106; GSK3352589; NMS-E668 and TAS0286/HM05.

Additional examples of RET-targeting therapeutic agents (eg, a first RET kinase inhibitor or a second RET kinase inhibitor) include 5-amino-3-(5-cyclopropylisoxazol-3-yl)-1-isopropyl-1H-pyrazole-4-carboxamide; 3-(5-cyclopropylisoxazol-3-yl)1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine; 3-((6,7-dimethoxyquinazolin-4-yl)amino)-4-fluoro-2methylphenol; N-(5-(t-butyl)isoxazol-3-yl)-2-(4-(imidazo[1,2-a]pyridin-6-yl)phenyl)acetamide; N-(5(t-butyl)isoxazol-3-yl)-2-(3-(imidazo[1,2-b]pyridazin-6-yloxy)phenyl)acetamide; Ν-(2-Φτορ-5trifluoromethylphenyl)-N'-{4'-[(2-benzamido)pyridin-4-ylamino]phenyl}urea; 2-amino-6-{[2-(4chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-thienyl)pyridine-3,5-dicarbonitrile and 3-arylureidobenzylidene indolin-2-ones.

Additional examples of other RET kinase inhibitors include those described in US Pat. For example, in some embodiments, another RET inhibitor is a compound of formula I

CI

J

SG X—to ' Λ-ORt o ¹ where R1 represents C ₆ -C ₂₄ alkyl or polyethylene glycol; or a pharmaceutically acceptable salt thereof. In some embodiments, another RET inhibitor is 4-{5-[bis-(chloroethyl)amino]-1-methyl-1H-benzimidazol-2-yl}butyric acid dodecyl ester.

Additional examples of other RET kinase inhibitors include those described in International Publication No. WO 2016127074, the contents of which are incorporated herein by reference. For example, in some embodiments, another RET inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

(O where rings A and B are each independently selected from aryl, heteroaryl, cycloalkyl and heterocyclyl; each L ¹ and L ² is independently selected from the bond, -(C1-C6-alkylene)-, -(C2-C6-alkenylene) -, -(C2-C6alkynylene)-, -(C1-C6-haloalkylene)-, -(C1-C6-heteroalkylene)-, -C(O)-, -O-, -S-, -S(O) , -S(O)2-, -N(R ¹ )-, -O-(C1-C6-alkylene)-, -(CrC ₆ -alkylene)-O-, -N(R ¹ )-C(O )-, -C(O)N(R1)-, -(C1-C6-alkylene)-N ¹ )-, -N(R1)-(CrC6alkylene)-, -N(R ¹ )-C(O) -(C1-C6-alkylene)-, -(C1-C6-alkylene)-N(R ¹ )-C(O)-, -C(O)-N(R ¹ )-(C1-C6-alkylene) -, -(C1-C6-alkylene)-C(O)-N(R ¹ )-, -N(R1)-S(O)2-, -S(O)2-N(R1)-, - N(R ¹ )-S(O)2-(C1-C6-alkylene)- and -S(O)2-N(R1)(C1- _C6 -αkylene)-; where each alkylene, alkenylene, alkynylene, haloalkylene and heteroalkylene is independently substituted with 0-5 R'groups; each RA and RB is independently selected from C1- _C6 alkyl, C1- _C6 alkoxy, halogen, C1- _C6 haloalkyl, C1- _C6 hydroxyalkyl, C1- _C6 heteroalkyl, and -N(R1)( R1); where each alkyl, alkoxy, haloalkyl, hydroxyalkyl and hydroxyalkyl is independently substituted with 0-5 R ^a groups; each ^RC and RD is independently selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogen, C1-C6 heteroalkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6-hydroxyalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl, heterocyclylalkyl, nitro, cyano, -C(O)R1, -OC(O)R1, -C(O)OR1, -(C1-C6 -alkylene)-C(O)R ¹ , -SR ¹ -S(O)2R ¹ , -S(O)2-N(R ¹ )(R ¹ ), -(C1-C6-αkylene)-S( O)2R ¹ , -(CrCgalkylene)-S(O)2-N(R ¹ )(R ¹ ), -N(R1)(R1) -C(O)-N(R ¹ )(R ¹ )- N(R ¹ )-C(O)R ¹ , -N(R1)-C(O)OR1, -(C1-C6-αkylene)N(R1)-C(O)R1, -N(R1)S (O)2R1 and -P(O)(R1)(R1); where each alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, haloalkyl, haloalkoxy, hydroxyalkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, heterocyclyl and heterocyclylalkyl are independently substituted with 0-5 R ^a groups; or 2 ^RC or 2 ^RD together with the carbon atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring independently substituted with 0-5 R ^a groups; each R ¹ is independently selected from hydrogen, hydroxyl, halogen, thiol, C1- _C6 alkyl, C1- _C6 thioalkyl, C1- _C6 alkoxy, C1- _C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6- heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl

- 88 041220 and heterocyclylalkyl, where each alkyl, thioalkyl, alkoxy, haloalkyl, hydroxyalkyl, heteroalkyl, cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl and heterocyclylalkyl are independently substituted with 0-5 R ^b groups, or 2 R ¹ together with atom(s), to to which they are attached form a cycloalkyl or heterocyclyl ring independently substituted with 0-5 R ^b groups; each R ^a and R ^b is independently C1-C6 alkyl, halogen, hydroxyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C1-C6 hydroxyalkyl, C1-C6 alkoxy, cycloalkyl, heterocyclyl, or cyano, wherein each alkyl , haloalkyl, heteroalkyl, hydroxyalkyl, alkoxy, cycloalkyl and heterocyclyl are independently substituted with 0-5 R'groups; each R' is C _g C6 alkyl, C _g C6 heteroalkyl, halo, hydroxyl, C _g C6 haloalkyl, C ₁ -C6 hydroxyalkyl, cycloalkyl, or cyano; or 2 R' together with the atom(s) to which they are attached form a cycloalkyl or heterocyclyl ring; m is 0, 1, 2 or 3; n is 0, 1 or 2; and p and q are each independently 0, 1, 2, 3, or 4.

For example, the RET inhibitor may be selected from the group consisting of:

- 89 041220

- 90 041220

- 91 041220

- 92 041220

or their pharmaceutically acceptable salts.

Additional examples of other RET kinase inhibitors include those described in International Publication No. WO 2016075224, the contents of which are incorporated herein by reference. For example, in some embodiments, another RET inhibitor is a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein

R1 and R2 are independently hydrogen or an optionally substituted group selected from straight or branched (C ₁ -C ₆ )alkyl, (C ₃ -C ₆ )cycloalkyl and COR', where R' is an optionally substituted group selected from straight chain or branched (C1C ₆ )alkyl and (C ₃ -C ₆ )cycloalkyl;

R3 is hydrogen or an optionally substituted group selected from straight or branched (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₂ -C ₆ ) alkynyl, (C ₃ -C ₆ )cycloalkyl, aryl, heteroaryl and 3-7 membered heterocyclyl ring;

R4 is hydrogen or an optionally substituted group selected from straight or branched (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, aryl, heteroaryl, or heterocyclyl;

A represents a 5- or 6-membered heteroaryl ring or a phenyl ring;

B is a 5- or 6-membered ring selected from heteroaryl, (C ₅ -C ₆ )cycloalkyl and a heterocyclyl ring, or a phenyl ring; where ring A and ring B are fused to each other to form a bicyclic system containing a 6-membered aromatic or 5-6-membered heteroaromatic ring fused to a 6-membered aromatic or 5-6-membered heteroaromatic, (C ₅ -C ₆ ) cycloalkyl or heterocyclyl ring;

Y is carbon or nitrogen;

X represents hydrogen, halogen, hydroxyl, cyano or an optionally substituted group selected from straight or branched (C ₁ -C ₆ )alkyl and (C ₁ -C ₆ )alkoxy; and R5 and R6 are independently hydrogen or an optionally substituted group selected from straight or branched (C ₁ -C ₆ )alkyl, (C ₃ -C ₆ )cycloalkyl, heterocyclyl, aryl, and heteroaryl.

Additional examples of other RET kinase inhibitors include those described in

- 93 041220 International Publication No. WO 2015079251, the contents of which are incorporated herein by reference. For example, in some embodiments, another RET inhibitor is a compound of formula (III) or a pharmaceutically acceptable salt or solvate thereof, wherein it

X is NH, NRx, O or S, where Rx is (1-3C) alkyl;

R ₁ is selected from halogen (for example, fluorine, chlorine or bromine), trifluoromethyl, (1-4C) alkyl (for example, methyl), (1-4C) alkoxy or (3-6C) cycloalkyl, where alkyl, alkoxy or cycloalkyl group optionally substituted with one or more fluorine atoms;

R ₂ is selected from hydrogen, halogen (eg fluorine, chlorine or bromine), hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C) alkyl (eg methyl), (3-8C) cycloalkyl or (1-4C) alkoxy (eg, OMe), where the alkyl, cycloalkyl or alkoxy group is optionally substituted with one or more fluorine atoms;

R ₃ is selected from hydrogen, halogen (eg fluorine, chlorine or bromine), hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C) alkyl (eg methyl), (3-8C) cycloalkyl or (1-4C) alkoxy (eg, OMe), where the alkyl, cycloalkyl or alkoxy group is optionally substituted with one or more fluorine atoms;

R ₄ is selected from hydrogen, halogen (eg fluorine, chlorine or bromine), hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C) alkyl (eg methyl), (3-8C) cycloalkyl or (1-4C) alkoxy (eg, OMe), where the alkyl, cycloalkyl or alkoxy group is optionally substituted with one or more fluorine atoms;

R5 is selected from hydrogen or the group defined by the formula

- O-L5-X5-Q5, where L5 is absent or represents a linear or branched (1-4C)alkylene;

X ₅ is absent or is -C(O)O-, -O-, -C(O)-, -OC(O)-, -CH(QR _5L )-, -N(Rj)-, -N( R _5L )C(O)-, -N(R _5L )-C(O)O-, -C(O)-N(R _5L )-, -S-, -SO-, -SO2-, -S (O) ₂ N(R _5L )- or -N(R _5L )SO ₂ -, where R _5L is selected from hydrogen or methyl; and Q5 is (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, (38C)cycloalkyl, (3-8C)cycloalkyl-(1-4C)alkyl, aryl, aryl-(1 -4C)alkyl, heteroaryl, heteroaryl-(14C)alkyl, heterocyclyl or heterocyclyl-(1-4C)alkyl;

R ₆ is selected from hydrogen or the group defined by the formula

- O-L6-X6-Q6, where L ₆ is absent or represents a linear or branched (1-4C)alkylene;

X6 is absent or selected from -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR6L)-, -N(R6L), -N(R6L)- C(O)-, -N(R6L)-C(O)O-, -C(O)-N(R6L)-, -S-, -SO-, -SO2-, -S(O)2N( R6L)- or -N(R6L)SO2-, where R6L is selected from hydrogen or (1-3C)alkyl;

Q ₆ is hydrogen, (1-8C) alkyl, (2-8C) alkenyl, (2-8C) alkynyl, (3-8C) cycloalkyl, (38C) cycloalkyl-(1-6C) alkyl, aryl, aryl- (1-6C)alkyl, heteroaryl, heteroaryl-(1-6C)alkyl, heterocyclyl, heterocyclyl-(1-6C)alkyl, or Q ₆ and R _L6 are bonded such that, together with the nitrogen atom to which they are attached, they form a heterocyclic ring;

where R ₆ is optionally substituted (for example, substituted in L ₆ and/or Q ₆ ) with one or more (1-6C)alkyl, (1-6C)alkanoyl, OR _6X , SR _6X , S(O)R6x, S(O ) ₂ R _6X , C(O)OR _6X or C(O)NR _6X R' _6X , where R _6X and R' _6X are independently hydrogen, (1-8C) alkyl, or R _6X and R' _6X are linked as follows that, together with the nitrogen atom to which they are attached, they form a heterocyclic ring; And

R ₇ is selected from hydrogen, (1-6C)alkoxy, or a group defined by the formula

- O-L7-X7-Q7-, where L7 is absent or represents a linear or branched (1-4C)alkylene;

X7 is absent or selected from -O-, -C(O)-, -C(O)O-, -OC(O)-, -CH(OR6L)-, -N(R7L)-, -N(R7L) -C(O)-, -N(R _7L )-C(O)O-, -C(O)-N(R _7L )-, -S-, -SO-, -SO2-, -S(O ) ₂ N(R _7L )-or -N(R _7L )SO ₂ -, where R _7L is selected from hydrogen or (1-3C) alkyl;

Q7 is hydrogen, (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl, (3-8C)cycloalkyl, (38C)cycloalkyl-(1-6C)alkyl, aryl, aryl-( 1-6C)alkyl, heteroaryl, heteroaryl-(1-6C)alkyl, heterocyclyl, heterocyclyl-(1-6C)alkyl, or Q7 and R7L are bonded so that together with the nitrogen atom to which they are attached, they form a heterocyclic ring ;

where R ₇ is optionally substituted (for example, substituted in L ₇ and/or Q ₇ ) with one or more halogens, hydroxyls, nitro, cyano, (1-8C) alkyls, (1-8C) alkanoyls, OR _7X , SR _7X , S (O)R _7X , S(O) ₂ R _7X , C(O)OR _7X or C(O)NR _7X R' _7X where R _7X and R' _7X are independently hydrogen, (1-8C) alkyl, or R _7X and R' _7X

- 94 041220 linked so that together with the nitrogen atom to which they are attached, they form a heterocyclic ring; or

R7 is optionally substituted with one or more groups selected from oxo, (1-4C)haloalkyl, (1-4C)hydroxyalkyl, C(O)R7y, or NR7 _y R'7 _y , where R7 _y and R'7 _y are independently hydrogen or (1-8C)alkyl.

Additional examples of other RET kinase inhibitors include those described in International Publication No. WO2017178845, the contents of which are incorporated herein by reference. For example, in some embodiments, another RET inhibitor is a compound of formula (IV) or a pharmaceutically acceptable salt thereof, wherein

R1 is selected from hydrogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, or a group of formula

-L-Y-Q, where L is absent or is (1-5C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

Y is absent or is O, S, SO, SO ₂ , N(R _a ), C(O), C(O)O, OS(O), C(O)N(R _a ), N(Ra) C(O), N(Ra)C(O)N(Rb), N(Ra)C(O)O, OC(O)N(Ra), S(O^N(Ra) or N(Ra) SO2, where Ra and R, are each independently selected from hydrogen or (1-4C)alkyl, and Q is hydrogen, (1-6C)alkyl, (26C)alkenyl, (2-6C)alkynyl, aryl, ( 3-10C)cycloalkyl, (3-10C)cycloalkenyl, heteroaryl or heterocyclyl, wherein Q is optionally further substituted with one or more substituent groups independently selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1- 4C) haloalkoxy, amino, (14C) aminoalkyl, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, NRcRd, ORc, C(O)Rc, C(O)ORc, OC(O)Rc, C(O )N(Rd)Rc, N(Rd)C(O)Rc, S(O)pRc (where p is 0, 1 or 2), SO2N(Rd)Rc, N(Rd)SO ₂ Rc, Si(Re )(R _d )R _c or (CH ₂ )qNR _c R _d (where q is 1, 2 or 3), where Rc, Rd and Re are each independently selected from hydrogen, (1-6C)alkyl or (3 -6C) cycloalkyl; or Rc and Rd are bonded so that together with the nitrogen atom to which they are attached, they form a 4-7 membered heterocyclic ring which is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (14C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy , (1-4C)alkylamino, amino, cyano or hydroxy; or Q is optionally substituted with a group of formula

- Li-Lqi-Wi, where L1 is absent or represents (1-3C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

- 95 041220

LQ absent or selected from O, S, SO, SO2, N(R _f ), C(O), C(O)O, OC(O), C(O)N(R _f ), N(R _f ) C(O),

N(R _f )C(O)N(R _g ), N(R _f )C(O)O, OC(O)N(R _f ), S(O) ₂ N(R _f ) or N(R _f )SO ₂ where R _f and Rg are each independently selected from hydrogen or (1-2C) alkyl; And

W1 is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl, (3-8C)cycloalkyl, (38C)cycloalkenyl, heteroaryl or heterocyclyl; where W1 is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, (14C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, aryl, heteroaryl, heterocyclyl, (3-6C) cycloalkyl, NRhRi, ORh, C(O)Rh, C(O)ORh, OC(O)Rh, C(O )N(Ri)Rh, N(Ri)C(O)Rh, S(O) _r Rh (where r is 0, 1 or 2), SO2N(Ri)Rh, N(Ri)SO2Rh or (CH2) _s NRiRh (where s is 1, 2, or 3); where Rh and Rj are each independently selected from hydrogen, (1-4C)alkyl or (36C)cycloalkyl;

each R _1a and R _1b are selected from H, (1-4C) alkyl, halogen, (1-4C) haloalkyl, (1-4C) haloalkoxy, (1-4C) alkoxy, (1-4C) alkylamino, amino, cyano , hydroxy, carboxy, carbamoyl, sulfamoyl or mercapto;

W is selected from O, S or NR _W1 where R _W1 is selected from H or (1-2C) alkyl; X1, X ₂ , X ₃ and X ₄ are independently selected from CH, CR ₂ or N;

R ₂ is selected from hydrogen, halo, (1-4C)alkyl, (1-4C)alkoxy, (1-4C)haloalkyl, (14C)haloalkoxy, amino, cyano, nitro, aryl, heteroaryl, heterocyclyl, cycloalkyl, (24C )alkynyl, NRjRk, ORj, C(O)Rj, C(O)OR _j , OC(O)Rj, C(O)N(R _k )Rj, N(R _k )C(O)R _j , N (R _k )C(O)N(Rj), S(O) _r1 R _k (where r1 is 0, 1 or 2), SO ₂ N(Rj)R _k , N(Rj)SO ₂ R _k or ( CH ₂ ) _v NRjRk (where v is 1, 2 or 3); where Rj and R _k are each independently selected from hydrogen or (1-4C)alkyl; and wherein said (1-4C)alkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one or more substituents selected from halo, (1-4C)alkyl, (1-4C)alkoxy, (1-4C)haloalkyl, ( 1-4C) haloalkoxy, amino, cyano, nitro, phenyl, (2-4C) alkynyl, NR _j1 R _k1 , ORj1, C(O)R _jb C(O)OR _j1 , OC(O)R _j1 , C( O)N(R _k1 )R _j1 , N(R _k1 )C(O)R _j1 , S(O) _r2 Rh (where r ₂ is 0, 1 or 2), SO ₂ N(R _j1 )R _k1 , N(R _j1 )SO ₂ R _k1 or (CH ₂ ) _v1 NR _j1 R _k1 (where v1 is 1, 2 or 3); and where Rj1 and R _k1 each independently selected from hydrogen or (1-4C)alkyl; And

R3 is selected from halogen, (1-4C) alkyl, (1-4C) alkoxy, (1-4C) haloalkyl, (1-4C) haloalkoxy, amino, cyano, nitro, (2-4C) alkynyl, NRlR _m , OR , C(O)Rl, C(O)ORl, OC(O)Rl, C(O)N(Rm)RN(Rm)C(O)R or (CH2)yNRlR _m (where y is 1, 2 or 3); wherein said (1-4C)alkyl is optionally substituted with one or more substituents selected from amino, hydroxy, (1-2C)alkoxy, or halo; and wherein Rl and R _m are each independently selected from hydrogen or (1-4C) alkyl.

Additional examples of other RET kinase inhibitors include those described in International Publication No. WO 2017178844, the contents of which are incorporated herein by reference. For example, in some embodiments, another RET inhibitor is a compound of formula (V) or a pharmaceutically acceptable salt thereof, wherein

NO selected from the following:

- 96 041220

where ' means the attachment point;

R1 is selected from hydrogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, or a group of formula

-L-Y-Q, where L is absent or is (1-5C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

Y is absent or is O, S, SO, SO ₂ , N(Ra), C(O), C(O)O, OS(O), C(O)N(R _a ), N(Ra)C (O), N(Ra)C(O)N(Rb), N(Ra)C(O)O, OC(O)N(Ra), S(O)2N(Ra) or N(Ra)SO2 where Ra and Rb are each independently selected from hydrogen or (1-4C) alkyl; and Q is hydrogen, (1-6C) alkyl, (26C) alkenyl, (2-6C) alkynyl, aryl, (3-10C) cycloalkyl, (3-10C) cycloalkenyl, heteroaryl, or heterocyclyl; where Q is optionally further substituted with one or more substituent groups independently selected from (1-4C) alkyl, halogen, (1-4C) haloalkyl, (1-4C) haloalkoxy, amino, (14C) aminoalkyl, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, NRcRd, ORc, C(O)Rc, C(O)ORc, OC(O)R _c , C(O)N(Rd)Rc, N(Rd)C(O)Rc , S(O)yR^ (where y is 0, 1 or 2), SO2N(R _d )R _c , N(R _d )SO ₂ R _c , Si(Rd)(R _c )Re or (CH ₂ ) _z NRcRd (where z is 1, 2, or 3); where Rc, Rd and Re are each independently selected from hydrogen, (1-6C) alkyl or (3-6C) cycloalkyl; or Rc and Rd may be bonded such that, together with the nitrogen atom to which they are attached, they form a 4-7 membered heterocyclic ring which is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, ( 1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano or hydroxyl; or Q is optionally substituted with a group of formula ^-L 1 ^-L Q1 ^-Z b where L ₁ is absent or is (1-3C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

L _Q1 is absent or selected from O, S, SO, SO ₂ , N(R _f ), C(O), C(O)O, OS(O), C(O)N(R _f ), N(R _f )C(O), N(Rg)C(O)N(Rf), N(R _f )C(O)O, OC(O)N(R _f ), S(O) ₂ N(R _f ) or N(R _f )SO ₂ where Rf and Rg are each independently selected from hydrogen or (1-2C)alkyl; and Z1 is hydrogen, (1-6C)alkyl, aryl, aryl(12C)alkyl, (3-8C)cycloalkyl, (3-8C)cycloalkenyl, heteroaryl, or heterocyclyl; wherein Z1 is optionally substituted with one or more substituents selected from (1-4C) alkyl, halo, (1-4C) haloalkyl, (1-4C) haloalkoxy, (1-4C) alkoxy, (1-4C) alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, aryl, heteroaryl, heterocyclyl, (3-6C) cycloalkyl, NRhRi, ORh, C(O)Rh, C(O)ORh, OC(O)Rh, C (O)N(Ri)Rh, N(Ri)C(O)Rh, S(O)yaRh (where y ^a is 0, 1, or 2), SO2N(Ri)Rh, N(Ri)SO2Rh, or (CH2 )zaNRiRh (where z ^a is 1, 2 or 3); wherein Rh and Ri are each independently selected from hydrogen, (1-4C) alkyl, or (3-6C) cycloalkyl;

each R _1a and R _1b are selected from hydrogen, (1-4C)alkyl, halogen, (1-4C)haloalkyl, (14C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxy , carboxy, carbamoyl, sulfamoyl or mercapto;

W is selected from O, S or NRj, where Rj is selected from H or (1-2C)alkyl;

X1 and X ₂ are each independently selected from N or CRk;

where R _k is selected from hydrogen, halogen, (1-4C)alkyl, (1-4C)alkoxy, amino, (1-4C)alkylamino, (14C)dialkylamino, cyano, (2C)alkynyl, C(O)R _k1 , C(O)OR _k1 , OC(O)R _k1 , C(O)N(R _k2 )R _k1 , N(R _k2 )C(O)R _k1 , S(O) _yb R _k1 (where y ^b is 0, 1 or 2), SO ₂ N(R _k2 )R _k1 , N(R _k2 )SO ₂ R _k1 or (CH ₂ ) _zb NRkiRk ₂ (where z ^b is 1, 2 or

- 97 041220

3); where the specified (1-4C)alkyl is optionally substituted with one or more substituents selected from amino, hydroxy, (1-2C)alkoxy or halogen; And

R _k1 and R _k2 are each independently selected from hydrogen or (1-4C) alkyl; X ₃ is selected from N or CRm; Where

_Rm is selected from hydrogen, halogen, (1-4C)alkyl, (1-4C)alkoxy, amino, (1-4C)alkylamino, (14C)dialkylamino, cyano, (2C)alkynyl, C(O)R _m1 , C(O)OR _m1 , OC(O)R _m1 , C(O)N(R _m2 )R _m1 , N(R _m2 )C(O)R _m1 , S(O)ycRm1 (where ^y c is 0, 1 or 2), SO2N(Rm2)Rm1, N(Rm2)SO2Rm1 or (CH2) _zc NRm1 Rm2 (where zc is 1, 2 or 3); where the specified (1-4C)alkyl is optionally substituted with one or more substituents selected from amino, hydroxy, (1-2C)alkoxy or halogen; And

Rm1 and Rm2 are each independently selected from hydrogen or (1-4C)alkyl;

Ro is selected from halo, (1-4C)alkyl, (1-4C)alkoxy, amino, (1-4C)alkylamino, (14C)dialkylamino, cyano, (2C)alkynyl, C(O)Ro1, C(O) ORo1, OC(O)Ro1, C(O)N(Ro2)Ro1, N(Ro2)C(O)Ro1, S(O) _yd R _o1 (where y ^d is 0, 1 or 2), SO ₂ N (R _o2 )R _o1 , N(R _o2 )SO ₂ R _o1 or (CH ₂ ) _zd NR _o1 R _o2 (where z ^d is 1, 2 or 3); where the specified (1-4C)alkyl is optionally substituted with one or more substituents selected from amino, hydroxy, (1-2C)alkoxy or halogen; And

R _o1 and R _o2 are each independently selected from hydrogen or (1-4C) alkyl; R ₂ is selected from hydrogen, (14C)alkyl or a group of formula ^-L 2 ^-Y 2 ^-Q ₂ where L ₂ is absent or is (1-3C)alkylene optionally substituted with one or more substituents selected from (1-2C )alkyl or oxo;

Y ₂ is absent or represents C(O), C(O)O, C(O)N(Rp), where Rp is selected from hydrogen or (14C)alkyl; And

Q2 is hydrogen, (1-6C) alkyl, aryl, (3-8C) cycloalkyl, (3-8C) cycloalkenyl, heteroaryl or heterocyclyl; where Q2 is optionally further substituted with one or more substituent groups independently selected from (1-4C) alkyl, halogen, (1-4C) haloalkyl, (1-4C) haloalkoxy, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, NRqRr , ORq, where R _q and R _r are each independently selected from hydrogen, (1-4C) alkyl or (3-6C) cycloalkyl;

R ₃ is selected from the group of formula

-Y3-Q3 where Y ₃ is C(O), C(O)N(R _y ), C(O)N(R _y )O, N(R _y )(O)C, C(O) O, OS(O), N(R _y )C(O)N(R _y1 ), SO ₂ N(R _y ), N(R _y )SO ₂ , oxazolyl, triazolyl, oxadiazolyl, thiazolyl, imidazolyl, thiadiazolyl, pyridinyl, pyrazolyl, pyrrolyl or tetrazolyl, where R _y and R _y1 are independently selected from hydrogen or (1-2C) alkyl; And

Q3 is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl, (3-8C)cycloalkyl, (38C)cycloalkenyl, heteroaryl or heterocyclyl; where Q3 is optionally further substituted with one or more substituent groups independently selected from (1-4C) alkyl, halogen, (14C) haloalkyl, (1-4C) haloalkoxy, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, NR _z R _aa , OR _z , where R _z and R _aa are each independently selected from hydrogen, (1-4C) alkyl or (36C) cycloalkyl; or Q ₃ is optionally substituted with a group of formula ^-L 4 ^-IQ 4 ^-Z 4 ^where L ₄ is absent or is (1-3C)alkylene optionally substituted with one or more substituents selected from (1-2C)alkyl or oxo;

Lq ₄ absent or selected from O, S, SO, SO ₂ , N(R _ab ), C(O), C(O)O, OS(O), C(O)N(R _ab ), N(R _ab )C(O), N(Rac)C(O)N(Rab), N(Rab)C(O)O, OC(O)N(Rab), S(O)2N(Rab) or N( Rab)SO2, where Rab and Rac are each independently selected from hydrogen or (1-2C)alkyl; And

Z ₄ is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl, (3-8C)cycloalkyl, (38C)cycloalkenyl, heteroaryl or heterocyclyl; where Z4 is optionally substituted with one or more substituents selected from (1-4C)alkyl, halogen, (1-4C)haloalkyl, (1-4C)haloalkoxy, (14C)alkoxy, (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl, sulfamoyl, mercapto, ureido, aryl, heteroaryl, heterocyclyl, (3-6C) cycloalkyl, NR _ad R _ae , OR _ad , C(O)R _ad , C(O)OR _ad , OC(O )Rad, C(O)N(Rae)Rad, N(Rae)C(O)Rad, S(O)yeRad (where e ^is already 0, 1, or 2), SO2N(Rae)Rad, N(Rae) SO2Rad or (CH2)zeNRadRae (where z ^e is 1, 2, or 3); where Rad and R _ae each independently selected from hydrogen, (14C)alkyl or (3-6C)cycloalkyl; or

Q ₃ and R _y are bonded such that together with the nitrogen atom to which they are attached, they form a 4-7 membered heterocyclic ring which is optionally substituted with one or more substituents selected from (1-4C) alkyl, halogen, (1-4C )haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (14C)alkylamino, amino, cyano or hydroxyl;

with the proviso that only one or two Xx X ₂ or X ₃ may be N.

Additional examples of other RET kinase inhibitors include those described in International Publication No. WO 2017145050, the contents of which are incorporated herein by reference. For example, in some embodiments, another RET has Formula (VI) or is a pharmaceutically acceptable salt thereof.

- 98 041220

Additional examples of other RET kinase inhibitors include those described in International Publication No. WO 2016038552, the contents of which are incorporated herein by reference. For example, in some embodiments, another RET has Formula (VII) or formula (VIII) or is a pharmaceutically acceptable salt thereof.

(VIII)

Other therapeutic agents include RET inhibitors such as those described, for example, in US Pat. Nos. 10030005; 9738660; 9801880; 9682083; 9789100; 9550772; 9493455; 9758508; 9604980; 9321772; 9522910; 9669028; 9186318; 8933230; 9505784; 8754209; 8895744; 8629135; 8815906; 8354526; 8741849; 8461161; 8524709; 8129374; 8686005; 9006256; 8399442; 7795273; 7863288; 7465726; 8552002; 8067434; 8198298; 8106069; 6861509; 8299057; 9150517; 9149464; 8299057; and 7863288; US Publication No. 2018/0009818; 2018/0009817; 2017/0283404; 2017/0267661; 2017/0298074; 2017/0114032; 2016/0009709; 2015/0272958; 2015/0238477; 2015/0099721; 2014/0371219; 2014/0137274; 2013/0079343;

2012/0283261; 2012/0225057; 2012/0065233; 2013/0053370; 2012/0302567; 2011/0189167; 2016/0046636;

2013/0012703; 2011/0281841; 2011/0269739; 2012/0271048; 2012/0277424; 2011/0053934; 2011/0046370;

2010/0280012; 2012/0070410; 2010/0081675; 2010/0075916; 2011/0212053; 2009/0227556; 2009/0209496;

2009/0099167; 2010/0209488; 2009/0012045; 2013/0303518; 2008/0234267; 2008/0199426; 2010/0069395;

2009/0312321; 2010/0173954; 2011/0195072; 2010/0004239; 2007/0149523; 2017/0281632; 2017/0226100;

2017/0121312; 2017/0096425; 2017/0044106; 2015/0065468; 2009/0069360; 2008/0275054; 2007/0117800;

2008/0234284; 2008/0234276; 2009/0048249; 2010/0048540; 2008/0319005; 2009/0215761; 2008/0287427;

2006/0183900; 2005/0222171; 2005/0209195; 2008/0262021; 2008/0312192; 2009/0143399; 2009/0130229;

2007/0265274; 2004/0185547; and 2016/0176865; and in international publications No. WO 2018/149382; WO2018/136796; WO2017/079140; WO2017/145050; WO2017/097697; WO2017/049462; WO2017/043550; W02017/027883; WO2017/013160; WO2017/009644; WO 2016/168992; WO 2016/137060; WO2016/127074; WO2016/075224; W02016/038552; WO2015/079251; W02014/086284; WO2013/042137; WO2013/036232; WO2013/016720; W02012/053606; WO2012/047017; WO 2007/109045; W02009/042646; W02009/023978; W02009/017838; WO2017/178845; WO2017/178844; WO2017/146116; WO2017/026718; WO2016/096709; W02007/057397; W02007/057399; W02007/054357; WO 2006/130613; W02006/089298; WO2005/070431; WO2003/020698; WO2001/062273; WO2001/016169; WO 1997/044356; W02007/087245; WO2005/044835; WO2014/075035; and WO 2016/038519; and J. Med. Chem. 2012, 55(10), 4872-4876, the entire contents of which are incorporated herein by reference.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second RET inhibitor) is a compound of Formula II:

- 99 041220 or its pharmaceutically acceptable salt or solvate, where X ¹ represents CH, CCH ₃ , CF, CCl or N; X ² represents CH, CF or N; X ³ represents CH, CF or N; X ⁴ is CH, CF or N;

where zero, one or two X ¹ , X ² , X ³ and X ⁴ represent N;

A represents H, Cl, CN, Br, CH ₃ , CH ₂ CH ₃ or cyclopropyl;

B is hetAr ¹ ;

hetAr ¹ is a 5-membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, S and O, said heteroaryl ring being optionally substituted with one or more substituents independently selected from the group consisting of halo, C1-C6 alkyl, hydroxy-C1-C6-alkyl, fluoro-C1-C6-alkyl, difluoro-C1-C6-alkyl, trifluoro-C1-C6-alkyl, cyanoC1-C6-alkyl, (C1-C6-alkoxy)C1-C6-alkyl , (C1-C4-alkoxy)CH2C(=O)-, (C1-C4-alkoxy)C(=O)C1-C3alkyl, C3-C6-cycloalkyl, (R ^a R ^b N)C1-C ₆ -alkyl , (R ^a RbN)C(=O)C1-C ₆ -alkyl, (C1-C ₆ -alkyl 8O ₂ )C1-C ₆ alkyl, hetCyca and 4-methoxybenzyl;

R ^a and R ^b independently represent H or C 1 -C ₆ -alkyl;

hetCyca is a 4-6 membered heterocyclic ring containing a ring heteroatom selected from N and O, said heterocyclic ring being optionally substituted with halo, C1- _C6 alkyl, fluoro-C1- _C6 alkyl, difluoro-C1-C ₆ -alkyl, trifluoro-C1-C ₆ -alkyl, (C1-C ₆ alkoxy)C1-C ₆ -alkyl, di (C ₁ -C ₃ -alkyl) NCH ₂ C (= O), (C 1 -C ₆ -alkoxy)C(=O) or (C1-C6alkoxy)CH ₂ C(=O);

D is hetCyc ¹ , hetCyc ² , hetCyc ³ or hetCyc ⁹ ;

hetCyc ¹ is a 4-6 membered heterocyclic ring containing 1-2 ring atoms selected from N and O, said heterocyclic ring being optionally substituted with one or more substituents independently selected from the group consisting of C1-C3 alkyl, fluoro -C1-C3 alkyl, difluoro-C1-C ₃ -alkyl, trifluoro-C1-C ₃ -alkyl and OH, or said heterocyclic ring is substituted with a C3-C6 cycloalkylidene ring, or said heterocyclic ring is substituted with an oxo group;

hetCyc ² is a 7-8 membered heterocyclic ring containing 1-3 ring heteroatoms independently selected from N and O, said heterocyclic ring being optionally substituted with C1- _C3 alkyl;

hetCyc ³ is a 7-11 membered heterospirocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, said ring being optionally substituted with C ₁ C3 alkyl;

hetCyc ⁹ is a fused 9-10 membered heterocyclic ring containing 1-3 ring nitrogen atoms and optionally substituted with an oxo group;

E is:

(a) hydrogen, (b) OH, (c) R ^a R ^b N-, where R ^a is H or C1-C6 alkyl and R ^b is H, C1-C6 alkyl or phenyl;

(d) C ₁ -C ₆ -alkyl optionally substituted with one to three fluorine atoms, (e) hydroxy-C ₁ -C ₆ -alkyl- optionally substituted with one to three fluorine atoms, (f) C ₁ -C ₆ - alkoxy optionally substituted with one to three fluorine atoms, (g) hydroxy(C1-C ₆ alkoxy) optionally substituted with one to three fluorine atoms, (h) (C ₁ -C ₆ alkoxy)hydroxy C1-C ₆ alkyl -, optionally substituted with one to three fluorine atoms, (i) (C ₁ -C ₆ -alkyl)C(=O)-, optionally substituted with one to three fluorine atoms, (j) (hydroxy C1-C ₆ -alkyl)C (=O)- optionally substituted with one to three fluorine atoms, (k) (C1-C6-alkoxy)C(=O)-, (l) (C ₁ -C ₆ -alkoxy) (C ₁ -C ₆ - alkyl)C(=O)-, (m) HC(=O)-, ⁽ⁿ⁾ Sus ¹ , (o) Sus ¹ C(=O)-, (p) Sus ¹ (C1-C6-alkyl)C (=O)-, wherein said alkyl moiety is optionally substituted with one or more groups independently selected from the group consisting of OH, fluorine, C1-C3-alkoxy, and R ^c R ^d N-, where R ^c and Rd are independently H or C1-C6-alkyl, (q) hetCyc ⁴ , (r) hetCyc ⁴ C(=O)-, (s) he tCyc ⁴ (C1-C3-alkyl)C(=O)-, (t) (hetCyc ⁴ )C(=O)C1-C2-alkyl-, (u) hetCyc ⁴ C(=O)NH-, (v ) Ar ² , (w) Ar ² C(=O)-, (x) Ar ² C ₁ -C ₆ -alkyl-,

- 100 041220 (y) (Ar ² )hydroxy C ₂ -C6-alkyl-, (z) Ar ² (C1-C3-alkyl)C (=O)-, where the specified alkyl part is optionally substituted by one or two groups, independently selected from the group consisting of OH, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1-C6 alkyl, C1-C6 alkoxy, and R ^e RfN-, where R ^e and Rf are independently H or C1-C6 alkyl, or R ^e and Rf together with the nitrogen atom to which they are attached, form a 5-6 membered azacyclic ring, optionally containing an additional ring heteroatom selected from N and O, (aa) hetAr ² C (=O)-, (bb) (hetAr ² )hydroxy-C ₂ -C ₆ -alkyl-, (cc) hetAr ² (C1-C3-alkyl)C(=O)-, where the specified alkyl part is optionally substituted with one or two groups independently selected from the group consisting of OH, C1-C6 alkyl, hydroxy-C1-C6 alkyl, C1-C6 alkoxy, and R ^e RfN-, where R ^e and Rf are independently H or C1-C6- alkyl, or R ^e and R ^f together with the nitrogen atom to which they are attached form 5-6-h a ringed azacyclic ring optionally containing an additional ring heteroatom selected from N and O, (dd) R1R ² NC(=O)-, (ee) R ¹ R ² N(C ₁ -C ₃ -alkyl)C(=O) -, where said alkyl moiety is optionally substituted with phenyl, (ff) R ¹ R ² NC(=O)C1-C2-alkyl-, (gg) R1R ² NC(=O)NH-, (hh) CH3SO2(C1-C6 -alkyl)C(=O)-, (ii) (C1-C6-alkyl)SO2-, (jj) (C ₃ -C ₆ -cycloalkyl)CH ₂ SO ₂ -, (kk) hetCyc ⁵ -SO2-, (ll) R4R5NSO2-, (mm) R ⁶ C(=O)NH-, (nn) hetCyc ⁶ , (oo) hetAr ² C ₁ -C ₆ -alkyl-, (pp) (hetCyc ⁴ )C ₁ -C ₆ -alkyl-, (qq) (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl-, optionally substituted with 1-3 fluorine atoms, (rr) (C ₃ -C ₆ -cycloalkoxy)C ₁ -C ₆ -alkyl-, (ss) (C ₃ -C ₆ -cycloalkyl)C ₁ -C ₆ -alkyl-, where the specified cycloalkyl is optionally substituted with 1-2 fluorine atoms, (tt) (R ⁹ RhN)C1 -C6 -alkyl -, where R ⁹ and R ^h independently represent H or C1-C6-alkyl, (uu) Ar ² -O-, (vv) (C ₁ -C ₆ -alkyl-SO ₂ )C ₁ -C ₆ -alkyl -, (ww) (C ₁ -C ₆ -alkoxy)C (=O)NHC ₁ -C ₆ -alkyl-, (xx) (C ₃ -C ₆ -cycloalkoxy)C (=O) -, (yy) (C ₃ -C ₆ -Cycloalkyl)SO ₂ -, where the specified cycloalkyl is optionally substituted with C1-C ₆ -alkyl, (zz) Ag ⁴ CH2OC (=O) -, (aaa) (N-(C ₁ -C ₃ -alkyl)pyridinonyl)C ₁ -C ₃ -alkyl-, and (bbb) (Ar ⁴ SO2)C1 -C6 -alkyl-;

Sus ¹ is C ₃ -C ₆ -cycloalkyl, where (a) said cycloalkyl is optionally substituted with one or more substituents independently selected from the group consisting of OH, halogen, C ₁ -C ₆ alkoxy, CN, hydroxy-C1-C ₆ -alkyl, (C1-C ₆ -alkoxy)C1-C ₆ -alkyl and C1-C ₆ -alkyl optionally substituted with 1-3 fluorine atoms, or (b) said cycloalkyl is substituted with phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1- _C3 alkyl, C1- _C3 alkoxy and CF, or (c) said cycloalkyl is substituted with a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms, independently selected and N and O, and the specified heteroaryl ring is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C1-C ₃ -alkyl, C1-C ₃ -alkoxy and CF ₃ ;

Ar ² is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halo, C1-C6 alkyl, C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), fluoro-C1-C6 alkyl , difluoro-C1-C6-alkyl, trifluoro-C1-C6-alkyl, CN, 5-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, and RiR ^j N-, where R ¹ and R ^j are independently H or C1-C6 alkyl;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S and optionally substituted with one or more substituents independently selected from the group consisting of halo, C1- _C6 - alkyl, C1-C ₆ -alkoxy (optionally substituted with 1-3 fluorine atoms), fluoro-C1-C ₆ -alkyl, difluoro-C1-C ₆ -alkyl, trifluoro-C1-C ₆ -alkyl, hydroxy-C1-C ₆ -alkyl, (C ₃ -C ₆ ) cycloalkyl, (C 1 -C ₆ -alkoxy) C 1 -C ₆ -alkyl, CN, OH and R'RN-, where R' and R are independently H or C 1 -C ₃ -alkyl;

hetCyc ⁴ is (a) a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N, O and S, said S atom being optionally oxidized to

- 101 041220

SO2, (b) 7-8 membered bridged heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, (c) 6-12 membered fused bicyclic heterocyclic ring containing 1-2 ring heteroatoms independently selected of N and O, and optionally independently substituted with 1-2 C1- _C6 alkyl substituents, or (d) a 7-10 membered spirocyclic heterocyclic ring containing 1-2 ring heteroatoms independently selected and N and O, each of said heterocyclic rings are optionally substituted with one or more substituents independently selected from the group consisting of halogen, OH, CN, C ₁ -C ₆ alkyl (optionally substituted with 1-3 fluorine atoms), C 1 -C ₆ alkoxy, (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl, (C ₃ -C ₆ ) cycloalkyl, (C1-C6 alkyl) C (= O) -, 5-6-membered heterocyclic ring containing 1-2 ring heteroatoms, independently selected from N and O, and phenyl, said phenyl being optionally substituted with one or more substituents yami selected from halogen, C1-C ₆ -alkyl and C1-C ₆ -alkoxy;

hetCyc ⁵ is a 5-6 membered heterocyclic ring containing a ring heteroatom selected from O and N;

hetCyc ⁶ is a 5-membered heterocyclic ring containing one or two ring heteroatoms independently selected from N and O, said ring being substituted with an oxo group, said ring being further optionally substituted with one or more substituents independently selected from the group, consisting of OH and C1-C ₆ -alkyl;

R ¹ represents H, C1-C ₆ -alkyl or (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl;

R ² is H, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), (C1-C6 alkoxy)C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), Sus ³ , hydroxy-C1-C6- alkyl (optionally substituted with 1-3 fluorine atoms), C1-C ₆ alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C ₆ alkoxy)C(=O), hetCyc ⁷ , Ar ³ , Ar ³ C ₁ -C ₃ -alkyl-, hydroxy-C1-C ₆ -alkoxy or (3-6C cycloalkyl)CH2O-;

Sus ³ is a 3-6 membered carbocyclic ring optionally substituted with 1-2 groups independently selected from the group consisting of C1- _C6 alkoxy, OH and halo;

hetCyc ⁷ is a 5-6 membered heterocyclic ring containing a ring heteroatom selected from O and N, said ring being optionally substituted with C1- _C6 alkyl;

Ar ³ is phenyl optionally substituted with one or more substituents independently selected from halo, C1- _C3 alkyl, C1- _C3 alkoxy, fluoroC1- _C3 alkyl, difluoroC1- _C3 alkyl, and trifluoro-C 1 -C ₃ -alkyl;

R ⁴ and R ⁵ are independently H or C1-C ₆ alkyl;

R ⁶ is C1-C ₆ alkyl, hydroxy-C1-C ₆ alkyl, C1-C ₆ alkoxy, (C ₁ -C ₆ alkoxy)C ₁ -C ₆ alkyl, phenyl, or hetCyc ⁸ ;

hetCyc ⁸ is a 5-6 membered heterocyclic ring containing a ring heteroatom selected from O and N, said heterocyclic ring being optionally substituted with C1-C6 alkyl; And

Ar ⁴ is phenyl optionally substituted with one or more halogen atoms.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second RET inhibitor) is a compound of Formula III:

or a pharmaceutically acceptable salt or solvate thereof, wherein X ¹ is CH or N; X ² represents CH or N; X ³ represents CH or N; X ⁴ is CH or N; where one or two X ¹ , X ² , X ³ and X ⁴ represent N; A is CN; B is hetAr ¹ ;

hetAr ¹ is a 5-membered heteroaryl ring containing 1-3 ring nitrogen atoms, said heteroaryl ring being optionally substituted with one or more substituents independently selected from the group consisting of halo, C1-C6-alkyl, hydroxy-C1-C6- alkyl, fluoroC1-C6-alkyl, difluoro-C1-C6-alkyl, trifluoro-C1-C6-alkyl, cyano-C1-C6-alkyl, (C1-C6-alkoxy)C1-C6-alkyl, (C1-C4-alkoxy) CH2C(=O)-, (C1-C4-alkoxy)C(=O)C1-C3-alkyl, C3-C6-cycloalkyl, (R ^a R ^b N)C1-C6 alkyl, (R ^a R ^b N)C (=O)C _r C ₆ -alkyl, (C ₁ -C ₆ -alkyl-SO ₂ )C ₁ -C ₆ -alkyl and 4-methoxybenzyl;

R ^a and R ^b independently represent H or C 1 -C ₆ -alkyl; D is hetCyc ¹ ;

hetCyc ¹ is a 4-6 membered heterocyclic ring containing 1-2 ring nitrogen atoms, wherein said heterocyclic ring is optionally substituted with one or more substituents independently selected from the group consisting of C1- _C3 alkyl, fluoro-C1-C ₃ -alkyl, difluoro-C1-C ₃ -alkyl, trifluoro-C1-C ₃ -alkyl and OH, or said heterocyclic ring is substituted with C ₃ - 102 041220

a C6-cycloalkylidene ring, or said heterocyclic ring is substituted with an oxo group;

E is:

(w) Ar ² C(=O)-, (x) Ar ² C ₁ -C ₆ -alkyl-, (z) Ar ² (C1-C3-alkyl)C(=O)-, where the specified alkyl part is optional substituted with one or two groups independently selected from the group consisting of OH, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1-C6 alkyl, C1-C6 alkoxy, and R ^e R ^f N- , where R ^e and R ^f are independently H or C1-C6 alkyl, or R ^e and R ^f together with the nitrogen atom to which they are attached, form a 5-6 membered azacyclic ring, optionally containing an additional ring heteroatom selected of N and O, (cc) hetAr ² (C1-C3-alkyl)C(=O)-, wherein said alkyl moiety is optionally substituted with one or two groups independently selected from the group consisting of OH, C1-C6-alkyl, hydroxy-C1-C6-alkyl, C1-C6-alkoxy and R ^e R ^f N-, where R ^e and R ^f independently represent H or C1-C6-alkyl, or R ^e and R ^f together with the nitrogen atom to which they attached, form a 5-6-membered azacyclic ring, optionally containing additional a positive ring heteroatom selected from N and O, (dd) R1R ² NC(=O)-, (oo) hetAr ² C ₁ -C ₆ -alkyl-,

Ar ² is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halo, C1-C6 alkyl, C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), fluoro-C1-C6 alkyl , difluoro-C1-C6-alkyl, trifluoro-C1-C6-alkyl, CN, a 5-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O, and RiRjN-, where R ¹ and Rj independently represent is H or C1-C6 alkyl;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S and optionally substituted with one or more substituents independently selected from the group consisting of halo, C1- _C6 - alkyl, C1-C ₆ -alkoxy (optionally substituted with 1-3 fluorine atoms), fluoro-C1-C ₆ -alkyl, difluoro-C1-C ₆ -alkyl, trifluoro-C1-C ₆ -alkyl, hydroxy-C1-C ₆ -alkyl, (C ₃ -C ₆ )cycloalkyl, (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl, CN, OH and R'RN-, where R' and R are independently H or C1 -C ₃ -alkyl;

R ¹ represents H, C1-C ₆ -alkyl or (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl; And

R ² is H, C1-C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), (C1-C6 alkoxy)C1-C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1-C ₆ - alkyl (optionally substituted with 1-3 fluorine atoms), C1- _C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1- _C6 alkoxy)C(=O), hydroxy-C1- _C6 alkoxy or (3-6C cycloalkyl) CH ₂ O.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second RET inhibitor) is selected from the group consisting of: (S)-4-(6-(4-(2-hydroxy-3-phenylpropanoyl)piperazin1-yl)pyridine -3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(1-methyl1H-pyrazol-4-yl)-4-(6-(4-(2-(pyridin-2-yl)acetyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1, 5a]pyridine-3-carbonitrile; 4-(6-(4-(2,6-difluorobenzoyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1Hpyrazol-4-yl)pyrazolo[1,5-a]pyridine -3-carbonitrile 2,2,2-trifluoroacetate; 4-(5-(3-cyano-6-(1-methyl1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-N,N-diethylpiperαzine- 1-carboxamide; 1(5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-а]pyridin-4-yl)pyridin-2-yl)-N-(2- methoxy-3methylbutyl)piperidine-4-carboxamide; 4-(6-(4-(2-(5-Fluoropyridin-2-yl)acetyl)piperazin-1yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[ 1,5-a]pyridine-3-carbonitrile bis(2,2,2trifluoroacetate); 4-(6-(4-(2,6-difluorobenzyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4yl)pyrazolo[1,5-a] pyridine-3-carbonitrile; 4-(6-(4-(2-methoxybenzyl)piperazin-1-yl)pyridin-3-yl)-6(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine- 3-carbonitrile; 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(4(pyridin-2-ylmethyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-а] pyridine-3-carbonitrile; 4-(6-(4-((6methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1, 5a]pyridine-3-carbonitrile; or their pharmaceutically acceptable salts or solvates.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second RET inhibitor) is a compound of Formula IV:

IV or a pharmaceutically acceptable salt or solvate thereof, wherein

X ¹ , X ² , X ³ and X ⁴ independently represent CH, CF, CCH ₃ or N, where zero, one or two X ¹ , X ² , X ³ and X ⁴ represent N;

- 103 041220

A represents H, CN, Cl, CH ₃ -, CH3CH2-, cyclopropyl, -CH2CN or -CH(CN)CH ₃ ;

B is:

(a) hydrogen, (b) C ₁ -C ₆ -alkyl, optionally substituted with 1-3 fluorine atoms, (c) hydroxy-C ₂ -C ₆ -alkyl-, where the alkyl part is optionally substituted with 1-3 fluorine atoms or C ₃ C ₆ -cycloalkylidene ring, (d) dihydroxy-C ₃ -C ₆ -alkyl-, where the alkyl part is optionally substituted with C ₃ -C ₆ - cycloalkylidene ring, (e) (C ₁ -C ₆ -alkoxy)C ₁ - C ₆ -alkyl- optionally substituted with 1-3 fluorine atoms, (f) (R ¹ R ² N)C ₁ -C ₆ -alkyl-, where the specified alkyl part is optionally substituted by an OH group, and while R ¹ and R ² are independently H or C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms);

(g) hetAr ¹ C1-C3-alkyl-, where hetAr ¹ is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S and optionally substituted with one or more independently selected C1 -C6-alkyl substituents;

(h) (C ₃ -C ₆ cycloalkyl)C ₁ -C ₃ -alkyl-, wherein said cycloalkyl is optionally substituted with an OH group, (i) (hetCyc ^a )C ₁ -C ₃ -alkyl-, (j) hetCyca- , (k) C ₃ -C ₆ -cycloalkyl-, where the specified cycloalkyl is optionally substituted by an OH group, (l) (C ₁ -C ₄ -alkyl)C (=O)O-C ₁ -C ₆ -alkyl-, where each C1- _C4 alkyl and C1- _C6 alkyl moiety is optionally and independently substituted with 1-3 fluorine atoms, or (m) (R ¹ R ² N)C(=O)C1-C6-alkyl-, where R ¹ and R ² are independently H or C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms);

hetCyca- is a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents independently selected from OH, C1-C ₆ -alkyl (optionally substituted with 1- 3 fluorine atoms), hydroxy-C1-C ₆ -alkyl-, C1-C ₆ -alkoxy, (C1-C ₆ -alkyl) C (= O) -, (C1-C ₆ -alkoxy) C1-C ₆ - alkyl- and fluoro, or where hetCyca is substituted with an oxo group;

ring D is (i) a saturated 4-7 membered heterocyclic ring containing two ring nitrogen atoms, (ii) a saturated 7-8 membered bridged heterocyclic ring containing two ring nitrogen atoms and optionally containing a third ring heteroatom representing oxygen , (iii) a saturated 7-11 membered heterospirocyclic ring containing two ring nitrogen atoms, or (iv) a saturated 9-10 membered bicyclic fused heterocyclic ring containing two ring nitrogen atoms, each of said rings being optionally substituted by (a) one to four groups independently selected from halo, OH, C1- _C3 alkyl which is optionally substituted with 1-3 fluorine atoms, or C1- _C3 alkoxy which is optionally substituted with 1-3 fluorine atoms, (b) C ₃ -C ₆ -cycloalkylidene ring, or (c) oxo group;

E is:

(a) hydrogen, (b) C ₁ -C ₆ -alkyl, optionally substituted with 1-3 fluorine atoms, (c) (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl-, optionally substituted with 1-3 fluorine atoms, (d) (C ₁ -C ₆ -alkyl)C(=O)-, where the specified alkyl part is optionally substituted by 1-3 fluorine atoms or Deputy R ^g R ^h N-, where R ^g and R ^h independently represent is H or C1- _C6 alkyl, (e) (hydroxy-C2-C6-alkyl)C(=O)- optionally substituted with 1-3 fluorine atoms, (f) (C1-C6-alkoxy)C(= O)-, (g) (C ₃ -C ₆ -cycloalkyl)C(=O)-, where the specified cycloalkyl is optionally substituted with one or more substituents independently selected from C1-C ₆ -alkyl, C1-C ₆ -alkoxy, OH and (C1- _C6 alkoxy)C1- _C6 alkyl- or said cycloalkyl is substituted with a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N and O, (h) Ar ¹ C ₁ -C ₆ -alkyl-, (i) Ar ¹ (C1-C6-alkyl)C(=O)-, where the specified alkyl part is optionally substituted by an OH group, hydroxy-C1-C6-alkyl-, C1-C6-alkoxy, R ^m R ⁿ N- or R ^m R ⁿ N-CH2-, where each R ^m and R ⁿ independently represents H or C 1 -C ₆ -alkyl, (j) hetAr ² C ₁ -C ₆ -alkyl-, where the specified alkyl part is optionally substituted with 1-3 fluorine atoms, (k) hetAr ² (C ₁ -C ₆ -alkyl)C(=O)-, where the specified alkyl part is optionally substituted by an OH group, hydroxy-C1-C ₆ -alkyl- or C1-C ₆ -alkoxy, (l) hetAr ² C(= O)-, (m) hetCyc ¹ C(=O)-, (n) hetCyc ¹ C ₁ -C ₆ -alkyl-, (o) R ³ R ⁴ NC(=O)-, (p) Ar1N(R ³ )C(=O)-, (q) hetAr ² N(R ³ )C(=O)-,

- 104 041220 (r) (C ₁ -C ₆ -alkyl)SO ₂ -, where the specified alkyl part is optionally substituted by 1-3 fluorine atoms, (s) Ar ¹ SO2-, (t) hetAr ² SO2-, (u) N-(C ₁ -C ₆ -alkyl)pyridinonyl, (v)Ar ¹ C(=O)-;

(w) Ar'OC(=O)-, (x) (C ₃ -C ₆ -cycloalkyl)(C ₁ -C ₆ -alkyl)C(=O)-, (y) (C ₃ -C ₆ - cycloalkyl)(C ₁ -C ₆ -alkyl)SO ₂ -, where the specified alkyl part is optionally substituted by 13 fluorine atoms, (z) Ar ¹ (C 1 -C 6 -alkyl)SO 2 -, (aa) hetCyc'-OC (=O )-, (bb) hetCyc'CH2C(=O)-, (cc) hetAr ² or (dd) C ₃ -C ₆ cycloalkyl;

Ar ¹ is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), R ^e R ^f N-, where R ^e and R ^f independently represent H, C1-C6-alkyl, (R ^p R ^q N)C1-C6-alkoxy-, where R ^p and R ^q independently represent is H or C1-C6-alkyl, and (hetAr ^a )C ₁ -C ₆ -alkyl-, where hetAra is a 5-6 membered heteroaryl ring containing 1-2 ring nitrogen atoms, or Ar ¹ is a phenyl ring fused with a 5-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, or a 9-10 membered bicyclic heteroaryl ring containing 1-3 ring nitrogen atoms, where hetAr ² is optional substituted with one or more substituents independently selected from the group consisting of halo, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1- C6alkoxy)C1-C6-alkyl- (optionally substituted with 1-3 fluorine atoms), R ^e R ^f N-, where R ^e and R ^f independently represent H or C1-C ₆ -alkyl, OH, (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkoxy - and C ₃ -C ₆ -cycloalkyl;

hetCyc ¹ is a 4-6 membered saturated heterocyclic ring containing 1-2 ring heteroatoms independently selected from N, O and S, wherein said heterocyclic ring is optionally substituted with one or more substituents independently selected from C ₁ -C ₆ alkoxy and halogen;

R ³ is H or C1-C6 alkyl; and R ⁴ is C1- _C6 alkyl.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second RET inhibitor) is a compound of formula V:

or a pharmaceutically acceptable salt and solvate thereof, wherein

X ¹ , X ² , X ³ and X ⁴ independently represent CH or N, where zero, one or two X ¹ , X ² , X ³ and X ⁴ represent N;

A is CN; B is:

(b) C ₁ -C ₆ -alkyl optionally substituted with 1-3 fluorine atoms, (c) hydroxy-C ₂ -C ₆ -alkyl-, where the alkyl part is optionally substituted with 1-3 fluorine atoms or C ₃ C ₆ -cycloalkylidene ring, (e) (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl-, optionally substituted by 1-3 fluorine atoms, (f) (R ¹ R ² N)C1-C6 -alkyl-, where the specified the alkyl moiety is optionally substituted with an OH group, wherein R ¹ and R ² are independently H or C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms);

(g) hetAr ¹ C1-C3-alkyl-, where hetAr ¹ is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S and optionally substituted with one or more independently selected C1 -C ₆ -alkyl substituents; or (i) (hetCyc ^a )C ₁ -C ₃ -alkyl-, hetCyca- is a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents , independently selected from OH, C1- _C6 -alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1- _C6 -alkyl-, C1-C6-alkoxy, (C1-C6-alkyl)C (=O)-, (C ₁ -C ₆ -alkoxy)C ₁ -C6-alkyl- and fluorine, or where hetCyc ^a is substituted with oxo;

ring D is (i) a saturated 4-7 membered heterocyclic ring containing two ring nitrogen atoms, or (ii) a saturated 7-9 membered bridged heterocyclic ring containing two ring nitrogen atoms and optionally containing a third ring heteroatom, which is oxygen, each of said rings being optionally substituted with (a) one to four groups independently selected from halogen, OH, C1- _C3 alkyl which is optionally substituted with 1-3 fluorine atoms, or C1- _C3 alkoxy which is optionally substituted with 1 -3 fluorine atoms, (b) a C ₃ -C ₆ cycloalkylidene ring, or (c) an oxo group;

E is:

(h) Ar ¹ C ₁ -C ₆ -alkyl-, (j) hetAr ² C1-C6 -alkyl-, where the alkyl part is optionally substituted with 1-3 fluorine atoms, or (l) hetAr ² C(=O)-,

Ar ¹ is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), R ^e R ^f N-, where Re and R ^f independently represent H or C1-C6-alkyl, (R ^p R ^q N)C1-C6-alkoxy-, where R ^p and R ^q independently represent H or C1-C6-alkyl, and (hetAr ^a )C ₁ -C ₆ -alkyl-, where hetAra is a 5-6 membered heteroaryl ring containing 1-2 ring nitrogen atoms, or Ar ¹ is a phenyl ring, fused with a 5-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O; and hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, or a 9-10 membered bicyclic heteroaryl ring containing 1-3 ring nitrogen atoms, where hetAr ² optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1 -C6alkoxy)C1-C6-alkyl- (optionally substituted with 1-3 fluorine atoms), R ^e R ^f N-, where R ^e and R ^f independently represent H or C1-C6-alkyl, OH, (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkoxy- and C ₃ -C ₆ -cycloalkyl.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second RET inhibitor) is selected from the group consisting of: 4-(6-(4-benzylpiperazin-1-yl)pyridin-3-yl)-6-(2morpholinoethoxy) pyrazolo[1,5-a]pyridine-3-carbonitrile; 6-(2-hydroxyethoxy)-4-(6-(6-((6methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo [1,5-a]pyridine3-carbonitrile; (R)-6-(2-hydroxypropoxy)-4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1 yl)pyridin-3-yl)pyrazolo[1,5-a ]pyridine-3-carbonitrile; 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3 -yl)pyrazolo[1,5-a]pyridine3-carbonitrile; 6-(2-methoxyethoxy)-4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3yl)pyrazolo[1,5-a]pyridine-3- carbonitrile; 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1, 5-a]pyridine-3carbonitrile; 6-(2-(dimethylamino)ethoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6diazabicyclo[3.1.1]heptan-3-yl)pyridin-3- yl) pyrazolo[1,5-a]pyridine-3-carbonitrile; 4-(6-(6-((6methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2morpholinoethoxy)pyrazolo[1 ,5-a]pyridine-3-carbonitrile; 4-(6-(6-((6-Methoxypyridin-3-yl)methyl)-3,6diazabicyclo [3.1.1]heptan-3-yl)pyridin-3-yl)-6-((1-methyl- 1 H-imidazol-4-yl)methoxy)pyrazolo[1,5a]pyridine-3-carbonitrile; and 6-ethoxy-4-(5-(6-((5-fluoro-6-methoxypyridin-3-yl)methyl)-3,6diazabicyclo[3.1.1]heptan-3-yl)pyrazin-2-yl) pyrazolo[1,5-a]pyridine-3-carbonitrile; or their pharmaceutically acceptable salts or solvates.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second RET inhibitor) is a compound of Formula VI:

or a pharmaceutically acceptable salt or solvate thereof, wherein

X ¹ , X ² , X ³ and X ⁴ independently represent CH, CCH ₃ , CF or N, where zero, one or two X ¹ , X ² , X ³ and X ⁴ represent N;

A represents H, CN, Cl, methyl, ethyl or cyclopropyl;

B is:

(a) hydrogen, (b) C ₁ -C ₆ -alkyl optionally substituted with 1-3 fluorine atoms,

- 106 041220 (c) hydroxy-C ₂ -C6-alkyl-, where the alkyl part is optionally substituted with C ₃ -C ₆ -cycloalkylidene ring, (d) dihydroxy-C ₃ -C ₆ -alkyl-, where the alkyl part is optionally substituted with C ₃ -C ₆ - cycloalkylidene ring, (e) (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl-, optionally substituted with 1-3 fluorine atoms, (f) (R ¹ R ² N)C1-C6 -alkyl-, where R ¹ and R ² are independently selected from H, C1-C6 -alkyl (optionally substituted with 1-3 fluorine atoms), (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl-, (C1 -C ₆ -alkyl)C(=O)- and (C1-C ₆ -alkoxy)C(=O)-;

(g) hetAr ¹ C1-C3-alkyl-, where hetAr ¹ is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S and optionally substituted with one or more independently selected C1 -C6-alkyl substituents;

(h) (C ₃ -C ₆ -cycloalkyl)C ₁ -C ₃ -alkyl-, where said cycloalkyl is optionally substituted with an OH group, (i) (hetCyc ^a )C ₁ -C ₃ -alkyl-, (j) hetCyca, (k) (R ¹ R ² N)C(=O)C1-C6-alkyl-, where R ¹ and R ² are independently selected from H and C1-C6-alkyl;

(l) (R1R2N)C(=O)-, where R ¹ and R ² are independently selected from H and C1-C ₆ -alkyl, or (m) hetCyc ^a C(=O)C ₁ -C ₆ -alkyl- ;

hetCyca- is a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents independently selected from OH, C1-C ₆ -alkyl (optionally substituted with 1- 3 fluorine atoms), hydroxy-C1-C ₆ -alkyl-, halogen, (C1-C ₆ -alkyl)C (= O) -, C1-C ₆ -alkoxy, oxo and (C1-C ₆ -alkoxy)C (=O)-;

ring D is:

(i) a saturated monocyclic 4-7 membered heterocyclic ring containing one ring heteroatom which is nitrogen, (ii) a saturated 7-8 membered bridged heterocyclic ring containing one ring heteroatom which is nitrogen, or (iii) a saturated 7-11 membered heterospirocyclic ring system containing one ring heteroatom which is nitrogen;

each R ^a is independently C1- _C6 alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1- _C6 alkyl, or (C1- _C6 alkoxy)C1- _C6 alkyl-;

^Rb is:

(a) hydroxy, (b) cyclopropyl, (c) hetCyc ^b CH2-, (d) R'R'NC(=O)CH ₂ OCH ₂ - where Ri and R ^j are independently H or C1-C ₆ - alkyl, (e) RcRdN-, (f) R ^c RdNCH2-, (g) C ₁ -C ₆ -alkoxy-, (h) (C ₁ -C4-alkyl)-C(=O)NH-, where the specified the alkyl moiety is optionally substituted with hetCyc ^b , hetAra, C ₁ -C ₆ alkoxy or R'RN-, or said alkyl moiety is optionally substituted with two substituents independently selected from R'RN- and OH, where each R' and R are independently is hydrogen or C1-C ₆ -alkyl, (i) (R'RN)C ₁ -C ₆ -alkoxy(CH ₂ ) _n -, where n is 0 or 1, and R' and R independently represent hydrogen or C1-C ₆ alkyl, (j) hetCyc ^b (C ₁ -C ₃ alkyl) OCH ₂ -, (k) hetCyc ^b C(=O)NH- or (l) hetAr ^a C(=O)NH- ;

hetCycb is a 4-6 membered heterocyclic ring, a 7-8 membered bridged heterocyclic ring, or a 7-10 membered heterospirocyclic ring, each ring containing 1-2 ring heteroatoms independently selected from N and O, wherein hetCyc ^b is optionally substituted one or more substituents independently selected from OH, fluorine, C1- _C6 alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1- _C6 alkyl- (optionally substituted with 1-3 fluorine atoms), (C1- C ₆ -alkoxy)C1-C ₆ -alkyl-, (C1-C ₆ -alkoxy)C(=O)-, C1-C ₆ -alkoxy and R'RN-, where R' and R independently represent hydrogen or C1- _C6 alkyl;

hetAr ^a is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, wherein hetAr ^a is optionally substituted with one or more substituents independently selected from the group consisting of halo, CN, C ₁ -C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms) and C1-C ₆ -alkoxy (optionally substituted with 1-3 fluorine atoms),

R ^c is hydrogen or C1- _C6 alkyl;

R ^d is hydrogen, C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), (C1C6-alkoxy)C(=O)-, hydroxy-C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), (hydroxy-C1C6-alkyl)C(=O)-, (C1-C6-alkyl)C(=O)-, (R ^k R ^l N)C1-C ₆ -alkyl-, where R ^k and R ¹ are independently are H or C1-C6-alkyl, R ^m R ⁿ NC(=O)C1-C ₆ -alkyl-, where R ^m and R ⁿ independently represent H or C1-C ₆

- 107 041220 alkyl, PhCH ₂ -, where the specified phenyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), C 1 -C ₆ -alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C ₆ -alkoxy)C1-C ₆ alkyl- (optionally substituted with 1-3 fluorine atoms), C ₃ -C ₆ -cycloalkyl, hydroxy-C1-C ₆ -alkyl, (C ₁ -C ₆ -alkyl)SO ₂ -, R ^e RfN- and (R ^e R ^f N)C1-C6-alkyl-, where each R ^e and Rf independently represents H or C1-C6 -alkyl, (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkyl- or hetCycc, where hetCycc is a 4-6 membered heterocyclic ring containing a ring heteroatom selected from N and O and optionally substituted with C1 -C ₆ -alkyl;

n is 0, 1.2, 3, 4, 5, or 6;

m is 0 or 1;

E is:

(a) hydrogen, (b) hydroxy, (c) C 1 -C ₆ -alkyl optionally substituted with 1-3 fluorine atoms, (d) Ar ¹ C ₁ -C ₆ -alkyl-, where the specified alkyl part is optionally substituted with 1- 3 fluorine atoms, (e) hetAr ² C ₁ -C ₆ -alkyl-, (f) (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ -alkoxy-, (g) Ar1O-, (h) hetAr ² -O-, (i) Ar1NR ^g -, where R ^g is H or C1-C6 alkyl, (j) hetAr ² NR ^g -, where R ^g is H or C1-C6 alkyl, (k) R ³ C(=O)NR ^g -, where R ^g represents H or C1-C6-alkyl; (l) Ar1C(=O)NR ^g - where R ^g is H or C1-C6 alkyl, (m) hetAr ² C(=O)NR ^g (CH2)p- where p is 0 or 1, and R ^g is H or C1-C6 alkyl, (n) R ⁴ R ⁵ NC(=O)-, (o) Ar1NR ^g C(=O)-, where R ^g is H or C1-C ₆ -alkyl, (p) hetAr ² NR ^g C(=O)-, where R ^g represents H or C1-C ₆ -alkyl, (q) Ar ¹ (C ₁ -C ₆ -alkyl)C (=O) -, where the specified alkyl part is optionally substituted by an OH group, hydroxy(C1-C ₆ -alkyl), C1-C ₆ -alkoxy or NH2, (r) hetCyc ⁵ C(=O)-, (s) R ⁴ R ⁵ NC (=O)NR ^g -, where R ^g represents H or C1-C ₆ -alkyl, or (t) (C ₁ -C ₆ alkyl)sO ₂ -;

(u) Ar ¹ (C1-C6 alkyl)C(=O)NR ^g - where R ^g is H or C1-C6 alkyl, (v) hetAr ⁴ C(=O)NR ^g - where R ^g is H or C1-C ₆ -alkyl, (w) hetAr ² -S(=O)-, (x) (C ₃ -C ₆ -cycloalkyl)CH ₂ SO ₂ -, (y) Ar ¹ (C ₁ -C ₆ -alkyl)sO ₂ -, (z) hetAr ² SO2-, (aa) Ar ¹ , (bb) hetAr ² , (cc) hetCyc ⁵ , (dd) C ₁ -C ₆ -alkoxy, (her ) Ar ¹ (C ₁ -C ₆ -alkyl) -O-, (ff) hetAr ² (C ₁ -C ₆ -alkyl) -O-, (gg) hetAr ² -OC ₁ -C ₆ -alkyl-, ( hh) Ar ¹ (C1-C6 alkyl)NR ^g -, where R ^g is H or C1-C6 alkyl, (ii) hetAr ² -S-, (jj) Ar ² SO2NR ^g (CH2)p-, where p is 0 or 1 and R ^g is H or C1-C ₆ alkyl, (kk) (C1-C6 alkoxy)C(=O)-, (ll) (C ₁ -C ₆ alkyl) NR ^g C(=O)O- where R ^g is H or C1-C ₆ alkyl, (mm) (C ₁ -C ₆ alkyl)NR ^g SO2- where R ^g is H or C1- C6-alkyl, (nn) hetCyc ⁵ C(=O)NR ^g -, where R ^g represents H or C1-C ₆ -alkyl, (oo) Q-NR ^h (C1-C3-alkyl)C (=O )NR ^g -, where R ^g and R ^h are independently H or C 1 -C 6 alkyl, Q is is H, C1-C ₆ -alkyl or (C1-C ₆ -alkyl)OC(=O)(pp)

where R ^g and R ^h are independently H or C1-C ₆ -alkyl, Q is H, C1-C6 alkyl or (C1-C ₆ -alkyl)OC(=O)-, and r is 1, 2, 3 or 4, (qq)

- 108 041220

R ^h O where R ^g and R ^h independently represent H or C 1 -C ₆ -alkyl, and Q represents H, C 1 -C 6 alkyl or (C 1 -C ₆ -alkyl) OC (=O) -, (rr)

where R ^g is H or C1-C ₆ alkyl and Q is H, C1-C ₆ alkyl or (C1-C6 alkyl)OC(=O)-, or (ss) R ^g R ^h N-, where R ^g and R ^h are independently H or C1-C ₆ -alkyl, (tt) (C ₃ -C ₆ -cycloalkyl)C(=O)NR ^s -, where cycloalkyl is optionally and independently substituted with one or more halogen atoms , (uu) (C ₁ -C ₆ alkyl)C(=O)NR ^s CH2-, where R ^g is H or C1-C6 alkyl, or (vv) C ₁ -C ₆ alkyl)SO ₂ NR ^s -, where R ^g represents H or C 1 -C ₆ -alkyl;

Ar ¹ is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halo, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C6-alkoxy)C1-C6-alkyl- (optionally substituted with 1-3 fluorine atoms), C3-C6-cycloalkyl, hydroxy-C1-C6-alkyl, (C1-C6-alkyl)SO2-, R ^e RfN- and (R ^e R ^f N)C1-C ₆ -alkyl-, where each R ^e and Rf independently represents H or C1-C6-alkyl;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, or a 9-10 membered bicyclic heteroaryl containing 1-2 ring nitrogen atoms, where hetAr ² is optionally substituted one or more substituents independently selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), C 1 -C ₆ -alkoxy (optionally substituted with 1-3 fluorine atoms), ( C1- _C6 -alkoxy)C1- _C6- alkyl- (optionally substituted with 1-3 fluorine atoms) and hydroxy- _C1 - _C6 -alkoxy-;

hetCyc ⁵ is a 4-6 membered saturated heterocyclic ring containing 1-2 ring heteroatoms independently selected from N, O and S, wherein said heterocyclic ring is optionally substituted with one or more substituents independently selected from C1-C6-alkoxy and oxo ;

R ³ is C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1C6-alkyl-, C1-C6-alkoxy, C3-C6-cycloalkyl, (C3-C6-cycloalkyl)CH2-, (C3 -C6-cycloalkyl)O-, (C3-C6cycloalkyl)CH2O-, hetCyc ⁷ O-, Ph-O- or (C1-C6-alkoxy)C1- _C6 -alkyl-; where each of the specified C ₃ -C ₆ cycloalkyl fragments is optionally substituted with C ₁ -C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), C1 -C ₆ -alkoxy, OH or R'RN-, where R' and R are independently are hydrogen or C1- _C6 alkyl;

R ⁴ is H or C1-C ₆ alkyl;

R ⁵ is Ar ² , hetAr ³ , Ar ² CH2-, hetCyc ⁶ -CH2-, hydroxy-C1-C ₆ -alkyl-, (C ₃ -C ₆ cycloalkyl)CH ₂ - or C1-C ₆ -alkyl, optionally substituted with 1-3 fluorine atoms;

Ar ² is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C6-alkoxy)C1-C6-alkyl- (optionally substituted with 1-3 fluorine atoms), C3-C6-cycloalkyl and R ^g R ^h N-, where R ^g and R ^h are independently H or C1 -C ₆ alkyl, or Ar ² is phenyl fused to a 6-membered heterocyclic ring containing a ring nitrogen atom and optionally substituted with C ₁ -C ₆ alkyl;

hetAr ³ is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S and optionally substituted with one or more substituents independently selected from the group consisting of halo, CN, C ₁ - C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), C 1 -C ₆ -alkoxy (optionally substituted with 1-3 fluorine atoms) and (C 1 -C ₆ alkoxy)C 1 -C ₆ -alkyl- (optionally substituted with 1-3 fluorine atoms);

hetAr ⁴ is pyridin-4(1H)-onyl or pyridin-2(1H)-onyl optionally substituted with one or more substituents independently selected from C1-C6 alkyl and halo;

hetCyc ⁶ is a 5-7 membered heterocyclic ring containing 1-3 ring heteroatoms independently selected from N, O and S; and hetCyc ⁷ is a 5-7 membered heterocyclic ring containing 1-3 ring heteroatoms independently selected from N, O and S.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second

- 109 041220 RET inhibitor) is a compound of formula VII:

VII or a pharmaceutically acceptable salt or solvate thereof, wherein

X ¹ , X ² , X ³ and X ⁴ independently represent CH or N, where zero, one or two X ¹ , X ² , X ³ and X ⁴ represent N;

A is CN; B is:

(b) C ₁ -C ₆ -alkyl optionally substituted with 1-3 fluorine atoms, (c) hydroxy-C ₂ -C ₆ -alkyl-, where the alkyl part is optionally substituted with a C ₃ -C ₆ -cycloalkylidene ring, or (i ) (hetCyc ^a )C ₁ -C ₃ -alkyl-;

hetCyca- is a 4-6 membered heterocyclic ring containing 1-2 ring heteroatoms independently selected from N and O and optionally substituted with one or more substituents independently selected from OH, C1-C ₆ -alkyl (optionally substituted with 1- 3 fluorine atoms), hydroxy-C ₁ -C ₆ -alkyl-, halogen, (C1-C ₆ -alkyl) C (= O) -, C1-C ₆ -alkoxy, oxo and (C ₁ -C ₆ -alkoxy )C(=O)-;

ring D is a saturated monocyclic 4-7 membered heterocyclic ring containing one ring heteroatom which is nitrogen;

each R ^a is independently C1- _C6 alkyl (optionally substituted with 1-3 fluorine atoms);

R ^b is (a) hydroxy;

n is 0 or 1;

m is 0 or 1;

E is:

(e) hetAr ² C ₁ -C ₆ -alkyl-, (h) hetAr ² -O-, (k) R ³ C(=O)NR ^g -, where R ^g represents H or C 1 -C ₆ -alkyl , (l) Ar1C(=O)NR ^g -, where R ^g is H or C1-C ₆ -alkyl, or (m) hetAr ² C(=O)NR ^g (CH2)P-, where p is 0 or 1 and R ^g is H or C1-C6 alkyl;

Ar ¹ is phenyl optionally substituted with one or more substituents independently selected from the group consisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1-C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C1-C6-alkoxy)C1-C6alkyl- (optionally substituted with 1-3 fluorine atoms), C3-C6-cycloalkyl, hydroxy-C1-C6-alkyl, (C1-C6-alkyl)SO2-, R ^e R ^f N- and (R ^e R ^f N)C1-C ₆ -alkyl-, where each R ^e and R ^f independently represents H or C ₁ -C ₆ -alkyl;

hetAr ² is a 5-6 membered heteroaryl ring containing 1-3 ring heteroatoms independently selected from N, O and S, or a 9-10 membered bicyclic heteroaryl containing 1-2 ring nitrogen atoms, where hetAr ² is optionally substituted one or more substituents independently selected from the group consisting of halogen, CN, C1- _C6 alkyl (optionally substituted with 1-3 fluorine atoms), C1- _C6 alkoxy (optionally substituted with 1-3 fluorine atoms), (C ₁ -C ₆ -alkoxy)C ₁ -C ₆ alkyl- (optionally substituted with 1-3 fluorine atoms) and hydroxy-C ₁ -C ₆ -alkoxy-; And

R ³ is C1-C6-alkyl (optionally substituted with 1-3 fluorine atoms), hydroxy-C1C6-alkyl-, C1-C6-alkoxy, C3-C6-cycloalkyl, (C3-C6-cycloalkyl)CH2-, (C3 -C6-cycloalkyl)O-, (C3-C6cycloalkyl)CH2O-, hetCyc ⁷ O-, Ph-O- or (C1-C ₆ -alkoxy)C ₁ -C ₆ -alkyl-; where each of these C ₃ -C ₆ cycloalkyl fragments is optionally substituted with C 1 -C ₆ -alkyl (optionally substituted with 1-3 fluorine atoms), C 1 -C ₆ -alkoxy, OH or R'RN-, where R' and R independently represent is hydrogen or C1- _C6 alkyl.

In some embodiments, the RET inhibitor (e.g., the first RET inhibitor or the second RET inhibitor) is selected from the group consisting of N-(1-(5-(3-cyano-6-(2-hydroxy-2methylpropoxy)pyrazolo[1,5 -a]pyridin-4-yl)pyridin-2-yl)-4-methylpiperidin-4-yl)benzamide; 6ethoxy-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine3-carbonitrile; 6-(2-Hydroxy-2-methylpropoxy)-4-(6-(3-(pyridin-2-yloxy)azetidin-1-yl)pyridin-3yl)pyrazolo[1,5-a]pyridine-3-carbonitrile ; 6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-((6-methoxypyridazin-3-yl)oxy)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5- a] pyridine-3-carbonitrile; (S)-6-(2hydroxy-2-methylpropoxy)-4-(6-(3-(pyridin-2-yloxy)pyrrolidin-1-yl)pyridin-3-yl)pyrazolo[1,5а]pyridine-3 -carbonitrile; N-(1-(5-(3-cyano-6-((3-fluoro-1-methylazetidin-3-yl)methoxy)pyrazolo[1,5a]pyridin-4-yl)pyridin-2-yl)- 4-methylpiperidin-4-yl)-5-fluoro-2-methylbenzamide; 3-chloro-N-(1-(5-(3-110 041220 cyano-6-((3-fluoro-1-methylazetidin-3-yl)methoxy)pyrazolo[1,5-a]pyridin-4-yl )pyridin-2-yl)-4methylpiperidin-4-yl)picolinamide; N((38.48)-1-(5-(3-cyano-6-ethoxypyrazolo[1,5-a]pyridin-4yl)pyridin-2-yl)-3-hydroxypiperidin-4-yl)-3- methyl butanamide; 6-(2-hydroxy-2-methylpropoxy)-4(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a] pyridine-3carbonitrile; and 3-chloro-M-((38.48)-1-(5-(3-cyano-6-ethoxypyrazolo[1,5-a]pyridin-4-yl)pyrazin-2-yl)-3hydroxypiperidin-4 -yl)picolinamide; or their pharmaceutically acceptable salts or solvates.

Non-limiting examples of therapeutic agents that target receptor tyrosine kinases (e.g., Trk) include afatinib, cabozantinib, cetuximab, crizotinib, dabrafenib, entrectinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, pazopanib, panitumumab, pertuzumab, trasuzuminib , I-((38,4R)-4-(3-fluorophenyl)-I-(2methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methylpyrimidin-5-yl)-1 -phenyl-1H-pyrazol-5yl)urea, AG 879, AR-772, AR-786, AR-256, AR-618, AZ-23, AZ623, D8-6051, G6 6976, GNF-5837, GTx-186 , GW 441756, LOXO-101, MGCD516, PLX7486, RXDX101, VM-902A, TPX-0005 and T8R-011. Additional therapeutic agents targeting Trk include those described in US Pat. Nos. 8,450,322; 8513263; 8933084; 8791123; 8946226; 8450322; 8299057; and 8912194; US Publication No. 2016/0137654; 2015/0166564; 2015/0051222; 2015/0283132; and 2015/0306086; in international publications No. WO 2010/033941; WO2010/048314; WO2016/077841; WO 2011/146336; WO2011/006074; WO2010/033941; WO2012/158413; WO2014078454; W02014078417; WO2014078408; W02014078378; WO2014078372; W02014078331; W02014078328; W02014078325; W02014078323; W02014078322; W02015175788; W02009/013126; WO 2013/174876; WO2015/124697; WO2010/058006; WO2015/017533; WO2015/112806; WO 2013/183578; and WO 2013/074518, the entire contents of which are incorporated herein by reference.

Additional examples of Trk inhibitors are provided in US Pat. , in US patent No. 8642035, in international publication WO 2009092049, in US patent No. 8691221, in international publication WO2006131952, the entire contents of which are incorporated herein by reference. Exemplary Trk inhibitors include GNF-4256, described in Cancer Chemother. Pharmacol. 75(1): 131-141, 2015; and GNF-5837 (N-[3-[[2,3-dihydro-2-oxo-3-(1H-pyrrol2-ylmethylene)-1H-indol-6-yl]amino]-4-methylphenyl]-M' -[2-fluoro-5-(trifluoromethyl)phenyl]urea) described in AC8 Med. Chem. Lett. 3(2): 140-145, 2012, the entire contents of which are incorporated herein by reference.

Additional examples of Trk inhibitors include those described in US Publication No. 2010/0152219, US Pat. No. 8,114,989, and International Publication No. WO 2006/123113, the entire contents of which are incorporated herein by reference. Exemplary Trk inhibitors include AZ623 described in Cancer 117(6):1321-1391, 2011; AZD6918 described in Cancer Biol. Ther. 16(3):477-483, 2015; AZ64 described in Cancer Chemother. Pharmacol. 70:477-486, 2012; AZ-23 ((8)-5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1Hpyrazol-3-yl)pyrimidin-2,4-diamine) described in Mol. Cancer Ther. 8:1818-1827, 2009; and AZD7451; the entire contents of which are incorporated herein by reference.

The Trk inhibitor may include agents described in US Pat. Nos. 7,615,383; 7384632; 6153189; 6027927; 6025166; 5910574; 5877016; and 5844092, the entire contents of which are incorporated herein by reference.

Additional examples of Trk inhibitors include CEP-751 described in Int. J. Cancer 72:672-679, 1997; CT327 described in Acta Derm. Venereol. 95:542-548, 2015; the compounds described in International Publication No. WO 2012/034095; compounds described in US patent No. 8673347 and in international publication No. WO 2007/022999; compounds described in US patent No. 8338417; the compounds described in International Publication No. WO 2016/027754; compounds described in US patent No. 9242977; the compounds described in US Publication No. 2016/0000783; sunitinib (N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4dimethyl-1H-pyrrole-3-carboxamide), described in PLo8 One 9:e95628, 2014; the compounds described in International Publication No. WO 2011/133637; compounds described in US patent No. 8637256; compounds described in the Expert publication. Opin. Ther. Pat. 24(7):731-744, 2014; compounds described in the Expert Opin publication. Ther. Pat. 19(3):305-319, 2009; (R)-2-phenylpyrrolidine-substituted imidazopyridazines, e.g. GNF-8625, (R)-1-(6-(6-(2-(3-fluorophenyl)pyrrolidin-1yl)imidazo[1,2-b]pyridazine-3 -yl)-[2,4'-bipyridin]-2'-yl)piperidin-4-ol described in AC8 Med. Chem. Lett. 6(5):562-567, 2015; GTx-186 and others described in PLo8 One 8(12):e83380, 2013; K252a ((98-(9α, 10β, 12a))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl9,12-epoxy-1H-diindolo[ 1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one) described in Mol. Cell Biochem. 339(1-2):201-213, 2010; 4-aminopyrazolylpyrimidines, e.g. AZ-23 (((8)5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl) pyrimidine-2,4-diamine) described in J. Med. Chem. 51(15):4672-4684, 2008; RHA-739358 (danusertib), described in

- 111 041220 publications Mol. Cancer Ther. 6:3158, 2007; Go 6976 (5,6,7,13-tetrahydro-13-methyl-5-oxo-12Nindolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile) described in J. Neurochem . 72:919924, 1999; GW441756 ((3Z)-3-[(1-methylindol-3-yl)methylidene]-1H-pyrrolo[3,2-b]pyridin-2-one) described in IJAE Publication 115:117, 2010; milciclib (PHA-848125AC) described in J.

Carcinog. 12:22, 2013; AG-879 ((2E)-3-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-2-cyano-2propanethioamide); altiratinib (N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-2,5difluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); cabozantinib (N-(4-((6,7dimethoxyquinolin-4-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropan-1,1-dicarboxamide); lestaurtinib ((5S,6S,8R)-6-hydroxy-6-(hydroxymethyl)-5-methyl-7,8,14,15-tetrahydro-5H-16-oxa-4b,8a,14-triaza-5, 8methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-ac-indacen-13(6H)-one); dovatinib (4-amino-5-fluoro-3-[6(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]quinolin-2(1H)-one mono-2-hydroxypropanoate hydrate); sitravatinib (N-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7yl)oxy)phenyl )-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide); ONO-5390556; regorafenib (4-[4({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide hydrate; and VSR-902A; all of the above references are included in this document by reference in its entirety.

The ability of a Trk inhibitor to act as an inhibitor of TrkA, TrkB and/or Trk C can be tested using the assays described in Examples A and B in US Pat. No. 8,513,263, the contents of which are incorporated herein by reference.

In some embodiments, the receptor tyrosine kinase inhibitor is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. For example, EGFR inhibitors may include osimertinib (merelectinib, Tagrisso), erlotinib (Tarceva), gefitinib (Iressa), cetuximab (Erbitux), necitumumab (Portrazza), neratinib (Nerlynx), lapatinib (Tykerb), panitumumab (Vectibix), and vandetanib ( Caprelsa). In some embodiments, the EGFR inhibitor is osimertinib.

In some embodiments, signal transduction pathway inhibitors include inhibitors of the Ras-Raf-MEK-ERK pathway (e.g., binimetinib, selumetinib, encorafinib, sorafenib, trametinib, and vemurafenib), inhibitors of the PI3K-Akt-mTOR-S6K pathway (e.g., everolimus, rapamycin, perifosine, temsirolimus) and other kinase inhibitors such as baricitinib, brigatinib, capmatinib, danusertib, ibrutinib, milciclib, quercetin, regorafenib, ruxolitinib, semaxanib, AP32788, BLU285, BLU554, INCB39110, INCB40093, INCB50465, INCB527CD28 -088, NMS-1286937, PF 477736 ((R)-amino-N-[5,6-dihydro-2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-1Hpyrrolo[4,3 ,2-ef][2,3]benzodiazepin-8-yl]cyclohexaneacetamide), PLX3397, PLX7486, PLX8394,

PLX9486, PRN1008, PRN1371, RXDX103, RXDX106, RXDX108 and TG101209 (N-tert-butyl-3-(5-methyl-2(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-ylamino)benzenesulfonamide) .

Non-limiting examples of checkpoint inhibitors include ipilimumab, tremelimumab, nivolumab, pidilizumab, MPDL3208A, MEDI4736, MSB0010718C, BMS-936559, BMS-956559, BMS-935559 (MDX-1105), AMP-224, and pembrolizumab.

In some embodiments, the cytotoxic chemotherapeutic agents are selected from arsenic trioxide, bleomycin, cabazitaxel, capecitabine, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, etoposide, fluorouracil, gemcitabine, irinotecan, lomustine, methotrexalicin C, mytomtrexate , paclitaxel, pemetrexed, temozolomide and vincristine.

Non-limiting examples of therapeutic agents that target angiogenesis include aflibercept and bevacizumab.

The term immunotherapy refers to an agent that modulates the immune system. In some embodiments, immunotherapy can enhance the expression and/or activity of an immune system regulator. In some embodiments, immunotherapy may reduce the expression and/or activity of an immune system regulator. In some embodiments, immunotherapy may involve and/or enhance the activity of an immune cell.

In some embodiments, the immunotherapy is cellular immunotherapy (eg, adoptive T cell therapy, dendritic cell therapy, natural killer cell therapy). In some embodiments, the cellular immunotherapy is sipuleucel-T (APC8015; Provenge™; Plosker (2011) Drugs 71(1): 101-108). In some embodiments, cellular immunotherapy includes cells that express a chimeric antigen receptor (CAR). In some embodiments, the cellular immunotherapy is a CAR-T cell therapy. In some embodiments, the CAR-T cell therapy is thisagenleucleucel (Kymriah™).

In some embodiments, the immunotherapy is an antibody therapy (eg, a monoclonal antibody, a conjugated antibody). In some embodiments, the antibody therapy is bevacizumab (Mvasti™, Avastin®), trastuzumab (Herceptin®),

- 112 041220 avelumab (Bavencio®), rituximab (MabThera™, Rituxan®), edrecolomab (Panorex), daratumumab (Darzalex®), olaratumab (Lartruvo™), ofatumumab (Arzerra®), alemtuzumab (Campath®), cetuximab (Erbitux ®), oregovomab, pembrolizumab (Keytruda®), dinutiximab (Unituxin®), obinutuzumab (Gazyva®), tremelimumab (CP-675,206), ramucirumab (Cyramza®), ublituximab (TG-1101), panitumumab (Vectibix®), elotuzumab (Empliciti™), avelumab (Bavencio®), necitumumab (Portrazza™), cirmtuzumab (UC961), ibritumomab (Zevalin®), isatuximab (SAR650984), nimotuzumab, fresolimumab (GC1008), lirilumab (INN), mogamulizumab (Poteligeo®) , ficlatuzumab (AV-299), denosumab (Xgeva®), ganitumab, urelumab, pidilizumab, or amatuximab.

In some embodiments, the immunotherapy is an antibody drug conjugate. In some embodiments, the antibody drug conjugate is gemtuzumab ozogamicin (Mylotarg™), inotuzumab ozogamicin (Besponsa®), brentuximab vedotin (Adcetris®), ado-trastuzumab emtansine (TDM-1; Kadcyla®), mirvetuximab soravtansine (IMGN853) or anetumab ravtansine.

In some embodiments, the immunotherapy comprises blinatumomab (AMG103; Blincyto®) or midostaurin (Rydapt).

In some embodiments, the immunotherapy includes a toxin. In some embodiments, the immunotherapy is denileukin diftitox (Ontak®).

In some embodiments, the immunotherapy is a cytokine therapy. In some embodiments, the cytokine therapy is interleukin 2 (IL-2) therapy, interferon alpha (IFNa) therapy, granulocyte colony stimulating factor (G-CSF) therapy, interleukin 12 (IL-12) therapy, interleukin 15 (IL-15), interleukin 7 (IL-7) therapy, or erythropoietin-alpha (EPO) therapy. In some embodiments, the IL-2 therapy is an aldesleukin (Proleukin®). In some embodiments, the IFNa therapy is IntronA® (Roferon-A®). In some embodiments, the GCSF therapy is filgrastim (Neupogen®).

In some embodiments, the immunotherapy is an immune checkpoint inhibitor. In some embodiments, the immunotherapy includes one or more immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumab (Yervoy®) or tremelimumab (CP-675206). In some embodiments, the PD-1 inhibitor is pembrolizumab (Keytruda®) or nivolumab (Opdivo®). In some embodiments, the PD-L1 inhibitor is atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi™).

In some embodiments, the immunotherapy is an mRNA-based immunotherapy. In some embodiments, the mRNA-based immunotherapy is CV9104 (see, e.g., Rausch et al. (2014) Human Vaccin Immunother 10(11): 3146-52; and Kubleretal. (2015) J. Immunother Cancer 3:26) .

In some embodiments, the immunotherapy is Bacillus Calmette-Guerin (BCG) therapy.

In some embodiments, the immunotherapy is an oncolytic viral therapy. In some embodiments, the oncolytic viral therapy is talimogen lagerparepvec (T-VEC; Imlygic®).

In some embodiments, the immunotherapy is a cancer vaccine. In some embodiments, the cancer vaccine is a human papillomavirus (HPV) vaccine. In some embodiments, the HPV vaccine is Gardasil®, Gardasil9®, or Cervarix®. In some embodiments, the cancer vaccine is a hepatitis B virus (HBV) vaccine. In some embodiments, the HBV vaccine is Engerix-B®, Recombivax HB®, or GI-13020 (Tarmogen®). In some embodiments, the cancer vaccine is Twinrix® or Pediarix®. In some embodiments, the cancer vaccine is BiovaxlD®, Oncophage®, GVAX, ADXS11-001, ALVAC-CEA, PROSTVAC®, Rindopepimut®, CimaVax-EGF, lapuleucel-T (APC8024; Neuvenge™), GRNVAC1, GRNVAC2, GRN- 1201, Hepcortespenlyzymuth-L (Hepko-V5), DCVAX®, SCIB1, BMT CTN 1401, PrCa VBIR, PANVAC, ProstAtak®, DPX-Survivac or Viagenpumatucel-L (HS-110).

In some embodiments, the immunotherapy is a peptide vaccine. In some embodiments, the peptide vaccine is nonlipepimut-S (E75) (NeuVax™), IMA901, or SurVaxM (SVN53-67). In some embodiments, the cancer vaccine is an immunogenic individual neoantigen vaccine (see, e.g., Ott et al. (2017) Nature 547: 217-221; Sahin et al. (2017) Nature 547: 222-226). In some embodiments, the cancer vaccine is RGSH4K or NEO-PV-01. In some embodiments, the cancer vaccine is a DNA-based vaccine. In some implementations

- 113 041220 DNA-based vaccine is a mammaglobulin-A DNA-based vaccine (see, for example,

Kim et al. (2016) Oncolmmunology 5(2): e1069940).

In some embodiments, immune system targeting agents are selected from aldesleukin, interferon alfa-2b, ipilimumab, lambrolizumab, nivolumab, prednisone, and sipuleucel-T.

Non-limiting examples of radiation therapy include radioiodide therapy, external beam radiation therapy, and radium-223 therapy.

Additional kinase inhibitors include those described in, for example, US Pat. Nos. 7,514,446; 7863289; 8026247; 8501756; 8552002; 8815901; 8912204; 9260437; 9273051; US Publication No. 2015/0018336; in international publications No. WO 2007/002325; WO2007/002433; WO2008/080001; W02008/079906; WO2008/079903; W02008/079909; WO2008/080015; W02009/007748; WO2009/012283; WO 2009/143018; WO2009/143024; WO WO 2009/014637; 2009/152083; WO2010/111527; WO 2012/109075; WO 2014/194127; WO2015/112806; WO 2007/110344; WO2009/071480; WO2009/118411; WO2010/031816; WO2010/145998; WO2011/092120; WO2012/101032; WO 2012/139930; WO 2012/143248; WO2012/152763; WO2013/014039; WO 2013/102059; WO2013/050448; WO2013/050446; WO2014/019908; WO2014/072220; WO2014/184069; and WO 2016/075224, the entire contents of which are incorporated herein by reference.

Additional examples of kinase inhibitors include those described, for example, in WO 2016/081450; WO2016/022569; WO2016/011141; WO2016/011144; WO2016/011147; WO 2015/191667; WO2012/101029; WO2012/113774; WO 2015/191666; WO2015/161277; WO2015/161274; WO 2015/108992; WO2015/061572; WO2015/058129; WO2015/057873; WO2015/017528; WO/2015/017533; WO 2014/160521; and WO 2014/011900, the entire contents of which are incorporated herein by reference.

Additional examples of kinase inhibitors include luminespib (AUY-922, NVP-AUY922) (5(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide) and dorampimod (BIRB-796) (1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4ylethoxy)naphthalene-1-yl]urea) .

Accordingly, this document also provides a method of treating cancer, comprising administering to a patient in need thereof, a pharmaceutical combination for treating cancer, which contains (a) a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, (b) additional a therapeutic agent and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate or sequential use in the treatment of cancer, wherein amounts of a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, and the additional therapeutic agent together are effective for cancer treatment.

In some embodiments, the additional therapeutic agent(s) includes any of the above therapeutic agents or therapeutic agents that are standard treatments for cancer, wherein the cancer is characterized by dysregulation of the RET gene, RET protein, or expression or activity or level of any of them.

Said additional therapeutic agents may be administered with one or more doses of a compound of formulas I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition thereof, in the same or different dosage forms, by the same or different routes of administration, and /or on the same or different routes of administration in accordance with standard pharmaceutical practice known to those skilled in the art.

This document also provides (i) a pharmaceutical combination for the treatment of cancer in a patient in need thereof, which contains (a) a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, (b) at least one additional therapeutic agent (e.g., any of the illustrative additional therapeutic agents described herein or known in the art), and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate, or sequential use in the treatment of cancer, wherein amounts of a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form and additional therapeutic agent together are effective for the treatment of cancer; (ii) a pharmaceutical composition containing such a combination; (iii) the use of such a combination for the preparation of a medicament for the treatment of cancer; and (iv) an industrial package or product containing such a combination as a combined preparation for simultaneous, separate or sequential use; and a method for treating cancer in a patient in need thereof. In one embodiment, the patient is a human. In some embodiments, the cancer is RET-associated cancer. For example, RET-associated cancer having one or more RET inhibitor resistance mutations.

The term pharmaceutical combination in this context refers to a pharmaceutical therapy resulting from mixing or combining more than one active ingredient, and

- 114 041220 includes both fixed and non-fixed combinations of active ingredients. The term fixed combination means that a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and at least one additional therapeutic agent (e.g., a chemotherapeutic agent) is administered to a patient simultaneously, in the form of a single composition or dosage form. The term loose combination means that a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and at least one additional therapeutic agent (e.g., a chemotherapeutic agent) are formulated as separate compositions or dosage forms so that they can be administered to a patient in need of it, simultaneously, in parallel or sequentially at various intermediate time intervals, and such an introduction provides an effective content of two or more compounds in the patient's body. This also applies to cocktail therapies, such as the administration of three or more active ingredients.

Accordingly, this document also provides a method of treating cancer, comprising administering to a patient in need thereof, a pharmaceutical combination for treating cancer, which contains (a) a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, (b) additional a therapeutic agent and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate or sequential use in the treatment of cancer, wherein amounts of a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, and the additional therapeutic agent together are effective for cancer treatment. In one embodiment, the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and the additional therapeutic agent are administered simultaneously as separate dosage forms. In one embodiment, the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and an additional therapeutic agent are administered as separate dosage forms sequentially, in any order, in a total therapeutically effective amount, for example, in daily or intermediate doses. In one embodiment, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and an additional therapeutic agent are administered simultaneously as a combined dosage form. In some embodiments, the cancer is RET-associated cancer. For example, RET-associated cancer having one or more RET inhibitor resistance mutations. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is osimertinib. In some embodiments, one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered to a patient prior to administration of said pharmaceutical composition. In some embodiments, the cancer is lung cancer (eg, RET-associated lung cancer).

Also provided herein is a method of treating a RET-mediated disease or disorder in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition. In some embodiments, the RET-mediated disease or disorder is a dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them. For example, dysregulation of the RET gene, RET kinase, or expression or activity or level of any of these includes one or more RET inhibitor resistance mutations. A disease or disorder mediated by RET may include any disease, disorder or pathological condition associated directly or indirectly with the expression or activity of RET, including overexpression and/or an abnormal level of activity. In another embodiment, the disease is cancer (eg, RET-associated cancer). In one embodiment, the cancer is any cancer or RET-associated cancer described herein. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is osimertinib. In some embodiments, one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered to a patient prior to administration of said pharmaceutical composition. In some embodiments, the cancer is lung cancer (eg, RET-associated lung cancer).

Although the genetic basis of tumorigenesis may vary according to different types of cancer, the cellular and molecular mechanisms required for the occurrence of metastasis are the same for all types of solid tumors. During the metastatic cascade, cancer cells lose their inhibitory growth responses, undergo changes in adhesiveness, and produce enzymes that can degrade extracellular matrix components. This leads to detachment of tumor cells from the original tumor, infiltration into the bloodstream through the newly formed vascular network, migration and extravasation of tumor cells in favorable distant foci,

- 115 041220 where they can form colonies. Many genes have been identified as promoters or suppressors of metastases. For example, overexpression of glial cell-derived neurotrophic factor (GDNF) and its receptor tyrosine kinase RET correlates with cancer proliferation and metastasis. See, for example, Zeng, Q. et al. J. Int. Med. Res. (2008) 36(4): 656-64.

Accordingly, this document also provides methods of suppressing, preventing, facilitating the prevention or reducing the symptoms of cancer metastasis in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, or its pharmaceutical composition. Such methods may be used in the treatment of one or more of the cancers described herein. See, for example, US Publication No. 2013/0029925; International Publication No. WO 2014/083567; and US Pat. No. 8,568,998. See also, for example, Hezam K et al., Rev Neurosci 2018 Jan 26;29:93-98; Gao L, et al., Pancreas 2015 Jan; 44:134-143; Ding K et al., J Biol Chem 2014 Jun 6; 289:16057-71; and Amit M et al., Oncogene 2017 Jun 8; 36:3232-3239. In some embodiments, the cancer is RET-associated cancer. In some embodiments, a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is used in combination with an adjuvant therapy or other therapeutic agent, including a chemotherapeutic agent such as a kinase inhibitor. For example, a first or second RET kinase inhibitor. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is osimertinib. In some embodiments, one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered to a patient prior to administration of said pharmaceutical composition. In some embodiments, the cancer is lung cancer (eg, RET-associated lung cancer).

The term metastasis is a known term and refers to the formation of an additional tumor (eg, a solid tumor) at a site remote from the primary tumor in a subject or patient, the additional tumor containing the same or similar cancer cells as the primary tumor.

Also provided are methods for reducing the risk of developing metastasis or additional metastasis in a patient suffering from RET-associated cancer, which includes: selecting, identifying or diagnosing the patient as having RET-associated cancer, and administering a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable thereof. salt, amorphous or polymorphic form to a patient who has been selected, identified or diagnosed as having RET-associated cancer. Also provided are methods for reducing the risk of developing metastasis or additional metastasis in a patient suffering from RET associated cancer, which comprises administering a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, to a patient suffering from RET associated cancer. The reduction in the risk of metastasis or additional metastasis in a patient suffering from RET-associated cancer can be compared with the risk of metastasis or additional metastasis in a patient before treatment, or compared with a patient or group of patients with a similar or the same RET-associated cancer who are not taking treatment or are taking another treatment. In some embodiments, the RET-associated cancer is RET-associated cancer having one or more RET inhibitor resistance mutations. In some embodiments, the additional therapeutic agent is crizotinib. In some embodiments, the additional therapeutic agent is osimertinib. In some embodiments, one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered to a patient prior to administration of said pharmaceutical composition. In some embodiments, the cancer is lung cancer (eg, RET-associated lung cancer).

The expression "risk of metastasis" means the risk that a subject or patient with a primary tumor will develop an additional tumor (e.g., a solid tumor) in a lesion distant from the primary tumor in the body of the subject or patient for a certain period of time, and the additional tumor contains the same or similar cancer cells as the primary tumor. Methods for reducing the risk of metastasis in a subject or patient suffering from cancer are described herein.

The expression risk of developing additional metastasis means the risk that a subject or patient having a primary tumor and one or more additional tumors in lesions distant from the primary tumor (with one or more additional tumors containing the same or similar cancer cells as the primary tumor ), one or more additional tumors will develop at a distance from the primary tumor, the additional tumors containing the same or similar cancer cells as the primary tumor. This document describes ways to reduce the risk of developing additional metastasis.

In some embodiments, the presence of one or more inhibitor resistance mutations

- 116 041220

RET in the tumor causes tumor resistance to treatment with the first RET inhibitor. Methods are described below that are useful when the RET inhibitor resistance mutation results in higher resistance of the tumor to treatment with the first RET inhibitor. For example, provided herein are methods for treating a subject suffering from cancer, which include: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and administering to the identified subject a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered in combination with a first RET inhibitor. Also provided are methods of treating a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations, which comprises administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered in combination with a first RET inhibitor. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

For example, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to said subject a therapeutically effective amount of a first RET inhibitor, the first RET inhibitor being selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU -667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-( 4-methyl6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one inhibitor resistance mutation. RET; or (e) administering additional doses of the first RET inhibitor from step (b) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation.

In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to said subject a therapeutically effective amount of a first RET inhibitor, the first RET inhibitor being selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU -667 ((1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3yl)ethyl)-1-methoxy-4-(4- methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one inhibitor resistance mutation. RET; or (e) administering additional doses of the first RET inhibitor from step (b) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation.

In some embodiments, the compound of formula I-IV is a polymorph. In some embodiments, the compound is the Form A polymorph of the Formula I compound. In some embodiments, the compound is the Form 1 polymorph of the Formula II compound. In some embodiments, the compound is the Form 2 polymorph of the compound of Formula II. In some embodiments, the compound is the form 7 polymorph of the compound of formula II. In some embodiments, the compound is the Form 8 polymorph of the Formula II compound. In some embodiments, the compound is the Form A polymorph of the compound of formula III. In some embodiments, the compound is Form A polymorph of the compound of formula IV. In some embodiments, the compound is the Form B polymorph of the compound of formula IV.

In some embodiments, a compound of Formula I-IV is a pharmaceutically acceptable salt. In some embodiments, the compound is the chloride salt of a compound of formula I. In some embodiments, the compound is the bromide salt of a compound of formula I. In some embodiments, the compound is the L malate salt of a compound of formula I. In some embodiments, the compound is the D-malate salt a compound of formula I. In some embodiments, the compound is the phosphate salt of a compound of formula II. In some embodiments, the phosphate salt is a sesqui phosphate salt (eg, PO ₄ :free base 1.4:1).

In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1. 1 and/or one or more point mutations/inserts/deletions in the RET kinase protein from Table. 2 and 2a in a sample obtained from a subject; and (b) administering to said subject a therapeutically effective amount of a first RET inhibitor, the first RET inhibitor being selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU -667 ((1S,4R)-N-((S)-1(6-(4-fluoro-1 H-pyrazol-1 -yl)pyridin-3-yl)ethyl)-1-methoxy-4-( 4-methyl-6-((5-methyl-1H-pyrazol-3yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179W6, GSK3352589 and NMS-E668. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation of Table 1. 3 or 4; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one inhibitor resistance mutation. RET; or (e) administering additional doses of the first RET inhibitor from step (b) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation.

In some embodiments, the compound of formula I-IV is a polymorph. In some embodiments, the compound is the Form A polymorph of the Formula I compound. In some embodiments, the compound is the Form 1 polymorph of the Formula II compound. In some embodiments, the compound is the Form 2 polymorph of the compound of Formula II. In some embodiments, the compound is the form 7 polymorph of the compound of formula II. In some embodiments, the compound is the Form 8 polymorph of the Formula II compound. In some embodiments, the compound is Form A polymorph of the compound of formula III. In some embodiments, the compound is Form A polymorph of the compound of formula IV. In some embodiments, the compound is the Form B polymorph of the compound of formula IV.

In some embodiments, a compound of Formula I-IV is a pharmaceutically acceptable salt. In some embodiments, the compound is the chloride salt of a compound of formula I. In some embodiments, the compound is the bromide salt of a compound of formula I. In some embodiments, the compound is the L malate salt of a compound of formula I. In some embodiments, the compound is the D-malate salt a compound of formula I. In some embodiments, the compound is the phosphate salt of a compound of formula II. In some embodiments, the phosphate salt is a sesqui phosphate salt (eg, PO ₄ :free base 1.4:1).

In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to said subject a therapeutically effective amount of a first RET inhibitor, the first RET inhibitor being selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU -667 ((1S,4R)-N-((3)-1-(6-(4-fluoro1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4- methyl-6-((5-methyl-1H-pyrazol-3yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has a V804M, G810S, or G810R RET inhibitor resistance mutation; and (d) administering to said subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, selected from the group consisting of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering additional doses of the first RET inhibitor from step (b) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation.

In some embodiments, the compound of formula I-IV is a polymorph. In some embodiments, the compound is the Form A polymorph of the Formula I compound. In some embodiments, the compound is the Form 1 polymorph of the Formula II compound. In some embodiments, the compound is the Form 2 polymorph of the compound of Formula II. In some embodiments, the compound is the form 7 polymorph of the compound of formula II. In some embodiments, the compound is the Form 8 polymorph of the Formula II compound. In some embodiments, the compound is Form A polymorph of the compound of formula III. In some embodiments, the compound is Form A polymorph of the compound of formula IV. In some embodiments, the compound is the Form B polymorph of the compound of formula IV.

In some embodiments, a compound of Formula I-IV is a pharmaceutically acceptable salt. In some embodiments, the compound is the chloride salt of a compound of formula I. In some embodiments, the compound is the bromide salt of a compound of formula I. In some embodiments, the compound is the L malate salt of a compound of formula I. In some embodiments, the compound is the D-malate salt a compound of formula I. In some embodiments, the compound is the phosphate salt of a compound of formula II. In some embodiments, the phosphate salt is a sesqui phosphate salt (eg, PO ₄ :free base 1.4:1).

As another example, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to the subject a second RET inhibitor, the second RET inhibitor being selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU667 ((1S, 4R)-N-((3)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6- ((5-methyl1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010,

GSK3179106, GSK3352589 and NMS-E668, alone or in combination with another anti-cancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject. ; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to the subject a second RET inhibitor, the second RET inhibitor being selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 (( 1S,4R)-N-((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl- 6-((5methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010,

GSK3179106, GSK3352589 and NMS-E668, alone or in combination with another anti-cancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1. 1 and/or one or more point mutations/inserts/deletions in the RET kinase protein from Table. 2 and 2a in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation of Table 1. 3 or 4; and (d) administering to the subject a second RET inhibitor, the second RET inhibitor being selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 (( 1S,4R)-N-((S)-1-(6-(4-fluoro-1Hpyrazol-1 -yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6- ((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668, as monotherapy or in combination with another an anticancer agent if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has a V804M, G810S, or G810R RET inhibitor resistance mutation; and (d) administering to the subject a second RET inhibitor, the second RET inhibitor being selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 (( 1 S,4R)-N-((S)-1 -(6-(4-fluoro-1 H-pyrazol-1 -yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4- methyl-6-((5-methyl-1Hpyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668, as monotherapy or in in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

As another example, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a second therapeutic agent, the second therapeutic agent being selected from the group consisting of crizotinib and osimertinib, either alone or in combination with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, if said the subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1. 1 and/or one or more point mutations/inserts in the RET kinase protein from Table. 2 in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation of Table 1. 3 or 4; and (d) administering to said subject a second therapeutic agent, the second therapeutic agent being selected from the group consisting of crizotinib and osimertinib, either alone or in combination with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, if said the subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments listed above, the RET-associated cancer is lung cancer.

In some embodiments, the presence of one or more inhibitor resistance mutations

- 120 041220

RET in the tumor causes tumor resistance to treatment with the first RET inhibitor. Methods are described below that are useful when the RET inhibitor resistance mutation results in higher resistance of the tumor to treatment with the first RET inhibitor. For example, provided herein are methods for treating a subject suffering from cancer, which include: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and administering to the identified subject a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered in combination with a first RET inhibitor. Also provided are methods of treating a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations, which comprises administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered in combination with a first RET inhibitor. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

In some embodiments provided herein, circulating tumor DNA can be used to monitor a patient's responsiveness to a particular therapy (e.g., a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form ). For example, prior to initiation of treatment using the therapy described herein (eg, using a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof), a biological sample can be obtained from a subject and determine the level of circulating tumor DNA in a biological sample. Such a sample is considered a sample in the initial state. The subject may then be administered one or more doses of the therapy described herein (e.g., a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof) and circulating tumor DNA levels may be monitored. (eg, after the first dose, second dose, third dose, etc., or after one week, two weeks, three weeks, four weeks, etc.). If the level of circulating tumor DNA is lower than in the sample at baseline (eg, decrease from 1% to about 99%, decrease from 1% to about 95%, decrease from 1% to about 90%, decrease from 1% to about 85 %, reduction from 1% to about 80%, reduction from 1% to about 75%, reduction from 1% to a reduction of about 70%, reduction from 1% to a reduction of about 65%, reduction from 1% to a reduction of about 60%, 1% reduction to about 55% reduction, 1% reduction to approximately 50% reduction, 1% reduction to approximately 45% reduction, 1% reduction to approximately 40% reduction, 1% reduction to approximately 35% reduction, decrease from 1% to a decrease of about 30%, decrease from 1% to a decrease of about 25%, decrease from 1% to a decrease of about 20%, decrease from 1% to a decrease of about 15%, decrease from 1% to a decrease of about 10%, 1% to about 5%, decrease from about 5% to about 99%, decrease from about 10% to about 99%, decrease from about 15% to about 99%, decrease from about 20% to about 99%, decrease from about 25% to about 99%, decrease from about 30% to about 99%, decrease decrease from about 35% to about 99%, decrease from about 40% to about 99%, decrease from about 45% to about 99%, decrease from about 50% to about 99%, decrease from about 55% to about 99%, about 60% to about 99% decrease, about 65% to about 99% decrease, about 70% to about 99% decrease, about 75% to about 95% decrease, about 80% to about 99% decrease, about 90% to about 99% decrease, about 95% to about 99% decrease, about 5% to about 10% decrease, about 5% to about 25% decrease, about 10% to about 30% decrease, decrease from about 20% to about 40%, decrease from about 25% to about 50%, decrease from about 35% to about 55%, decrease from about 40% to about 60%, decrease from about 50% to about 75%, a decrease from about 60% to about 80%, or a decrease from about 65% to about 85%, etc.), this is indicative of responsiveness to therapy. In some embodiments, the level of circulating tumor DNA is reduced such that the level is below the detection limit of the instrument. In some embodiments, the level of circulating tumor DNA in a biological sample obtained from a patient (n) is compared with a sample taken immediately before it (n-1). If the level of circulating tumor DNA in sample n is lower than in sample n-1 (eg, decrease from 1% to about 99%, decrease from 1% to about 95%, decrease from 1% to about 90%, decrease from 1% to about 85%, decrease from 1% to about 80%, decrease from 1% to about 75%, decrease from 1% to decrease about 70%, decrease from 1% to decrease about 65%, decrease from 1% to decrease about 60%, decrease from 1% to about 55% decrease, decrease from 1% to about 50% decrease, decrease from 1% to about 45% decrease, decrease from 1%

- 121 041220 to around 40% reduction, 1% reduction to approximately 35% reduction, 1% reduction to approximately 30% reduction, 1% reduction to approximately 25% reduction, 1% reduction to approximately 20% reduction, reduction 1% to decrease about 15%, decrease from 1% to decrease about 10%, decrease from 1% to about 5%, decrease from about 5% to about 99%, decrease from about 10% to about 99%, decrease from about 15% to about 99%, decrease from about 20% to about 99%, decrease from about 25% to about 99%, decrease from about 30% to about 99%, decrease from about 35% to about 99%, decrease from about 40% to about 99%, decrease from about 45% to about 99%, decrease from about 50% to about 99%, decrease from about 55% to about 99%, decrease from about 60% to about 99%, decrease from about 65% to about 99%, decrease from about 70% to about 99%, decrease from about 75% to about 95%, decrease from about 80% to about 99%, decrease from about 90% to about 99%, decrease from about 95% to about 99%, decrease from about 5% to about 10%, decrease from about 5% to approx. about 25%, decrease from about 10% to about 30%, decrease from about 20% to about 40%, decrease from about 25% to about 50%, decrease from about 35% to about 55%, decrease from about 40% to about 60%, a decrease from about 50% to a decrease of about 75%, a decrease from about 60% to a decrease of about 80%, or a decrease from about 65% to a decrease of about 85%, etc.), then this is an indicator of responsiveness to therapy . In some embodiments, the level of circulating tumor DNA is reduced such that the level is below the detection limit of the instrument. If responsive to therapy, the subject can be administered one or more doses of therapy and continue to monitor circulating tumor DNA.

If the level of circulating tumor DNA in the sample is higher than at baseline (eg, increase from 1% to about 99%, increase from 1% to about 95%, increase from 1% to about 90%, increase from 1% to about 85 %, 1% to about 80% increase, 1% to about 75% increase, 1% to about 70% increase, 1% to about 65% increase, 1% to about 60% increase, 1% increase to about 55% increase, 1% increase to about 50% increase, 1% increase to about 45% increase, 1% increase to about 40% increase, 1% increase to about 35% increase, 1% increase to about 30% increase, 1% increase to about 25% increase, 1% increase to about 20% increase, 1% increase to about 15% increase, 1% increase to about 10% increase, increase from 1% to about 5% increase, increase from about 5% to about 99%, increase from about 10% to about 99%, increase from about 15% to about 99%, increase from about 20% to about 99%, increase from about 25% to about 99%, increase from about 30% to about 99%, increase from about 35% to about 99%, increase from about 40% to about 99%, increase from about 45% to about 99%, increase from about 50% to about 99%, increase from about 55% to about 99%, increase from about 60% to about 99%, increase from about 65% to about 99%, increase from about 70% to about 99%, increase from about 75% to about 95%, increase from about 80% to about 99%, increase from about 90% to about 99%, increase from about 95% to about 99%, increase from about 5% to about 10%, increase from about 5% to about 25%, increase from about 10% to about 30%, increase from about 20% to about 40%, increase from about 25% to about 50%, increase from about 35% to about 55%, increase from about 40% to about 60%, increase from about 50% to about 75%, increase from about 60% to about 80%, or increase from about 65% to about 85%, etc. .), this may indicate resistance to therapy. If the level of circulating tumor DNA in sample n is higher than in sample n-1 (eg, increase from 1% to about 99%, increase from 1% to about 95%, increase from 1% to about 90%, increase from 1% to about 85%, increase 1% to about 80%, increase from 1% to about 75%, increase from 1% to about 70%, increase from 1% to about 65%, increase from 1% to about 60%, increase 1% to about 55% increase 1% to about 50% increase 1% to about 45% increase 1% to about 40% increase 1% to about 35% increase 1% to about 30%, increase from 1% to about 25%, increase from 1% to about 20%, increase from 1% to about 15%, increase from 1% to about 10%, increase from 1% to about 5%, increase from about 5% to about 99%, increase from about 10% to about 99%, increase from about 15% to about 99%, increase from about 20% to about 99%, increase from about 25% to about 99%, increase from about 30% to about 99%, increase from about 35% to about 99%, increase from about 40% to about about 99%, increase from about 45% to about 99%, increase from about 50% to about 99%, increase from about 55% to about 99%, increase from about 60% to about 99%, increase from about 65% to about 99%, increase from about 70% to about 99%, increase from about 75% to about 95%, increase from about 80% to about 99%, increase from about 90% to about 99%, increase from about 95% to about 99%, increase from about 5% to about 10%, increase from about 5% to about 25%, increase from about 10% to about 30%, increase from about 20% to about 40%, increase from about 25% to about 50%, increase from about 35% to about 55%, increase from about 40% to about 60%, increase from about 50% to about 75%, increase from about 60% to about 80%, or increase from about 65% to about 85%, etc.), this may indicate resistance to therapy. In suspected therapy resistance, the subject may be referred for one or more of imaging, biopsy, surgery, or other diagnostic tests. In some embodiments, when resistance to therapy is suspected, a subject may be administered (as monotherapy or in combination with prior therapy) a compound capable of treating resistance to an RET inhibitor (e.g., a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or a polymorphic form as proposed herein). See, for example, Cancer Discov; 7(12); 1368-70 (2017); And

cancer discov; 7(12); 1394-403 (2017).

In some embodiments provided herein, a protein biomarker may be used to monitor a patient's responsiveness to a particular therapy (e.g., a first RET inhibitor, a second RET inhibitor, or a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form) . For example, prior to initiation of treatment using the therapy described herein (eg, using a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof), a biological sample can be obtained from a subject and determine the level of a protein biomarker in a biological sample. Such a sample is considered a sample in the initial state. The subject may then be administered one or more doses of the therapy described herein (e.g., a first RET inhibitor, a second RET inhibitor, or a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof) and levels of a protein biomarker ( for example, after the first dose, second dose, third dose, etc., or after one week, two weeks, three weeks, four weeks, etc.). If the protein biomarker level is lower than the sample at baseline (e.g., decrease from 1 to about 99%, decrease from 1 to about 95%, decrease from 1 to about 90%, decrease from 1 to about 85%, decrease from 1 to about 80%, decrease from 1% to about 75%, decrease from 1% to decrease about 70%, decrease from 1% to decrease about 65%, decrease from 1% to decrease about 60%, decrease from 1% to decrease about 55%, decrease from 1% to decrease about 50%, decrease from 1% to decrease about 45%, decrease from 1% to decrease about 40%, decrease from 1% to decrease about 35%, decrease from 1% to decrease about 30%, 1% reduction to approximately 25% reduction, 1% reduction to approximately 20% reduction, 1% reduction to approximately 15% reduction, 1% reduction to approximately 10% reduction, 1 to approximately 5% reduction, about 5 to about 99% decrease, about 10 to about 99% decrease, about 15 to about 99% decrease, about 20 to about 99% decrease, about 25 to about 99% decrease, about 30 to about 30% decrease about 99%, reduction from about 35 to about about 99%, decrease from about 40% to about 99%, decrease from about 45% to about 99%, decrease from about 50% to about 99%, decrease from about 55% to about 99%, decrease from about 60% to about 99%, decrease about 65 to about 99%, about 70 to about 99% decrease, about 75 to about 95% decrease, about 80 to about 99% decrease, about 90 to about 99% decrease, about 95 to about 95% decrease 99%, decrease from about 5% to about 10%, decrease from about 5% to about 25%, decrease from about 10% to about 30%, decrease from about 20% to about 40%, decrease from about 25% to about 50%, decrease from about 35% to about 55%, about 40% to about 60% decrease, about 50% to about 75% decrease, about 60% to about 80% decrease, or about 65% to about 85% decrease, etc.), then this is indicative of responsiveness to therapy. In some embodiments, the level of the protein biomarker is reduced such that the level is below the detection limit of the instrument. In some embodiments, the level of a protein biomarker in a biological sample obtained from a patient (n) is compared with a sample taken immediately before it (n-1). If the level of the protein biomarker in sample n is lower than in sample n-1 (e.g., decrease from 1 to about 99%, decrease from 1 to about 95%, decrease from 1 to about 90%, decrease from 1 to about 85%, 1% to about 80% decrease, 1% to about 75% decrease, 1% decrease to about 70% decrease, 1% decrease to about 65% decrease, 1% decrease to about 60% decrease, 1% decrease to about 55% decrease, decrease from 1% to about 50% decrease, decrease from 1% to about 45% decrease, decrease from 1% to about 40% decrease, decrease from 1% to about 35% decrease, decrease from 1% to a decrease of about 30%, a decrease from 1% to a decrease of about 25%, a decrease from 1% to a decrease of about 20%, a decrease from 1% to a decrease of about 15%, a decrease from 1% to a decrease of about 10%, a decrease from 1 to about 5%, decrease from about 5% to about 99%, decrease from about 10% to about 99%, decrease from about 15% to about 99%, decrease from about 20% to about 99%, decrease from about 25% to about 99%, decrease from about 30 to about 99%, reduction from about 35 to about 99%, decrease from about 40% to about 99%, decrease from about 45% to about 99%, decrease from about 50% to about 99%, decrease from about 55% to about 99%, decrease from about 60% to about 99%, about 65 to about 99% decrease, about 70 to about 99% decrease, about 75 to about 95% decrease, about 80 to about 99% decrease, about 90 to about 99% decrease, about 95 to about 95% decrease about 99%, decrease from about 5% to about 10%, decrease from about 5% to about 25%, decrease from about 10% to about 30%, decrease from about 20% to about 40%, decrease from about 25% to about 50%, decrease about 35 to about 55%, about 40 to about 60%, about 50% to about 75%, about 60% to about 80%, or about 65% to about 85%, and etc.), then this is an indicator of susceptibility to therapy. In some embodiments, the level of the protein biomarker is reduced such that the level is below the detection limit of the instrument. In the case of responsiveness to therapy, the subject can be administered one or more doses of therapy and continue to monitor the protein biomarker.

If the protein biomarker level in the nth sample is above baseline (e.g., increase from 1 to about 99%, increase from 1 to about 95%, increase from 1 to about 90%, increase from 1 to about 85%, increase from 1 to about 80%, increase from 1 to about 75%, increase from 1 to about 70%,

- 123 041220 1 to about 65% increase, 1 to about 60% increase, 1 to about 55% increase, 1 to about 50% increase, 1 to about 45% increase, 1 to about 40% increase , increase from 1 to about 35%, increase from 1 to about 30%, increase from 1 to about 25%, increase from 1 to about 20%, increase from 1 to about 15%, increase from 1 to about 10%, increase 1 to about 5%, about 5 to about 99% increase, about 10 to about 99% increase, about 15 to about 99% increase, about 20 to about 99% increase, about 25 to about 99% increase %, increase from about 30% to about 99%, increase from about 35% to about 99%, increase from about 40% to about 99%, increase from about 45% to about 99%, increase from about 50% to about 99%, increase from about 55 to about 99%, about 60 to about 99% increase, about 65 to about 99% increase, about 70 to about 99% increase, about 75 to about 95% increase, about 80 to about 99% increase , increase about 90 to about 99% increase, about 95 to about 99% increase, about 5 to about 10% increase, about 5 to about 25% increase, about 10 to about 30% increase, about 20 to about 20% increase about 40%, about 25 to about 50% increase, about 35 to about 55% increase, about 40 to about 60% increase, about 50 to about 75% increase, about 60 to about 80% increase, or increase from about 65 to about 85%, etc.), this may indicate resistance to therapy. If the level of the protein biomarker in sample n is higher than in sample n-1 (e.g., increase from 1 to about 99%, increase from 1 to about 95%, increase from 1 to about 90%, increase from 1 to about 85%, increase from 1 to about 80%, increase from 1 to about 75%, increase from 1 to about 70%, increase from 1 to about 65%, increase from 1 to about 60%, increase from 1 to about 55%, increase from 1 to about 50%, increase from 1 to about 45%, increase from 1 to about 40%, increase from 1 to about 35%, increase from 1 to about 30%, increase from 1 to about 25%, increase from 1 to about 20%, 1 to about 15% increase, 1 to about 10% increase, 1 to about 5% increase, about 5 to about 99% increase, about 10 to about 99% increase, about 15 to about 15% increase about 99%, about 20 to about 99% increase, about 25 to about 99% increase, about 30 to about 99% increase, about 35 to about 99% increase, about 40 to about 99% increase, increase from about 45 to approx. about 99%, about 50 to about 99% increase, about 55 to about 99% increase, about 60 to about 99% increase, about 65 to about 99% increase, about 70 to about 99% increase, increase about 75 to about 95%, about 80 to about 99% increase, about 90 to about 99% increase, about 95 to about 99% increase, about 5 to about 10% increase, about 5 to about 5% increase 25%, increase from about 10% to about 30%, increase from about 20% to about 40%, increase from about 25% to about 50%, increase from about 35% to about 55%, increase from about 40% to about 60%, increase from about 50% to about 75%, an increase from about 60% to about 80%, or an increase from about 65% to about 85%, etc.), this may indicate resistance to therapy. In suspected therapy resistance, the subject may be referred for one or more of imaging, biopsy, surgery, or other diagnostic tests. In some embodiments, when resistance to therapy is suspected, a subject may be administered (as monotherapy or in combination with prior therapy) a compound capable of treating resistance to an RET inhibitor (e.g., a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, as suggested in this document).

In some embodiments, one or more protein biomarkers are monitored. The specific protein biomarkers to be monitored may depend on the type of cancer and can be readily determined by one of skill in the art. Non-limiting examples of protein biomarkers include: CA 125, carcinoembryonic antigen (CEA), calcitonin, adrenocorticotropic hormone (ACTH), cortisol, CA 19-9, prolactin, hepatocyte growth factor, osteopontin, myeloperoxidase, tissue inhibitor of metalloproteinase 1, angiopoietin-1 (Ang -1), cytokeratin 19 (CK-19), tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase-3-like protein-1 (YKL-40), galectin-3 (GAL-3), CYFRA 21-1 (cytokeratins), EPCAM (epithelial cell adhesion molecule), ProGRP (progastrin-releasing peptide) and CEACAM (carcinoembryonic antigen). See, for example, Cohen JD, Li L, Wang Y, et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. science; published online January 18, 2018. pii: eaar3247. DOI: 10.1126/science.aar3247; Fawaz M Makki et al. Serum biomarkers of papillary thyroid cancer. J Otolaryngol Head Neck Surg. 2013; 42(1): 16; and Tatiana N. Zamay et al. Current and Prospective Protein Biomarkers of Lung Cancer. Cancers (Basel). Nov 2017; 9(11): 155. In some embodiments, the biomarkers include one or more of CEA, calcitonin, thyroglobulin, ACTH, and cortisol. In some embodiments, the cancer is medullary thyroid cancer and the protein biomarkers include CEA and calcitonin. In some embodiments, the cancer is non-medullary thyroid cancer and the protein biomarker comprises thyroglobulin. In some embodiments, the biomarkers are ACTH and cortisol (eg, if the patient is suffering from Cushing's disease due to their cancer).

Also provided herein are methods of treating RET-associated cancer in a subject that comprise (a) administering one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a first inhibitor kinase RET to a subject who has

- 124 041220 identified or diagnosed as having a RET-associated cancer (e.g., any type of RET-associated cancer described herein) (e.g., identified or diagnosed as having a RET-associated cancer using any of the illustrative methods described herein, or known in the art); (b) after step (a) determining the level of circulating tumor DNA in a biological sample (eg, in a biological sample containing blood, serum or plasma) obtained from the subject; (c) administering a therapeutically effective amount of a second RET inhibitor or a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, alone or in combination with another anticancer agent, to a subject identified as having approximately the same or elevated levels of circulating tumor DNA compared to a reference level of circulating tumor DNA (eg, any reference level of circulating tumor DNA described herein). In some examples of these methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in the biological sample obtained from the subject prior to step (a). Some embodiments of the proposed methods further include determining the level of circulating tumor DNA in a biological sample obtained from a subject prior to step (a). In some examples of the proposed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (e.g., the average level of circulating tumor DNA in a group of subjects having the same RET-associated but receiving an ineffective treatment or placebo, or not yet receiving a therapeutic treatment, or the level of circulating tumor DNA in a subject having the same RET-associated cancer but receiving an ineffective treatment or placebo, or not yet receiving therapeutic treatment In some examples of the proposed methods, the first RET inhibitor is selected from the group: cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667 and BLU6864.

Also provided herein are methods of treating RET-associated cancer in a subject which comprises administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, to a subject (i) identified or diagnosed as having RET-associated cancer. (e.g., any type of RET-associated cancer described herein) (e.g., who has been identified or diagnosed as having RET-associated cancer using any of the illustrative methods described herein or known in the art), (ii) who has previously administered one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a second RET kinase inhibitor, and (ii) who, after administration of one or more doses of a second RET kinase inhibitor the presence of approximately the same or increased levels of circulating tumor DNA was found, compared with yu with a reference level of circulating tumor DNA (for example, with any reference level of circulating tumor DNA described in this document or known in the art). In some embodiments of the proposed methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample (e.g., a biological sample containing blood, plasma, or serum) obtained from a subject prior to administration of one or more doses of a second RET kinase inhibitor. Some embodiments of the proposed methods further include determining the level of circulating tumor DNA in a biological sample obtained from a subject prior to administration of one or more doses of a second RET kinase inhibitor. In some examples of the proposed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (for example, the average level of circulating tumor DNA in a group of subjects having the same RET-associated, but receiving an ineffective treatment or placebo, or not yet receiving therapeutic treatment, or the level of circulating tumor DNA in a subject having the same RET-associated cancer but receiving a failed treatment or placebo or not yet receiving therapy In some embodiments of such methods, the second RET kinase inhibitor is selected from the group consisting of: cabozantinib, vandetanib , alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667 and BLU6864.

Also provided herein are methods of treating RET-associated cancer in a subject, comprising: (a) administering one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy to a subject identified or diagnosed as having RET-associated cancer (eg, any type of RET-associated cancer described herein) (eg, a subject identified or diagnosed as having RET-associated cancer using any method described herein or known in the art); (b) after step (a) determining the level of circulating tumor DNA in a biological sample (eg, in a biological sample containing blood, serum or plasma) obtained from the subject; (c) the introduction of a therapeutic effect

- 125 041220 an effective amount of a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form and an additional therapeutic agent or treatment (for example, any additional therapeutic agents or treatments for RET-associated cancer described herein or known in this field technique) to a subject identified as having approximately the same or elevated levels of circulating tumor DNA as compared to a reference level of circulating tumor DNA (e.g., any exemplary reference level of circulating tumor DNA described herein or known in the art). In some embodiments of the proposed methods, the additional therapeutic agent is a second RET kinase inhibitor (e.g., a RET kinase inhibitor selected from the group: cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667, and BLU6864). In some examples of any proposed methods, the additional therapeutic agent or treatment includes one or more of: radiation therapy, a chemotherapeutic agent (for example, any illustrative chemotherapeutic agent described herein or known in the art), a checkpoint inhibitor (for example, any illustrative checkpoint inhibitor as described herein or known in the art), surgery (e.g., at least partial tumor resection), and one or more other kinase inhibitors (e.g., any exemplary kinase inhibitor as described herein or known in this technical field). In some examples of these methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample (eg, a biological sample containing blood, serum or plasma) obtained from the subject prior to step (a). In some examples of the proposed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (for example, the average level of circulating tumor DNA in a group of subjects having the same RET-associated, but receiving an ineffective treatment or placebo, or not yet receiving therapeutic treatment, or the level of circulating tumor DNA in a subject who has the same RET-associated cancer but is receiving a failed treatment or placebo, or not yet receiving therapeutic treatment.

Also provided herein are methods for treating RET-associated cancer in a subject, comprising: administering a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and an additional therapeutic agent or treatment to a subject (i) who has an identified or diagnosed with RET-associated cancer (e.g., any type of RET-associated cancer described herein) (e.g., a subject identified or diagnosed with RET-associated cancer using any of the methods described herein or known in of the art), (ii) who has previously received one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy, and (ii) who, after administration of one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy, the presence of the same or an increased level of circulating tumor DNA is identified, in comparison with the reference level of circulating tumor DNA (for example, with any illustrative reference level circulating tumor DNA described herein). In some embodiments of such methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample obtained from a subject prior to administration of one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy. Some embodiments of the proposed methods further include determining the level of circulating tumor DNA in a biological sample obtained from a subject prior to administration of one or more doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy. In some examples of the proposed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (for example, the average level of circulating tumor DNA in a group of subjects having the same RET-associated, but receiving an ineffective treatment or placebo, or not yet receiving therapeutic treatment, or the level of circulating tumor DNA in a subject having the same RET-associated cancer but receiving an ineffective treatment or placebo, or not yet receiving a therapeutic treatment In some embodiments of the proposed method, the additional therapeutic agent is a second RET kinase inhibitor (e.g., a second RET kinase inhibitor selected from the group of cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667 and BLU6864). apeutic agent

- 126 041220 or treatment includes one or more of radiation therapy, a chemotherapeutic agent (for example, any exemplary chemotherapeutic agent described herein or known in the art), a checkpoint inhibitor (for example, any exemplary checkpoint inhibitor described in herein or known in the art), surgery (eg, at least partial tumor resection), and one or more other kinase inhibitors (eg, any kinase inhibitor described herein or known in the art).

Also provided herein are methods for selecting treatment for a subject, which include: selecting a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, for a subject (i) who has been identified or diagnosed as having RET-associated cancer ( for example, any RET-associated cancer described herein) (for example, for a subject identified or diagnosed as having RET-associated cancer using any of the methods described herein or known in the art), (ii) who has previously received one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a second RET kinase inhibitor (e.g., any of the RET kinase inhibitors described herein or known in the art), and (ii) who, after administration of one or more doses of a second RET kinase inhibitor, is identified the presence of approximately the same or increased level of circulating tumor DNA compared to the reference level of circulating tumor DNA was detected. In some embodiments of any of the proposed methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample (e.g., a biological sample containing blood, serum, or plasma) obtained from a subject prior to administration of one or more doses of a second RET kinase inhibitor. Some embodiments of the proposed methods further include determining the level of circulating tumor DNA in a biological sample obtained from a subject prior to administration of one or more doses of a second RET kinase inhibitor. In some examples of the proposed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (e.g., the average level of circulating tumor DNA in a group of subjects having the same RET-associated but receiving an ineffective treatment or placebo, or not yet receiving a therapeutic treatment, or level of circulating tumor DNA in a subject having the same RET-associated cancer but receiving a failed treatment or placebo or not yet receiving therapy In some embodiments of such methods, the second RET kinase inhibitor is selected from the group of cabozantinib, vandetanib, alectinib, apatinib , sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667 and BLU6864.

Also provided herein are methods for selecting a treatment for a subject, which includes selecting a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and an additional therapeutic agent or treatment for a subject (i) identified or diagnosed as having RET-associated cancer (e.g., any RET-associated cancer described herein or known in the art) (e.g., for a subject diagnosed or identified as having RET-associated cancer using any of the methods described herein or known in the art) art), (ii) who has previously received one or more doses (e.g., two or more, three or more, four or more, five or more, or ten or more) of the compounds of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form as monotherapy, and (ii) in which, after administration one or more doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, identified the presence of the same or an increased level of circulating tumor DNA compared to the reference level of circulating tumor DNA. In some embodiments of such methods, the reference level of circulating tumor DNA is the level of circulating tumor DNA in a biological sample (e.g., a biological sample containing blood, serum, or plasma) obtained from a subject prior to administration of one or more doses of a compound of Formula I-IV, or its pharmaceutically acceptable salt, amorphous or polymorphic form as monotherapy. Some embodiments further include determining the level of circulating tumor DNA in a biological sample obtained from a subject prior to administration of one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy. In some examples of the proposed methods, the reference level of circulating tumor DNA is a threshold level of circulating tumor DNA (for example, the average level of circulating tumor DNA in a group of subjects having the same RET-associated, but receiving an ineffective treatment or placebo, or not yet receiving therapeutic treatment, or the level of circulating tumor DNA in the body of a subject who has the same RET-associated cancer, but is receiving ineffective treatment or placebo,

- 127 041220 or not yet receiving therapeutic treatment. In some embodiments of any of the proposed methods, the additional therapeutic agent is a second RET kinase inhibitor (e.g., a second RET kinase inhibitor selected from the group: cabozantinib, vandetanib, alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib, BLU667 and BLU6864). In some embodiments of any of the methods described herein, the additional therapeutic agent or treatment includes one or more of radiation therapy, a chemotherapeutic agent (e.g., any of the examples of chemotherapeutic agents described herein or known in the art), an inhibitor checkpoint (e.g., any checkpoint inhibitor described herein or known in the art), surgery (e.g., at least partial tumor resection), and one or more other kinase inhibitors (e.g., any of the other kinase inhibitors, described in this document or known in the art).

Also provided herein are methods for determining the effectiveness of a treatment in a subject, which includes: (a) determining a first level of circulating tumor DNA in a biological sample (e.g., a biological sample containing blood, serum, or plasma) obtained from a subject identified or the presence of RET-associated cancer was diagnosed, at the first time point; (b) administering to the subject a treatment comprising one or more doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, after a first time point and before a second time point; (c) determining a second level of circulating tumor DNA in a biological sample (eg, a biological sample containing blood, serum or plasma) obtained from the subject at a second time point; and (d) determining that the treatment is effective in a subject found to have a reduced second level of circulating tumor DNA compared to a first level of circulating tumor DNA; or determining that the treatment is ineffective in a subject found to have approximately the same or increased second level of circulating tumor DNA as compared to the first level of circulating tumor DNA. In some embodiments of such methods, the first time point and the second time point have an interval of from about 1 week to about 1 year (e.g., from about 1 week to about 10 months, from about 1 week to about 8 months, from about 1 week to about 6 months, about 1 week to about 4 months, about 1 week to about 3 months, about 1 week to about 2 months, about 1 week to about 1 month, or about 1 week to about 2 weeks).

This document also provides methods for determining whether a subject has developed resistance to treatment, which include: (a) determining the first level of circulating tumor DNA in a biological sample (for example, in a biological sample containing blood, serum or plasma) obtained from the subject , which is identified or diagnosed as having RET-associated cancer, at the first time point; (b) administering to the subject a treatment comprising one or more (e.g., two or more, three or more, four or more, five or more, or ten or more) doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, whether amorphous or polymorphic forms after the first time point and before the second time point; (c) determining a second level of circulating tumor DNA in a biological sample obtained from a subject at a second time point; and (d) determining that the subject has not developed resistance to treatment if there is a decrease in the second level of circulating tumor DNA compared to the first level of circulating tumor DNA; or determining that the subject has developed resistance to treatment if the subject has about the same or increased second level of circulating tumor DNA compared to the first level of circulating tumor DNA. In some embodiments of such methods, the first time point and the second time point have an interval of from about 1 week to about 1 year (e.g., from about 1 week to about 10 months, from about 1 week to about 8 months, from about 1 week to about 6 months, about 1 week to about 4 months, about 1 week to about 3 months, about 1 week to about 2 months, about 1 week to about 1 month, or about 1 week to about 2 weeks).

Exemplary methods for determining circulating tumor DNA are described in Moati et al., Clin. Res. Hepatol. Gastroenterol. April 4, 2018; Oussalah et al., EBioMedicine March 28, 2018; Moon et al., Adv. drug deliv. Rev. April 4, 2018; Solassaol et al., Clin. Chem. Lab. Med. April 7, 2018; Arriola et al., Clin. Transl. oncol. April 5, 2018; Song et al., J. Circ. biomark. March 25, 2018; Aslibekyan et al., JAMA Cardiol. April 4, 2018; Isbell et al., J. Thorac. Cardiovasc. Surg. March 13, 2018; Boeckx et al., Clin. Colorectal Cancer February 22, 2018; Anunobi et al., J. Surg. Res. March 28, 2018; Tan et al., Medicine 97(13):eO197, 2018; Reithdorf et al., Transl. Androl. Urol. 6(6):1090-1110, 2017; Volckmar et al., Genes Chromosomes Cancer 57(3): 123-139, 2018; and Lu et al., Chronic Dis. Transl. Med. 2(4):223-230, 2016. Additional methods for determining the level of circulating tumor DNA are known in the art.

In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a resolution

- 128 041220 gene RET, RET kinase or expression, or activity, or the level of any of them in the sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a multikinase inhibitor, the multikinase inhibitor being selected from vandetanib or cabozantinib; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one inhibitor resistance mutation. RET; or (e) administering additional doses of the multikinase inhibitor from step (b) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation.

In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a first multikinase inhibitor, said multikinase inhibitor being selected from the group consisting of: vandetanib or cabozantinib; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a compound of formula IIV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering additional doses of the multikinase inhibitor from step (b) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation.

In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1. 1 and/or one or more point mutations/inserts/deletions in the RET kinase protein from Table. 2 and 2a in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a multikinase inhibitor, the multikinase inhibitor being selected from the group consisting of: vandetanib or cabozantinib; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation of Table 1. 3 or 4; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one inhibitor resistance mutation. RET; or (e) administering additional doses of the multikinase inhibitor from step (b) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation.

In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a multikinase inhibitor, said multikinase inhibitor being selected from the group consisting of vandetanib or cabozantinib; or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has a V804M, G810S, or G810R RET inhibitor resistance mutation; and (d) administering to said subject a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, selected from the group consisting of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering additional doses of the multikinase inhibitor from step (b) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation.

As another example, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further include (after (b)) (c) setting

- 129 041220 determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a multikinase inhibitor (eg, vandetanib or cabozantinib) alone or in combination with another anticancer agent if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods of treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or the expression or activity or level of any of them in a sample obtained from the subject. ; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to said subject a multikinase inhibitor (eg, vandetanib or cabozantinib) alone or in combination with another anticancer agent if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1. 1 and/or one or more point mutations/inserts/deletions in the RET kinase protein from Table. 2 and 2a in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation of Table 1. 3 or 4; and (d) administering to said subject a multikinase inhibitor (eg, vandetanib or cabozantinib) alone or in combination with another anticancer agent if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has a V804M, G810S, or G810R RET inhibitor resistance mutation; and (d) administering to said subject a multikinase inhibitor (eg, vandetanib or cabozantinib) alone or in combination with another anticancer agent if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

Also provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I -IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or with immunotherapy) or with anti-cancer therapy (eg, surgery or radiation), if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation. In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting

- 130 041220 deregulation of the RET gene, RET kinase or expression or activity or level of any of them in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation; and (d) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I -IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or with immunotherapy) or with anti-cancer therapy (eg, surgery or radiation), if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation. In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1. 1 and/or one or more point mutations/inserts/deletions in the RET kinase protein from Table. 2 and 2a in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, selected from the group consisting of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation of Table 1. 3 or 4; and (d) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I -IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or with immunotherapy) or with anti-cancer therapy (eg, surgery or radiation), if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation. In some embodiments, a second RET inhibitor selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N -((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl- 1 H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668 are introduced in step (d). In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) determining if a cancer cell in a sample obtained from a subject has a V804M, G810S, or G810R RET inhibitor resistance mutation; and (d) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) as monotherapy or in combination with another anti-cancer agent (e.g. a second RET inhibitor, a second compound of formula I -IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or with immunotherapy) or with anti-cancer therapy (eg, surgery or radiation), if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation. In some embodiments, a second RET inhibitor selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N -((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl- 1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668 are introduced in step (d).

Also provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) detecting at least one RET inhibitor resistance mutation in a cancer cell in a sample obtained from a subject; and (d) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I -IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, or immunotherapy) or anti-cancer therapy (eg, surgery or radiation). In some

- 131 041220 embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a downregulation of the RET gene, RET kinase, or the expression or activity or level of any of them in a sample obtained from subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) detecting at least one RET inhibitor resistance mutation in a cancer cell in a sample obtained from a subject; and (d) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula IIV or its pharmaceutically acceptable salt, amorphous or polymorphic form, or immunotherapy) or anti-cancer therapy (eg, surgery or radiation). In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting one or more fusion proteins from Table 1. 1 and/or one or more point mutations/inserts/deletions in the RET kinase protein from Table. 2 and 2a in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, selected from the group consisting of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) detecting at least one RET inhibitor resistance mutation of Table 1. 3 or 4 in a cancer cell in a sample obtained from a subject; and (d) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I -IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, or immunotherapy) or anti-cancer therapy (eg, surgery or radiation). In some embodiments, a second RET inhibitor selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N -((3)-1-(6-(4-fluoro1 H-pyrazol-1 -yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl -1 H-pyrazol-3yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668, are introduced in step (d). In some embodiments, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting a KIF5B-RET fusion protein in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed methods further comprise (after (b)) (c) detecting a V804M, G810S, or G810R RET inhibitor resistance mutation in a cancer cell in a sample obtained from a subject; and (d) administering to the subject additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) as monotherapy or in combination with another anti-cancer agent (e.g., a second RET inhibitor, a second compound of formula I -IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, or immunotherapy) or anti-cancer therapy (eg, surgery or radiation). In some embodiments, a second RET inhibitor selected from the group consisting of alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)-N -((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4(4-methyl-6-((5-methyl -1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide), BLU6864, DS-5010, GSK3179106, GSK3352589 and NMS-E668 are introduced in step (d).

This document further provides a method of treating lung cancer in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, crizotinib, osimertinib, or any combination thereof.

In some embodiments, the lung cancer is RET-associated cancer. For example, the proposed method may include: (a) detecting dysregulation of the RET gene, RET kinase, or expression, or activity, or the level of any of them in a sample obtained from a subject; and (b) administering to said subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation (e.g., MET deregulation, such as amplification MET gene); and (d) administering to said subject a second therapeutic agent, wherein the second therapeutic agent is crizotinib, either alone or in combination with a compound of Formula I-IV or a compound thereof.

- 132 041220 pharmaceutically acceptable salt, amorphous or polymorphic form, if the specified subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some such embodiments, the proposed method includes (a) the detection of one or more fusion proteins from table. 1 and/or one or more point mutations/inserts in the RET kinase protein from Table. 2 in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In further embodiments, the provided methods further comprise (after (b)) (c) determining whether a cancer cell in a sample obtained from a subject has at least one RET inhibitor resistance mutation (e.g., MET deregulation, such as amplification MET gene); and (d) administering to said subject a second therapeutic agent, the second therapeutic agent being crizotinib, either alone or in combination with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (e) administering to the subject additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (b) if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation.

In some embodiments, the lung cancer is an EGFR-associated cancer. For example, the proposed method may include: (a) detecting deregulation of the EGFR gene, EGFR kinase, or expression, or activity, or level of any of them in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of an EGFR inhibitor (eg, osimertinib). In some embodiments, the provided methods further comprise (after (b)) (c) determining if a cancer cell in a sample obtained from a subject has at least one dysregulation of the RET gene, RET kinase, or expression, or activity, or level any of them (eg, RET fusion protein); and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with an EGFR inhibitor (e.g., osimertinib), if the subject has a cancer cell that has at least one deregulation of the RET gene, RET kinase, or expression or activity or level of any of them (eg, RET fusion protein); or (e) administering to said subject additional doses of an EGFR inhibitor (e.g., osimertinib) from step (b) if the subject has a cancer cell that does not have a RET gene, RET kinase, or expression or activity or level of any of them downregulated. (eg RET fusion protein). In some such embodiments, the proposed method includes (a) detecting dysregulation of the EGFR gene, EGFR kinase, or expression or activity or level of any of them in a sample obtained from a subject; and (b) administering to the subject a therapeutically effective amount of osimertinib. In additional embodiments, the proposed method further comprises (after (b)) (c) determining if the cancer cell in the sample obtained from the subject has one or more of the fusion proteins from Table. 1 and/or one or more point mutations/inserts in the RET kinase protein from Table. 2; and (d) administering to said subject a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with osimertinib if the subject has a cancer cell that contains one or more of the fusion proteins from Table 1. 1 and/or one or more point mutations/inserts in the RET kinase protein from Table. 2; or (e) administering additional doses of osimertinib from step (b) to said subject if the subject has a cancer cell that does not contain one or more of the fusion proteins of Table 1. 1 and/or one or more point mutations/inserts in the RET kinase protein from Table. 2.

The term EGFR-associated cancer as used herein refers to cancers associated with or characterized by dysregulation of the EGFR gene, EGFR kinase, or expression, or activity, or level of any of them.

Expression of EGFR dysregulation, EGFR kinase, or expression or activity or level of any of them refers to a genetic mutation (e.g., translocation of the EGFR gene that results in the expression of a fusion protein, a deletion in the EGFR gene that results in the expression of an EGFR protein that contains a deletion of at least one amino acid, compared to the wild-type EGFR protein, or a mutation in the EGFR gene that results in the expression of an EGFR protein with one or more point mutations, or an alternative splice version of EGFR mRNA that produces an EGFR protein, resulting in a deletion of at least one amino acid in the EGFR protein compared to the wild-type EGFR protein) or to amplification of the EGFR gene that results in overexpression of the EGFR protein or autocrine activity due to overexpression of the EGFR gene in the cell, resulting in pathogenic upregulation of the kinase domain of the EGFR protein (e.g. , the constitutively active kinase domain of the EGFR protein) in the cell. As another example, dysregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of these may be a mutation in the EGFR gene that encodes for an EGFR protein that is constitutively active or has an increased activity.

- 133 041220 compared to a protein encoded by the EGFR gene that does not contain such a mutation. For example, dysregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of them may be the result of a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of EGFR that contains a functional kinase domain and a second portion of the EGFR protein. partner (i.e. non-EGFR). In some examples, dysregulation of an EGFR gene, EGFR protein, or expression or activity may result from a gene translocation of one EGFR gene with another non-EGFR gene. In some embodiments, the EGFR mutation is a T790M mutation. In some embodiments, the EGFR mutation is a C797S mutation.

The term wild-type EGFR describes a nucleic acid (e.g., EGFR gene or EGFR mRNA) or protein (e.g., EGFR protein) that occurs in a subject not suffering from EGFR-associated cancer (and optionally also not at increased risk of developing EGFR-associated cancer and/or not predisposed to EGFR-associated cancer), or which occur in a cell or tissue derived from a subject not suffering from EGFR-associated cancer (and optionally also not at increased risk of developing EGFR-associated cancer and/or not predisposed to EGFR-associated cancer ).

Also provided herein are methods for selecting treatment for a subject suffering from cancer, which include: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and the choice of treatment, which includes the introduction of the compounds of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered in combination with a first RET inhibitor. Also provided herein are methods of selecting a treatment for a subject suffering from cancer, which include: selecting a treatment that comprises administering a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, in an amorphous or polymorphic form, to a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations. Also provided are methods for selecting a subject suffering from cancer for treatment that does not include a first RET inhibitor as monotherapy, which includes: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and selecting an identified subject for treatment, which includes a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Also provided are methods of selecting a subject suffering from cancer for treatment that does not include a first RET inhibitor as monotherapy, which includes: selecting a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations for treatment, which comprises administering a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. In some embodiments, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

Also provided are methods for determining the likelihood that a subject suffering from a cancer (e.g., RET-associated cancer) will demonstrate a positive response to treatment with a first RET inhibitor as monotherapy, which includes: determining whether a cancer cell in a sample obtained from subject, one or more RET inhibitor resistance mutations; and determining that a subject having a cancer cell having one or more RET inhibitor resistance mutations has a reduced chance of responding positively (i.e., an increased chance of not responding) to treatment with the first RET inhibitor as monotherapy. Also provided are methods for determining the likelihood that a subject suffering from a cancer (e.g., RET-associated cancer) will demonstrate a positive response to treatment with a first RET inhibitor as monotherapy, which includes: determining whether a cancer cell in a sample obtained from subject, one or more RET inhibitor resistance mutations; and determining that a subject lacking a cancer cell that contains one or more RET inhibitor resistance mutations has an increased likelihood of responding positively to treatment with the first RET inhibitor as monotherapy compared to a subject having a cancer cell having one or more RET inhibitor resistance mutations. Also provided are methods for predicting the effectiveness of treatment with a first RET inhibitor as monotherapy in a subject suffering from cancer, which includes: determining whether a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and determining that treatment with the first RET inhibitor as monotherapy is less likely to be effective in a subject having a cancer cell in a sample obtained from a subject that contains one or more RET inhibitor resistance mutations. Methods for predicting the effects are also proposed.

- 134 041220 the effectiveness of treatment with the first RET inhibitor as monotherapy in a subject suffering from cancer, which include: determining that treatment with the first RET inhibitor as monotherapy is less likely to be effective in a subject having a cancer cell in a sample obtained from a subject that contains one or more RET inhibitor resistance mutations. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

Also provided are methods of treating a subject suffering from cancer, which include: (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a) determining whether the cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (c) administering to a subject a compound of Formula IIV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (d) administering additional doses of the first RET inhibitor from step (a) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation. In some embodiments, if additional doses of the first RET inhibitor from step (a) are administered to the subject, then the subject may also be administered another anti-cancer agent (e.g., a second RET inhibitor or a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or immunotherapy). In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (c), the other RET inhibitor may be the first RET inhibitor administered in step (a). In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations can include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

Also provided are methods of treating a subject suffering from cancer, which include: (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a) determining whether the cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (c) administering to the subject a second RET inhibitor, alone or in combination with another anticancer agent, if said subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (d) administering additional doses of the first RET inhibitor from step (a) to the subject if said subject has a cancer cell that does not have the RET inhibitor resistance mutation. In some embodiments, if additional doses of the first RET inhibitor from step (a) are administered to the subject, then another anticancer agent may also be administered to the subject. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof). In some embodiments, the additional anti-cancer agent is an immunotherapy.

Also provided are methods of treating a subject suffering from cancer (e.g., RET-associated cancer), which include: (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously treated with one or more doses of a first RET inhibitor, one or more RET inhibitor resistance mutations; and (b) administering to the subject a compound

- 135 041220 formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form as monotherapy or in combination with another anticancer agent, if the specified subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (c) administering additional doses of the previously administered first RET inhibitor to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation. In some embodiments, if the subject is administered additional doses of the first RET inhibitor previously administered to the subject, then another anti-cancer agent (e.g., a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form, or immunotherapy) may also be administered to the subject. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (b), the other anticancer agent may be the first RET inhibitor administered in step (a).

Also provided are methods of treating a subject suffering from cancer, which include: (a) determining whether a cancer cell in a sample obtained from a subject suffering from cancer and previously treated with one or more doses of a first RET inhibitor has one or more resistance mutations to an RET inhibitor; and (b) administering to the subject a second RET inhibitor, alone or in combination with another anticancer agent, if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (c) administering to the subject additional doses of the first RET inhibitor previously administered to that subject if the subject has a cancer cell that does not have the RET inhibitor resistance mutation. In some embodiments, if the subject is administered additional doses of the first RET inhibitor previously administered to the subject, then another anticancer agent may also be administered to the subject. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (b), the other anticancer agent may be the first RET inhibitor administered in step (a).

In some embodiments, the RET-associated cancer described herein may occur in a subject along with dysregulation of another gene, another protein, or the expression or activity or level of any of them.

For example, a RET-associated cancer that exhibits RET fusion may occur in a subject along with one or more of: dysregulation of the MET gene, MET protein, or expression or activity or level of any of these; deregulation of the PIK3CA gene, PIK3CA protein, or expression or activity or level of any of them; deregulation of the KRAS gene, KRAS protein, or expression or activity or level of any of them; deregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of them (eg, amplification of the EGFR gene); deregulation of the FGFR2 gene, FGFR2 protein, or expression or activity or level of any of them (eg, FGFR2 gene or FGFR2 protein fusion); deregulation of the CDK4 gene, CDK4 protein, or expression or activity or level of any of them (eg, amplification of the CDK4 gene); deregulation of the mTOR gene, mTOR protein, or expression or activity or level of any of them; deregulation of the CDKN2A gene, CDKN2A protein, or expression or activity or level of any of them (eg, deletions in the CDKN2A gene or CDKN2A protein); deregulation of the CDKN2B gene, CDKN2B protein, or expression or activity or level of any of them (eg, deletions in the CDKN2B gene or CDKN2B protein); deregulation of the NF1 gene, NF1 protein, or expression or activity or level of any of them; deregulation of the MYC gene, MYC protein, or expression or activity or level of any of them (eg, amplification in the MYC gene); - 136 041220 regulation of the MDM2 gene, MDM2 protein, or expression, or activity, or level of any of them (eg, amplification in the MDM2 gene); deregulation of the GNAS gene, GNAS protein, or expression or activity or level of any of them; deregulation of the BRCA2 gene, BRCA2 protein, or expression or activity or level of any of them.

In some embodiments, a RET-associated cancer that exhibits a mutation of the RET gene and/or RET protein may occur in a subject along with one or more of: dysregulation of the PIK3CA gene, PIK3CA protein, or the expression or activity or level of any of them; deregulation of the KRAS gene, KRAS protein, or expression or activity or level of any of them; deregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of them; dysregulation of the FGFR1 gene, FGFR1 protein, or expression, or activity, or level of any of them (eg, amplification of the FGFR1 gene; dysregulation of the FGFR2 gene, FGFR2 protein, or expression, or activity, or level of any of them (eg, amplification of the FGFR2 gene) ; downregulation of the FGFR3 gene, FGFR3 protein, or expression, or activity, or level of any of them (e.g., fusion of the FGFR3 gene or FGFR3 protein); downregulation of the ERBB2 gene, ERBB2 protein, or expression, or activity, or level of any of them (e.g. , ERBB2 gene amplification); and dysregulation of the KIT gene, KIT protein, or expression or activity or level of any of them.

In some embodiments, a RET-associated cancer that exhibits RET gene amplification may occur in a patient along with one or more additional kinase amplifications. For example, with amplification in the FGFR1 gene; with amplification in the FGFR2 gene; with amplification in the FGFR3 gene; with amplification in the FGFR4 gene; with amplification in the CDK4 gene; and with amplification in the CDK6 gene.

In some embodiments, if the RET-associated cancer described herein may occur in a subject along with dysregulation in another kinase, then the methods described herein may further comprise administering an additional therapeutic agent that targets and/or provides treatment deregulation in another kinase. For example, provided herein are methods for treating RET-associated cancer in a subject in need of such treatment, comprising (a) detecting dysregulation of the RET gene, RET kinase, or expression or activity or level of any of them in a sample obtained from the subject; and (b) administering to the subject a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the proposed method further comprises (c) detecting a dysregulation in another kinase in a sample obtained from a subject; and (d) administering to the subject a therapeutic agent that targets and/or provides treatment for a dysregulation in another kinase. In some embodiments, the administration of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered simultaneously, sequentially, or serially. In some embodiments, detection steps (a) and (c) may be performed simultaneously or sequentially in any order.

Additional therapeutic agents that target and/or provide treatment for dysregulation of another kinase may include any known inhibitor of the other kinase. Examples of such agents are presented below:

Exemplary PARP inhibitors include: 3-aminobenzamide (INO-1001), 5-aminoisoquinoline, ABT472, ABT767, AG140361, AG14032, ANG2864, ANG3186, AZD2281, AZD2461, BGP-15, BSI101, BSI122, CEP6800 prodrug CEP8983), CPH101 with CPH102, DR2313, E7016 (GPI-21016), E7449, GP16150, IMP4297, IMP04149, INO1002, INO1003, JPI283, JPI289, KU0687, KU58948, niraparibola (MK-4827), NT-4825 , ONO-1924H, ONO2231, pamiparib (BGB290), PJ-34, rucaparib (AG014699), SC10914, SOMCL9112, talazoparib (BMN-673), and veliparib (ABT888).

Exemplary CDK 4/6 inhibitors include: palbociclib (PD0332991), abemaciclib (LY2835219), ribociclib (LEE011), trilaciclib (G1T28), voruciclib, and G1T38.

Exemplary ERBB2 (HER2/neu) inhibitors include: afatinib, afatinib, dacomitinib (PF-00299804), DS8201-a, erlotinib, gefitinib, KU004, lapatinib, lapatinib ditosylate, MM-111, mubritinib (TAK-165), neratinib, pyrotinib (HTI-1001), tucatinib (ONT-380, ARRY-380), 7C3, cetuximab, HER2-BsAb, gersintuzumab, margetuximab, MI130004, NeuVax, paitumumab, pertuzumab, SYD985, trastuzumab, and trastuzumab emtansine.

Illustrative inhibitors of amplified ERBB2 (HER2/neu) include dacomitinib (PF-00299804), lapatinib, neratinib, pertuzumab, trastuzumab, and trastuzumab emtansine.

Exemplary EGFR inhibitors include: AC0010, afatinib, AP26113, ASP8273, avatinib, avitinib, AZD3759, BMS-690514, brigatinib, canertinib, Cap-701, CHMFL-EGFR-202, CUDC-101, dacomitinib, EAI045, EGF816, erlotinib, erlotinib, , gefitinib, GNS-1481, GNS-1486, G66976, HS10296, icotinib, KU004, lapatinib, nazartinib, neratinib, olmutinib (HM61713, BI 1482694), osimertinib, osimertinib (AZD9291), pelitinib, PF-06747772, PKC4pyropitinib HTI-1001), rocilentinib, vandetanib, varlitinib, XL647, 7C3, cetuximab, depatuxizumab mafodotin (AVT-414), matuzumab, nimotuzumab, panitumumab, and zalutumumab.

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Exemplary wild-type EGFR inhibitors include: afatinib, BMS-690514, canertinib,

CUDC-101, dacomitinib, erlotinib, gefitinib, lapatinib, neratinib, pelitinib, vandetanib, varlitinib, XL647, cetuximab, matuzumab, nimotuzumab, panitumumab, and zalutumumab.

Exemplary inhibitors of mutated EGFR include: AC0010, afatinib, AP26113, ASP8273, avatinib, avitinib, AZD3759, BMS-690514, brigatinib, canertinib, Cap-701, CHMFL-EGFR202, CUDC-101, dacomitinib, EAI045, ENS-148 GNS-1486, G66976, HS-10296, icotinib, nazartinib, neratinib, olmutinib (HM61713, BI 1482694), osimertinib (AZD9291), PF-06747775, PKC412, rocilentinib, vandetanib, varlitinib and cetuximab.

An exemplary inhibitor of amplified EGFR is depatuxizumab mafodotin (AVT-414).

Illustrative FGFR inhibitors include: ASP5878, AZD4547, BGJ398, BLU9931, brivatinib, cediranib, DEBIO 1347, derazantinib (ARQ-087), dovitinib (CHIR258), E7090, ENMD-2076, erdafitinib (JNJ-42756293), FGFIN-401 1, FRIN-1, INCB054828, L16H50, lenvatinib, lucitanib, LY2874455, nintedanib, NP603, orantinib (SU6668), pazopanib, PBI05204, PD173074, ponatinib, PRN1371, regorafenib, garartinib (BAY-1163876), S85,3077 SU5402, sunitinib, TAS-120, FP-1039, GAL-F2, GAL-FR21, GAL-FR22, GAL-FR23, GP369, hLD1.vb, LD1, MFGR1877S, MM-161, PRO001 and R3Mab.

Exemplary FGFR fusion inhibitors include: BGJ398, DEBIO 1347, derazantinib (ARQ087), E7090, erdafitinib (JNJ-42756293), lucitanib, and TAS-120.

Illustrative inhibitors of FGFR1, FGFR2 and FGFR3 include: AZD4547, BGJ398, DEBIO 1347, E7090, INCB054828, S49076, SOMCL-085 and TAS-120.

Exemplary FGF4 inhibitors include: BLU-554, BLU9931, NVP-FGF401 and hLD1.vb.

Illustrative inhibitors of amplified FGFR1 include: AZD4547, BGJ398, DEBIO 1347, derazantinib (ARQ-087), erdafitinib (JNJ-42756293), INCB054828, and lucitanib.

Illustrative inhibitors of amplified FGFR2 include: AZD4547, DEBIO 1347, derazantinib (ARQ-087), lucitanib, regorafenib, and TAS-120.

An exemplary inhibitor of amplified FGFR3 is AZD4547.

Exemplary MEK inhibitors include: AZD8330 (ARRY-424704), AZD6244 (ARRY142866), BI-847325, binimetinib, BIX02188, BIX02189, CH4987655, CH5126766, CI-1040, cobemetinib (GDC-0973), EBI-573 G8935, GDC-0623, Myricetin, Nobiletin, PD0325901, PD184161, PD318088, PD98059, PD334581, Pimasertib (AS-703026), Refametinib (RDEA119, BAY 869766), Selumentinib (AZD6244), TAK-327, and Tramethinib U0126.

Exemplary KRAS inhibitors include: 0375-0604, Switch Pocket II Covalent Quinazoline Compound (SUP), ARS-1620, AZD4785, and LP1.

Exemplary PI3K inhibitors include: 3-methyladenine, A66, alpelisib (BYL719), AMG319, apitolisib (GDC-0980, RG7422), AS-252424, AS-604850, AS-605240, AZD6842, AZD8186, AZD8835, BGT226 (2VP-BGT226 ), Buparlisib (VKM120), CAY10505, CH5132799, Copanlisib (BAY 80-6946), CUDC-907, CZC24832, Dactolisib (BEZ235, NVP-BEZ235), DS7423, Duvelisib (IPI-145, INK1197), GDC-0032, GDC -0084, GDC-0326, Gedatolisib (PF-05212384, PKI-5587), GNE-317, GS-9820, GSK1059615, GSK2292767, GSK2636771, HS-173, IC-87114, Idelalisib (CAL-101, GS-1101) , IPI-145, IPI-3063, IPI-549, LY294002, LY3023414, nemiralisib (GSK2269557), omipalisib (GSK2126458, GSK458), PF-04691502, PF-4989216, PI-103, PI-3065, pictilisib (GDC-0941 ), PIK-293, PIK-294, PIK-75, PIK-90, PIK-93, PIK-III, Pilaralisib (XL147), PKI-587, PP-110, PQR309, PQR309, PW-12, PX-866 , Quercetin, S14161, SAR245409 (XL765), SAR260301, SAR405, Serabelisib (INK-1117, MLN-1117, TAK1117), SF-1126, SF-2523, SN32976, Taselisib (GDC-0032), TV101110, TG100-115, TG100-7 13, TGR-1202, TGX-221, umbralisib (TGR-1202), voxtalisib (XL765, SAR245409), VPS34-IN1, VS-5584 (SB2343), WJD008, wortmannin and ZSTK474.

Exemplary KIT inhibitors include: AMG 706, amuvatinib (MP-470), APcK110, axitinib (AG-013736), AZD2932, dasatinib (BMS-354825), dovitinib (TKI-258, CHIR-258), EXEL-0862, imatinib, KI-328, masitinib (AB1010), midostaurin, MLN518, motesanib, N3-(6-aminopyridin-3-yl)-N1-(2-cyclopentylethyl)-4-methylisophthalamide, nilotinib, OSI-930, pazopanib (GW786034), pexidartinib ( PLX3397), PKC412, PLX647, PP1, quisartinib (AC220), regorafenib (BAY 73-4506), semaxinib (SU 5416), sitravatinib (MGCD516), sorafenib, STI571, SU11248, SU9529, sunitinib, telatinib, tivozanib (AV-951 ), tyrphostin AG 1296, VX-322 and WBZ_4.

Exemplary MDM2 inhibitors include: (-)-parthenolide, ALRN6924, AM-8553, AMG232, CGM-097, DS-3032b, GEM240, HDM201, HLI98, idasanutlin (RG-7338), JapA, MI-219, MI-219, MI-319, MI-77301 (SAR405838), MK4828, MK-8242, MX69, NSC 207895 (XI-006), Nutlin-3, Nutlin-3a, Nutlin3b, NVP-CFC218, NVP-CGM097, PXn727/822, RG7112 , RO2468, RO5353, RO5503781, Serdemethane (JNJ26854165), SP-141 and YH239-EE.

Illustrative inhibitors of amplified MDM2 include: AM-8553, AMG232, DS3032b, MI-77301 (SAR405838), NSC 207895 (XI-006), Nutlin-3a, NVP-CFC218, NVP-CGM097 and RG7112.

Exemplary MET inhibitors include: (-)-oleocanthal, ABBV-399, AMG-208, AMG-337,

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AMG-458, BAY-853474, BMS-754807, BMS-777607, BMS-794833, cabozantinib (XL184, BMS-907351), capmatinib (INCB28060), crizotinib (PF-02341066), DE605, foretinib (GSK1363089, XL880), Glezatinib (MGCD265), Golvatinib (E7050), INCB028060, JNJ-38877605, KRC-408, Merestinib (LY2801653), MK-2461, MK8033, NPS-1034, NVP-BVU972, PF-04217903, PHA-665752, S49076, Savolitinib (AZD6094, HMPL-504), SGX-523, SU11274, TAS-115, tepotinib (EMD 1214063, MSC2156119J), volitinib, CE355621, and onartuzumab.

Exemplary mTOR inhibitors include: anthracymycin, apitolisib (GDC-0980, RG7422), AZD-8055, BGT226 (NVP-BGT226), CC-223, CZ415, dactolisib (BEZ235, NVP-BEZ235), DS7423, everolimus (RAD001), GDC -0084, GDC-0349, Gedatolisib (PF-05212384, PKI-5587), GSK1059615, INK128, KU-0063794, LY3023414, MLN0128, Omipalisib (GSK2126458, GSK458), OSI-027, OSU-53, Palomide 529 , PF-04691502, PI-103, PKI-587, PP242, PQR309, ridafarolimus (AP-23573), sapanisertib (INK 128, MLN0128), SAR245409 (XL765), SF-1126, SF2523, sirolimus (rapamycin), SN32976, TAK228, temsirolimus (CCI-779, NSC 683864), thorin 1, torin 2, torkinib (PP242), mirolimus, vistusertib (AZD2014), voxtalisib (XL765, SAR245409), VS-5584, VS-5584 (SB2343), WAY- 600, WYE-125132 (WYE-132), WYE-354, WYE-687, XL388 and zotarolimus (AWT-578).

Exemplary MYC inhibitors include: 10058-F4, 10074-G5 and KSI-3716.

The expression dysregulation of a gene, protein, or the expression or activity or level of any of these refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing a kinase domain and a fusion partner, a mutation in a gene that results in the expression of a protein which contains a deletion of at least one amino acid relative to the wild-type protein, a mutation in a gene that results in the expression of a protein with one or more point mutations relative to the wild-type protein, a mutation in a gene that results in the expression of a protein with at least at least one inserted amino acid compared to the wild-type protein, a gene duplication that results in an increased level of the protein in the cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of the protein in the cell), alternative splice a version of an mRNA that results in a protein having at least one amino acid deletion in the protein compared to the wild-type protein), or increased expression (e.g., increased levels) of the wild-type protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, the dysregulation of a gene, protein, or the expression or activity or level of any of them may be a mutation in a gene that encodes a protein that is constitutively active or has increased activity compared to a protein encoded by a gene that does not contain such mutation. For example, dysregulation of a gene, protein, or the expression or activity or level of any of these may result from a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the protein that contains a functional kinase domain and a second portion of a partner protein ( i.e. which is not a primary protein). In some examples, dysregulation of a gene, protein, or expression, or activity, or level of any of them may be the result of a gene translocation of one gene with another gene.

Treatment of a patient suffering from cancer with a multikinase inhibitor (MKI) or a target-specific kinase inhibitor (eg, BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, or RAS inhibitor) can lead to dysregulation of the RET gene, RET kinase, or their expression or activity or level in a given cancer and/or resistance to a RET inhibitor. See, for example, Bhinge et al., Oncotarget 8:27155-27165, 2017; Chang et al., Yonsei Med. J. 58:9-18, 2017; and Lopez-Delisle et al., doi: 10.1038/s41388-017-0039-5, Oncogene 2018.

Treatment of a cancer patient with a RET inhibitor in combination with a multikinase inhibitor or a target-specific kinase inhibitor (eg, BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, or RAS inhibitor) may provide increased therapeutic efficacy compared to treating the same patient or the same patient with a RET inhibitor as monotherapy, or with a multi-kincase inhibitor or target-specific kinase inhibitor as monotherapy. See, for example, Tang et al., doi: 10.1038/modpathol.2017.109, Mod. Pathol. 2017; Andreucci et al., Oncotarget 7:80543-80553, 2017; Nelson-Taylor et al., Mol. Cancer Ther. 16:1623-1633, 2017; and Kato et al., Clin. Cancer Res. 23:1988-1997, 2017.

Provided herein are methods of treating a patient suffering from cancer (e.g., any cancer described herein) who has previously received a multikinase inhibitor (MKI) or a target-specific kinase inhibitor (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, or RAS inhibitor) (e.g., as monotherapy) which comprises: administering to a patient (i) a therapeutically effective dose of a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, whether amorphous or polymorphic form as monotherapy, or (ii) a therapeutically effective dose

- 139 041220 compounds of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form and a therapeutically effective dose of previously administered MKI or previously administered target-specific kinase inhibitor.

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) who has previously received an MKI or a target-specific kinase inhibitor (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, ALK inhibitor, ROS1 inhibitor). , MET inhibitor, aromatase inhibitor, RAF inhibitor, or RAS inhibitor) (for example, as monotherapy), which include: identifying a patient having a cancer cell that contains a deregulation of the RET gene, RET kinase, or expression, or activity, or level of any of them; and administering to the identified patient (i) a therapeutically effective dose of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy, or (ii) a therapeutically effective dose of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof form and therapeutically effective dose of the previously administered MKI or previously administered target-specific kinase inhibitor.

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) which comprises: administering to the patient a therapeutically effective amount of an MKI or a target-specific kinase inhibitor (e.g., BRAF inhibitor, EGFR inhibitor, MEK inhibitor, an ALK inhibitor, ROS1 inhibitor, MET inhibitor, aromatase inhibitor, RAF inhibitor, or RAS inhibitor) (eg, as monotherapy) for a first time period; after a specified period of time - identifying a patient having a cancer cell that has a deregulation of the RET gene, RET kinase, or their expression, or activity, or level; and administering to the identified patient (i) a therapeutically effective dose of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy, or (ii) a therapeutically effective dose of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof and a therapeutically effective dose of the previously administered MKI or previously administered target-specific kinase inhibitor.

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has a BRAF gene, BRAF kinase, or expression, or activity, or level of dysregulation, which comprises administering to the patient (i) a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of a BRAF inhibitor (eg, any BRAF inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that includes: identifying a patient having a cancer cell that has a BRAF gene, BRAF kinase, or expression or activity dysregulation, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a BRAF inhibitor (e.g., any BRAF inhibitor described herein or known in the art). technology).

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has dysregulation of the EGFR gene, EGFR protein, or their expression, or activity, or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of an EGFR inhibitor (eg, any EGFR inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that includes: identifying a patient having a cancer cell that is downregulated in the EGFR gene, EGFR protein, or their expression or activity, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of an EGFR inhibitor (e.g., any EGFR inhibitor described herein or known in the art). technology).

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has dysregulation of the MEK gene, MEK protein, or their expression, or activity, or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of a MEK inhibitor (eg, any MEK inhibitor described herein or known in the art).

Provided herein are methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that includes: identifying a patient having a cancer cell that has a downregulation of the MEK gene, MEK protein, or their expression, or activity, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a MEK inhibitor (e.g., any MEK inhibitor described herein or known in the art). technology).

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has dysregulation of the ALK gene, ALK protein, or their expression, or activity, or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of an ALK inhibitor (eg, any ALK inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that includes: identifying a patient having a cancer cell that is downregulated in the ALK gene, ALK protein, or their expression or activity, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of an ALK inhibitor (e.g., any ALK inhibitor described herein or known in the art). technology).

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has dysregulation of the ROS gene, ROS protein, or their expression, or activity, or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of an inhibitor of ROS (eg, any inhibitor of ROS described herein or known in the art).

Provided herein are methods of treating a patient suffering from a cancer (for example, any cancer described herein), which includes: identifying a patient having a cancer cell that has a ROS gene, ROS protein, or expression or activity dysregulation, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a ROS inhibitor (e.g., any ROS inhibitor described herein or known in the art). technology).

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has a dysregulation of the MET gene, MET protein, or their expression, or activity, or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of a MET inhibitor (eg, any MET inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that includes: identifying a patient having a cancer cell that is downregulated in the MET gene, MET protein, or their expression or activity, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a MET inhibitor (e.g., any MET inhibitor described herein or known in the art). technology).

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has a dysregulation of an aromatase gene, an aromatase protein, or their expression, or activity, or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of an aromatase inhibitor (eg, any aromatase inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from a cancer (e.g., any cancer described herein) that includes: identifying a patient having a cancer cell that has a dysregulation of an aromatase gene, an aromatase protein, or their expression or activity, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of an aromatase inhibitor (e.g., any aromatase inhibitor described herein or known in the art). technology).

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The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has dysregulation of the RAF gene, RAF protein, or their expression, or activity, or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of a RAF inhibitor (eg, any RAF inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from a cancer (for example, any cancer described herein), which includes: identifying a patient having a cancer cell that has a RAF gene, RAF protein, or expression or activity dysregulation, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a RAF inhibitor (e.g., any RAF inhibitor described herein or known in the art). technology).

The present invention provides methods for treating a patient suffering from a cancer (e.g., any cancer described herein) that has a dysregulation of the RAS gene, RAS protein, or their expression, or activity, or level, which comprises administering to the patient (i) a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof; and (ii) a therapeutically effective amount of a RAS inhibitor (eg, any RAS inhibitor described herein or known in the art).

Provided herein are methods of treating a patient suffering from a cancer (for example, any cancer described herein), which includes: identifying a patient having a cancer cell that has a RAS gene, RAS protein, or expression or activity dysregulation, or level; and administering to the identified patient (i) a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and (ii) a therapeutically effective amount of a RAS inhibitor (e.g., any RAS inhibitor described herein or known in the art). technology).

Expression of the dysregulation of a BRAF gene, a BRAF protein, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing a BRAF kinase domain and a fusion partner, a mutation in the BRAF gene that results in the expression of a BRAF protein that contains a deletion of at least one amino acid compared to the wild-type BRAF protein, a mutation in the BRAF gene that results in the expression of a BRAF protein with one or more point mutations compared to the wild-type BRAF protein, mutations in the gene BRAF that results in the expression of a BRAF protein with at least one inserted amino acid compared to a wild-type BRAF protein, a gene duplication that results in an increased level of the BRAF protein in the cell, or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) , which leads to an increased level of the BRAF protein in the cell), an alternative splice version of the BRAF mRNA, which leading to generation of a BRAF protein having at least one amino acid deletion in the BRAF protein compared to the wild-type BRAF protein), or increased expression (e.g., elevated levels) of the wild-type BRAF protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation autocrine/paracrine signaling (eg, compared to a control non-cancerous cell). As another example, dysregulation of the BRAF gene, BRAF protein, or expression or activity or level of any of them may be a mutation in the BRAF gene that encodes a BRAF protein that is constitutively active or has increased activity compared to the protein encoded by the gene. BRAF that does not contain this mutation. For example, dysregulation of a BRAF gene, a BRAF protein, or the expression or activity or level of any of them may result from a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the BRAF protein that contains a functional kinase domain and a second portion of the protein -partner (i.e. who is not a BRAF). In some examples, dysregulation of a BRAF gene, a BRAF protein, or the expression or activity or level of any of these may be the result of a gene translocation of one BRAF gene with another non-BRAF gene.

Non-limiting examples of a BRAF inhibitor include dabrafenib, vemurafenib (also referred to as RG7204 or PLX4032), sorafenib tosylate, PLX-4720, GDC-0879, BMS-908662 (Bristol-Meyers Squibb), LGX818 (Novartis), PLX3603 (Hofmann-LaRoche), RAF265 (Novartis), RO5185426 (HofmannLaRoche) and GSK2118436 (GlaxoSmithKline). Additional examples of a BRAF inhibitor are known in the art.

Expression of dysregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing an EGFR kinase domain and a fusion partner, a mutation in the EGFR gene, which results in the expression of an EGFR protein that contains a deletion of at least

- 142 041220 one amino acid compared to the wild-type EGFR protein, a mutation in the EGFR gene that results in the expression of the EGFR protein with one or more point mutations compared to the wild-type EGFR protein, a mutation in the EGFR gene that results in the expression of the EGFR protein with at least one inserted amino acid compared to the wild-type EGFR protein, a gene duplication that results in an increased level of EGFR protein in the cell, or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) that results in an increased level of EGFR protein in the cell), an alternative splice version of EGFR mRNA that results in an EGFR protein having at least one amino acid deletion in the EGFR protein compared to the wild-type EGFR protein), or increased expression (e.g., increased levels) of wild-type EGFR protein in mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, dysregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of them may be a mutation in the EGFR gene that encodes an EGFR protein that is constitutively active or has increased activity relative to the protein encoded by the gene. EGFR that does not contain this mutation. For example, dysregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of them may result from a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the EGFR protein that contains a functional kinase domain and a second portion of the protein. -partner (i.e. non-EGFR). In some examples, dysregulation of the EGFR gene, EGFR protein, or expression or activity or level of any of these may result from a gene translocation of one EGFR gene with another non-EGFR gene.

Non-limiting examples of an EGFR inhibitor include gefitinib, erlotinib, brigatinib, lapatinib, neratinib, icotinib, afatinib, dacomitinib, posiotinib, vandetanib, afatinib, AZD9291, CO-1686, HM61713, AP26113, CI-1033, PKI-166, GWKB -569, PDI-168393, AG-1478, CGP-59326A. Additional examples of an EGFR inhibitor are known in the art.

The expression of a dysregulation of a MEK gene, a MEK protein, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing a MEK kinase domain and a fusion partner, a mutation in the MEK gene that results in the expression of a MEK protein that contains a deletion of at least one amino acid compared to the wild-type MEK protein, a mutation in the MEK gene that results in the expression of a MEK protein with one or more point mutations compared to the wild-type MEK protein, mutations in the gene MEK that results in the expression of a MEK protein with at least one inserted amino acid compared to the wild-type MEK protein, a gene duplication that results in an increased level of the MEK protein in the cell, or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) , which leads to an increased level of the MEK protein in the cell), an alternative splice version of the MEK mRNA, which leads to the formation a MEK protein having a deletion of at least one amino acid in the MEK protein compared to the wild-type MEK protein), or overexpression (e.g., elevated levels) of the wild-type MEK protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/ paracrine signaling (eg, compared to a control non-cancerous cell). As another example, dysregulation of the MEK gene, MEK protein, or expression or activity or level of any of these may be a mutation in the MEK gene that encodes a MEK protein that is constitutively active or has increased activity relative to the protein encoded by the gene. MEK that does not contain such a mutation. For example, dysregulation of the MEK gene, MEK protein, or the expression or activity or level of any of them may result from a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the MEK protein that contains a functional kinase domain and a second portion of the protein -partner (i.e. not MEK). In some examples, dysregulation of the MEK gene, MEK protein, or expression or activity or level of any of these may result from a gene translocation of one MEK gene with another non-MEK gene.

Non-limiting examples of a MEK inhibitor include mekinist, trametinib (GSK1120212), cobimetinib (XL518), binimetinib (MEK162), selumetinib, PD-325901, CI-1040, PD035901, TAK-733, PD098059, U0126, AS703026/MSC1935 GDC-0973, BAY869766/RDEA119 and GSK1120212. Additional examples of a MEK inhibitor are known in the art.

Expression of the dysregulation of an ALK gene, an ALK protein, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing an ALK kinase domain and a fusion partner, a mutation in the ALK gene, which results in the expression of an ALK protein that contains a deletion of at least one amino acid compared to the wild-type ALK protein, a mutation in the ALK gene that results in the expression of an ALK protein with one or more point mutations compared to the wild-type ALK protein, a mutation in an ALK gene that results in the expression of an ALK protein with at least one inserted amino acid compared to a wild-type ALK protein, a gene duplication that results in an increased level of the ALK protein in the cell, or a mutation in a regulatory sequence (e.g.,

- 143 041220 promoter and/or enhancer), which leads to an increased level of the ALK protein in the cell), an alternative splice version of the ALK mRNA, which leads to the formation of the ALK protein, having a deletion of at least one amino acid in the ALK protein compared to the ALK protein wild-type), or increased expression (eg, elevated levels) of wild-type ALK protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/paracrine signaling (eg, compared to a control non-cancerous cell). As another example, dysregulation of the ALK gene, ALK protein, or expression or activity or level of any of these may be a mutation in the ALK gene, which encodes an ALK protein that is constitutively active or has increased activity relative to the protein encoded by the gene. ALK that does not contain such a mutation. For example, dysregulation of the ALK gene, ALK protein, or expression or activity or level of any of them may result from a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the ALK protein that contains a functional kinase domain and a second portion of the protein -partner (i.e. not ALK). In some examples, dysregulation of the ALK gene, ALK protein, or expression or activity or level of any of these may result from a gene translocation of one ALK gene with another non-ALK gene.

Non-limiting examples of an ALK inhibitor include crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa), dalantercept, ACE-041 (brigatinib) (AP26113), entrectinib (NMSE628), PF-06463922 (Pfizer), TSR-011 (Tesaro) , CEP-37440 (Teva), CEP-37440 (Teva), X-396 (Xcovery), and ASP-3026 (Astellas). Additional examples of an ALK inhibitor are known in the art.

The expression of a ROS1 gene dysregulation, a ROS1 protein, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing a ROS1 kinase domain and a fusion partner, a mutation in the ROS1 gene that results in the expression of a ROS1 protein that contains a deletion of at least one amino acid compared to the wild-type ROS1 protein, a mutation in the ROS1 gene that results in the expression of the ROS1 protein with one or more point mutations compared to the wild-type ROS1 protein, mutations in the gene ROS1 that results in the expression of a ROS1 protein with at least one inserted amino acid compared to the wild-type ROS1 protein, a gene duplication that results in an increased level of the ROS1 protein in the cell, or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) , which leads to an increased level of the ROS1 protein in the cell), an alternative splice version of ROS1 mRNA, which leads to the formation of a ROS1 protein having at least one amino acid deletion in the ROS1 protein compared to the wild-type ROS1 protein), or overexpression (e.g., increased levels) of the wild-type ROS1 protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation autocrine/paracrine signaling (eg, compared to a control non-cancerous cell). As another example, dysregulation of the ROS1 gene, the ROS1 protein, or the expression or activity or level of any of these may be a mutation in the ROS1 gene that encodes a ROS1 protein that is constitutively active or has increased activity relative to the protein encoded by the gene. ROS1 that does not contain such a mutation. For example, dysregulation of the ROS1 gene, ROS1 protein, or the expression or activity or level of any of them may result from a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the ROS1 protein that contains a functional kinase domain and a second portion of the protein -partner (i.e., not ROS1). In some examples, dysregulation of the ROS1 gene, ROS1 protein, or expression or activity or level of any of these may result from a gene translocation of one ROS1 gene with another non-ROS1 gene.

Non-limiting examples of a ROS1 inhibitor include crizotinib, etrectinib (RXDX-101), lorlatinib (PF-06463922), certinib, TPX-0005, DS-605, and cabozantinib. Additional examples of a ROS1 inhibitor are known in the art.

The expression of a dysregulation of a MET gene, a MET protein, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing a MET kinase domain and a fusion partner, a mutation in the MET gene that results in the expression of a MET protein that contains a deletion of at least one amino acid compared to the wild-type MET protein, a mutation in the MET gene that results in the expression of a MET protein with one or more point mutations compared to the wild-type MET protein, mutations in the gene MET that results in the expression of a MET protein with at least one inserted amino acid compared to the wild-type MET protein, a gene duplication that results in an increased level of the MET protein in the cell, or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) , which leads to an increased level of the MET protein in the cell), an alternative splice version of the MET mRNA, which leads to the formation a MET protein having a deletion of at least one amino acid in the MET protein compared to the wild-type MET protein), or overexpression (e.g., elevated levels) of the wild-type MET protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/ paracrine signaling (eg, compared to a control non-cancerous cell). As another example, dysregulation of the MET gene, MET protein or expression or activity or level of any of them

- 144 041220 may be a mutation in the MET gene that encodes a MET protein that is constitutively active or has increased activity compared to the protein encoded by the MET gene that does not contain such a mutation. For example, dysregulation of a MET gene, a MET protein, or the expression or activity or level of any of them may result from a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the MET protein that contains a functional kinase domain and a second portion of the protein -partner (i.e. not MET). In some examples, dysregulation of a MET gene, MET protein, or expression or activity or level of any of these may result from a gene translocation of one MET gene with another non-MET gene.

Non-limiting examples of a MET inhibitor include crizotinib, cabozantinib, JNJ-38877605, PF-04217903 (Pfizer), MK-2461, GSK 1363089, AMG 458 (Amgen), tivantinib, INCB28060 (Incyte), PF02341066 (Pfizer), E7050 (Eisai), BMS-777607 (Bristol-Meyers Squibb), JNJ-38877605 (Johnson & Johnson), ARQ197 (ArQule), GSK/1363089/XL880 (GSK/Exeilixis) and XL174 (BMS/Exelixis). Additional examples of a MET inhibitor are known in the art.

Expression of the dysregulation of an aromatase gene, an aromatase protein, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a mutation in the aromatase gene that results in the expression of an aromatase protein that contains a deletion of at least one amino acid relative to the protein wild-type aromatase, a mutation in the aromatase gene that results in the expression of an aromatase protein with one or more point mutations compared to the wild-type aromatase protein, a mutation in the aromatase gene that results in the expression of an aromatase protein with at least one inserted amino acid compared to wild-type aromatase protein, a gene duplication that results in an increased level of aromatase protein in the cell, or a mutation in a regulatory sequence (e.g., a promoter and/or enhancer) that results in an increased level of aromatase protein in the cell), an alternative splice version of the aromatase mRNA , which leads to the formation of the aromatase protein, which has a deletion at at least one amino acid in the aromatase protein compared to the wild-type aromatase protein), or increased expression (e.g., increased levels) of the wild-type aromatase protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/paracrine signaling (e.g., compared to a control non-cancerous cell). As another example, dysregulation of an aromatase gene, an aromatase protein, or the expression or activity or level of any of them may be a mutation in an aromatase gene that encodes an aromatase protein that is constitutively active or has increased activity compared to the protein encoded by the gene. aromatase that does not contain this mutation.

Non-limiting examples of an aromatase inhibitor include Arimidex (Anastrozole), Aromasin (Exemestane), Femara (Letrozole), Teslac (Testolactone) and Formestane. Additional examples of an aromatase inhibitor are known in the art.

The expression of a dysregulation of a RAF gene, a RAF protein, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing a RAF kinase domain and a fusion partner, a mutation in the RAF gene that results in the expression of a RAF protein that contains a deletion of at least one amino acid compared to the wild-type RAF protein, a mutation in the RAF gene that results in the expression of a RAF protein with one or more point mutations compared to the wild-type RAF protein, mutations in the gene RAF that results in the expression of a RAF protein with at least one inserted amino acid compared to wild-type RAF protein, a gene duplication that results in an increased level of RAF protein in the cell, or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) , which leads to an increased level of the RAF protein in the cell), an alternative splice version of the RAF mRNA, which leads to the formation RAF protein having a deletion of at least one amino acid in the RAF protein compared to the wild-type RAF protein), or increased expression (e.g., elevated levels) of the wild-type RAF protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/ paracrine signaling (eg, compared to a control non-cancerous cell). As another example, dysregulation of a RAF gene, a RAF protein, or the expression or activity or level of any of these may be a mutation in the RAF gene that encodes a RAF protein that is constitutively active or has increased activity relative to the protein encoded by the gene. RAF that does not contain such a mutation. For example, dysregulation of the RAF gene, RAF protein, or the expression or activity or level of any of them may result from a gene or chromosome translocation that results in the expression of a fusion protein containing a first portion of the RAF protein that contains a functional kinase domain and a second portion of the protein. -partner (i.e. who is not an RAF). In some examples, dysregulation of a RAF gene, RAF protein, or expression or activity or level of any of these may result from a gene translocation of one RAF gene with another non-RAF gene.

Non-limiting examples of a RAF inhibitor include sorafenib, vemurafenib, dabrafenib, BMS-908662/XL281, GSK2118436, RAF265, RO5126766 and RO4987655. Additional Inga Examples

- 145 041220 RAF beaters are known in the art.

The expression of a dysregulation of a RAS gene, a RAS protein, or the expression or activity or level of any of them refers to a genetic mutation (e.g., a chromosomal translocation that results in the expression of a fusion protein containing a RAS kinase domain and a fusion partner, a mutation in the RAS gene that results in the expression of a RAS protein that contains a deletion of at least one amino acid compared to the wild-type RAS protein, a mutation in the RAS gene that results in the expression of the RAS protein with one or more point mutations compared to the wild-type RAS protein, mutations in the gene RAS that results in the expression of a RAS protein with at least one inserted amino acid compared to the wild-type RAS protein, a gene duplication that results in an increased level of the RAS protein in the cell, or a mutation in a regulatory sequence (e.g., promoter and/or enhancer) , which leads to an increased level of the RAS protein in the cell), an alternative splice version of the RAS mRNA, which leads to the formation a RAS protein having a deletion of at least one amino acid in the RAS protein compared to the wild-type RAS protein), or overexpression (e.g., elevated levels) of the wild-type RAS protein in a mammalian cell due to aberrant cellular signaling and/or dysregulation of autocrine/ paracrine signaling (eg, compared to a control non-cancerous cell). As another example, dysregulation of the RAS gene, RAS protein, or expression or activity or level of any of these may be a mutation in the RAS gene that encodes a RAS protein that is constitutively active or has increased activity relative to the protein encoded by the gene. RAS that does not contain such a mutation. For example, dysregulation of a RAS gene, a RAS protein, or the expression or activity or level of any of them may result from a gene or chromosomal translocation that results in the expression of a fusion protein containing a first portion of the RAS protein that contains a functional kinase domain and a second portion of the protein -partner (i.e. non-RAS). In some examples, dysregulation of a RAS gene, RAS protein, or expression or activity or level of any of these may be the result of a gene translocation of one RAS gene with another non-RAS gene.

Non-limiting examples of an RAS inhibitor include Kobe0065 and Kobe2602. Additional examples of a RAS inhibitor are known in the art.

Non-limiting examples of multikinase inhibitors (MKIs) include dasatinib and sunitinib.

In some embodiments, provided herein are methods of treating a subject suffering from cancer, which include: (a) administering to the subject one or more doses of a first RET inhibitor or a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, for a specified period of time. period of time; (b) after (a) determining whether the cancer cell in the sample obtained from the subject has at least one deregulation of a gene, protein, or expression, or activity, or level of any of them, and the specified gene or protein is selected from the group , consisting of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA and HER2; and (c) administering 1) a second RET inhibitor as monotherapy or in combination with another anti-cancer agent, 2) administering additional doses of the first RET inhibitor or a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, in combination with the inhibitor, targeting a gene or protein (eg, with an inhibitor of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2), or 3) stopping the administration of the RET inhibitor from step a) and administering to the subject a gene-targeting inhibitor or a protein (for example, an inhibitor of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2) if said subject has a cancer cell that has at least one deregulation of a gene, protein, or their expression or activity, or level, wherein said gene or protein is selected from the group consisting of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2; or (d) administering additional doses of the first RET inhibitor from step (a) to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation. In some embodiments, one or more deregulations of a gene, protein, or expression, or activity, or level of any of them, wherein said gene or protein is selected from the group consisting of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA and HER2 provide increased resistance of a cancer cell or tumor to treatment with a first RET inhibitor or a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the tumor is an NSCLC tumor and one or more dysregulations of a gene, protein, or expression or activity, or level of any of these are selected from targeting mutations in EGFR or MET, targeting rearrangements affecting ALK or ROS1 , or activating mutations in KRAS. In some embodiments, the tumor is a thyroid tumor (not MTC) and one or more dysregulations of a gene, protein or expression or activity or level of any of these are selected from targeting mutations in BRAF or activating mutations in RAS. In some embodiments, the tumor is an MTC tumor and the one or more dysregulations of a gene, protein or expression or activity or level of any of these are selected from targeting mutations in ALK or activating mutations in RAS. In some embodiments, the tumor is a tumor of the pancreas

- 146 041220 gland, and one or more deregulation of a gene, protein, or expression, or activity, or level of any of them are activating mutations in KRAS. In some embodiments, the tumor is a tumor of the colon and rectum, and one or more dysregulations of a gene, protein, or expression, or activity, or level of any of these are selected from targeting mutations in BRAF or PIK3CA or an activating mutation in RAS. In some embodiments, the tumor is a breast tumor and the one or more dysregulations of a gene, protein or expression or activity or level of any of these are selected from targeting mutations in PIK3CA or alterations in HER2.

Methods for selecting treatment for a subject suffering from cancer are also provided, which include (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a) determining whether the cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (c) selecting for a subject a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if said subject has a cancer cell that contains one or more RET inhibitor resistance mutations ; or (d) selecting additional doses of the first RET inhibitor from step (a) for the subject if the subject has a cancer cell that does not contain the RET inhibitor resistance mutation. In some embodiments, when selecting additional doses of the first RET inhibitor from step (a), the proposed method may further include selecting doses of another anticancer agent for a given subject. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (c), the other RET inhibitor may be the first RET inhibitor administered in step (a).

Methods for selecting treatment for a subject suffering from cancer are also provided, which include (a) administering to the subject one or more doses of a first RET inhibitor over a period of time; (b) after (a) determining whether the cancer cell in the sample obtained from the subject has at least one RET inhibitor resistance mutation; and (c) selecting for the subject a second RET inhibitor as monotherapy or in combination with another anticancer agent if the subject has a cancer cell that contains one or more RET inhibitor resistance mutations; or (d) selecting additional doses of the first RET inhibitor from step (a) for the subject if the subject has a cancer cell that does not contain the RET inhibitor resistance mutation. In some embodiments, when selecting additional doses of the first RET inhibitor from step (a), the proposed method may further include selecting doses of another anticancer agent for a given subject. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be the first RET inhibitor administered in step (a).

Methods for selecting a treatment for a subject suffering from cancer are also provided, which include (a) determining whether a cancer cell in a sample obtained from a subject who suffers from cancer and has previously taken one or more doses of a first RET inhibitor has one or more mutations. RET inhibitor resistance; (b) selecting for the subject a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation ; or (c) selecting for the subject additional doses of the first RET inhibitor previously administered to the subject if the subject has a cancer cell that does not contain

- 147 041220 RET inhibitor resistance mutation. In some embodiments, if additional doses of the first RET inhibitor previously administered to the subject are selected for the subject, then the proposed method may further comprise selecting doses for the subject of another anticancer agent (e.g., a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic forms). In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments of step (c), the other RET inhibitor may be the first RET inhibitor administered in step (a).

Methods for selecting a treatment for a subject suffering from cancer are also provided, which include (a) determining whether a cancer cell in a sample obtained from a subject who suffers from cancer and has previously taken one or more doses of a first RET inhibitor has one or more mutations. RET inhibitor resistance; (b) selecting for the subject a second RET inhibitor as monotherapy or in combination with another anticancer agent if the subject has a cancer cell that contains at least one RET inhibitor resistance mutation; or (c) selecting for the subject additional doses of the first RET inhibitor previously administered to the subject if said subject has a cancer cell that does not contain the RET inhibitor resistance mutation. In some embodiments, if additional doses of the first RET inhibitor previously administered to the subject are selected for the subject, then the proposed method may further comprise selecting doses for the subject of another anticancer agent (e.g., a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic forms). In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and 4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be the first RET inhibitor administered in step (a).

Also provided are methods for determining a subject's risk of developing cancer that has a particular resistance to a first RET inhibitor, which includes: determining whether a cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and identifying a subject having a cell that has one or more RET inhibitor resistance mutations as a cancer-prone subject that has a certain resistance to the first RET inhibitor. Also provided are methods for determining a subject's risk of developing cancer that has a particular resistance to a first RET inhibitor, which includes: identifying a subject having a cell that contains one or more RET inhibitor resistance mutations as a subject with an increased likelihood of developing cancer having a particular resistance. to the first RET inhibitor. Also provided are methods for determining the presence of a cancer that has a particular resistance to a first RET inhibitor, which includes: determining whether a cell in a sample obtained from a subject has one or more RET inhibitor resistance mutations; and determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations is suffering from a cancer that has a certain resistance to the first RET inhibitor. Also provided are methods for determining the presence of a cancer that has a specific resistance to a first RET inhibitor in a subject, which includes: determining that a subject having a cancer cell that has one or more RET inhibitor resistance mutations is suffering from a cancer that has a specific resistance to the first RET inhibitor. In some embodiments, one or more RET inhibitor resistance mutations provide increased resistance of the cancer cell or tumor to treatment with the first RET inhibitor. In some embodiments, the one or more RET inhibitor resistance mutations include one or more of the RET inhibitor resistance mutations listed in Table 1. 3 and

4. For example, one or more RET inhibitor resistance mutations may include a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D.

In some embodiments of any of the methods described herein, the RET inhibitor resistance mutation that confers increased resistance to treatment with the first RET inhibitor in the cancer cell or tumor can be any of the RET inhibitor resistance mutations listed in Table 1. 3 or 4 (for example, a substitution at amino acid position 804, such as V804M, V804L, or V804E, or a substitution at amino acid position 810, such as G810S, G810R, G810C, G810A, G810V, and G810D).

In some embodiments, the presence of one or more RET inhibitor resistance mutations in a tumor results in greater tumor resistance to treatment with a compound of formula IIV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Methods useful when the RET inhibitor resistance mutation results in greater tumor resistance to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, are described below. For example, provided herein are methods for treating a subject suffering from cancer, which include: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and administering to the identified subject a treatment that does not include a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy (eg, a second RET kinase inhibitor). Also provided are methods of treating a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations, which comprises administering to said subject a treatment that does not include a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof in as monotherapy (eg, a second RET kinase inhibitor). In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

Also provided herein are methods for selecting treatment for a subject suffering from cancer, which include: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and selecting a treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy for the identified subject (eg, a second RET kinase inhibitor). Also provided herein are methods for selecting a treatment for a subject suffering from cancer, which include: selecting a treatment that does not include a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy (e.g., a second RET kinase inhibitor) for a subject identified as having a cancer cell that contains one or more RET inhibitor resistance mutations. Also provided are methods for selecting a subject suffering from cancer for treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy (e.g., a second RET kinase inhibitor), which includes: identifying a subject having a cancer cell that contains one or more RET inhibitor resistance mutations; and selecting an identified subject for treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy (eg, a second RET kinase inhibitor). Also provided are methods for selecting a subject suffering from cancer for treatment that does not include a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy (e.g., a second RET kinase inhibitor), which includes: selecting a subject, at which identified the presence of a cancer cell that contains one or more RET inhibitor resistance mutations, for treatment that does not include a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form as monotherapy. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

Also provided are methods for determining the likelihood that a subject suffering from cancer will demonstrate a positive response to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy, which includes: determining whether a cancer cell has a sample obtained from a subject, one or more RET inhibitor resistance mutations; and determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations has a reduced likelihood of responding positively to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy. Also provided are methods for determining the likelihood that a subject suffering from cancer will demonstrate a positive response to treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy, which includes: determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations has a reduced likelihood of responding positively to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy. Also provided are methods for predicting the effectiveness of treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy, in a subject suffering from cancer, which includes: determining that a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and determining that treatment with a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy is less likely to be effective in a subject having a cancer cell in a sample obtained from that subject that contains one or more resistance mutations to RET inhibitor. Also provided are methods for predicting the effectiveness of treatment with a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof as monotherapy in a subject suffering from cancer, which includes: determining that treatment with a compound of formula I-IV or a pharmaceutically acceptable salt thereof , amorphous or polymorphic form as monotherapy is less likely to be effective in a subject having a cancer cell in a sample obtained from that subject that contains one or more RET inhibitor resistance mutations. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

Also provided are methods of treating a subject suffering from cancer, which include: (a) administering one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, over a period of time; (b) after (a) determining that the cancer cell in the sample obtained from the subject has one or more RET inhibitor resistance mutations; and (c) administering a second RET inhibitor or a second compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent, to a subject who has a cancer cell that contains one or more resistance mutations. to an RET inhibitor; or (d) administering additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (a) to a subject having a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, if the subject is administered additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (a), then the subject may also be administered another anticancer agent or a second compound of formula I-IV or a pharmaceutically acceptable form thereof. salt, amorphous or polymorphic form. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, introduced in step (a).

Methods for treating a subject suffering from cancer are also provided, which include: (a) determining whether a cancer cell in a sample obtained from a subject who suffers from cancer and has previously received one or more doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form, one or more RET inhibitor resistance mutations; (b) administering a second RET inhibitor or a second compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, alone or in combination with another anticancer agent, to a subject having a cancer cell that contains one or more RET inhibitor resistance mutations ; or (c) administering additional doses of a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, previously administered to a subject having a cancer cell that does not contain the RET resistance mutation. In some embodiments, if additional doses of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (a) are administered to the subject, then another anticancer agent may also be administered to the subject. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the additional anti-cancer agent is

- 150 041220 is any anti-cancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, introduced in step (a).

Also provided herein are methods for selecting treatment for a subject suffering from cancer, which include: (a) administering to the subject one or more doses of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, over a period of time; (b) after (a) determining that the cancer cell in the sample obtained from the subject has one or more RET inhibitor resistance mutations; and (c) selecting a second RET inhibitor or a second compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent for a subject if said subject has a cancer cell that contains a resistance mutation to an RET inhibitor; or (d) selecting additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (a) in a subject if said subject has a cancer cell that does not contain a RET inhibitor resistance mutation. In some embodiments, if additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (a) are selected for a subject, then said method may also include additional selection of another anticancer agent. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, introduced in step (a).

Also provided herein are methods for selecting treatment for a subject suffering from cancer, which include: (a) determining if a cancer cell in a sample obtained from a subject who suffers from cancer and has previously taken one or more doses of a compound of formula I- IV or its pharmaceutically acceptable salt, amorphous or polymorphic form, one or more RET inhibitor resistance mutations; (b) selecting a second RET inhibitor or a second compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, as monotherapy or in combination with another anticancer agent for a subject if said subject has a cancer cell that contains an inhibitor resistance mutation RET; or (c) selecting additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof previously administered to a subject if said subject has a cancer cell that does not contain a RET resistance mutation. In some embodiments, if additional doses of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof from step (a) are selected for a subject, then said method may also include additional selection of another anticancer agent. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, the additional anticancer agent is any anticancer agent known in the art. For example, the additional anti-cancer agent is another RET inhibitor (eg, a second RET inhibitor). In some embodiments, the additional anti-cancer agent is an immunotherapy. In some embodiments, the other RET may be a compound of Formula IIV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, introduced in step (a).

Also provided are methods for determining the risk of a subject developing a cancer that has a certain resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, which includes: determining whether a cancer cell in a sample obtained from the subject has one or more RET inhibitor resistance mutations; and identifying the subject, if the subject has a cancer cell that contains one or more RET inhibitor resistance mutations, as having an increased likelihood of developing cancer that has some resistance to the compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Also provided are methods for determining a subject's risk of developing a cancer that has a certain resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, which includes: identifying a subject having

- 151 041220 a cell that contains one or more resistance mutations to an RET inhibitor, as having an increased likelihood of developing cancer, which has a certain resistance to a compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form. Also provided are methods for determining the presence of a cancer that has a certain resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, which includes: determining whether a cancer cell in a sample obtained from a subject has one or more resistance mutations to an RET inhibitor; and determining that a subject having a cancer cell that contains one or more RET inhibitor resistance mutations is suffering from a cancer having some resistance to a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. Also provided are methods for determining whether a subject has a cancer that has a certain resistance to a compound of formula IIV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, which includes: determining that a subject having a cancer cell that contains one or more inhibitor resistance mutations RET suffers from a cancer having some resistance to a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form. In some embodiments, one or more RET inhibitor resistance mutations render the cancer cell or tumor more resistant to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

In some embodiments of any of the methods described herein, the RET inhibitor resistance mutation that confers increased resistance to treatment with a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, can be any of the resistance mutations. to the RET inhibitor listed in Table. 3 or 4.

Methods for determining the degree of resistance of a cancer cell or tumor to an RET inhibitor (eg, any RET inhibitor described herein or known in the art) can be determined using methods known in the art. For example, the degree of resistance of a cancer cell to a RET inhibitor can be assessed by determining the IC ₅₀ of the RET inhibitor (eg, any RET inhibitor described herein or known in the art) on cancer cell viability. In other examples, the degree of resistance of a cancer cell to a RET inhibitor can be assessed by determining the growth rate of the cancer cell in the presence of the RET inhibitor (eg, any RET inhibitor) described herein. In other examples, the degree of tumor resistance to an RET inhibitor can be assessed by determining the mass or size of one or more tumors in a subject during treatment with an RET inhibitor (eg, any RET inhibitor described herein). In other examples, the degree of resistance of a cancer cell or tumor to a RET inhibitor can be indirectly assessed by determining the activity of a RET kinase containing one or more RET inhibitor resistance mutations (i.e., the same RET kinase expressed in the cancer cell or tumor in the subject's body). ). The degree of resistance of a cancer cell or tumor containing one or more RET inhibitor resistance mutations to the RET inhibitor is expressed relative to the degree of resistance of the cancer cell or tumor that does not contain the RET inhibitor resistance mutation (e.g., a cancer cell or tumor that does not contain the same RET inhibitor resistance mutation, a cancer cell or tumor that does not contain any RET inhibitor resistance mutation, or a cancer cell or tumor that expresses wild-type RET protein). For example, the established degree of resistance of a cancer cell or tumor containing one or more RET inhibitor resistance mutations can be greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%. %, more than about 6%, more than about 7%, more than about 8%, more than about 9%, more than about 10%, more than about 11%, more than about 12%, more than about 13%, more than about 14%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 110%, more than about 120%, more than about 130 %, more than about 140%, more than about 150%, more than about 160%, more than about 170%, more than about 180%, more than about 190%, more than about 200%, more than about 210%, more than about 220%, more than about 23 0%, greater than about 240%, greater than about 250%, greater than about 260%, greater than about 270%, greater than about 280%, greater than about 290%, or greater than about 300% of the degree of resistance of the cancer cell, or a tumor that does not contain a RET inhibitor resistance mutation (e.g., a cancer cell or tumor that does not contain the same RET inhibitor resistance mutations, a cancer cell or tumor that does not contain any RET inhibitor resistance mutations, or a cancer cell or tumor, which expresses the wild-type RET protein).

RET is thought to play an important role in the development and survival of afferent nociceptors in the skin and gut. RET kinase knockout mice lack enteric-loving neurons.

- 152 041220 and there are other anomalies of the nervous system, indicating the need for a functional protein product RET-kinase during development (Taraviras, S. et al., Development, 1999, 126:27852797). In addition, in population studies of patients with Hirschsprung's disease, characterized by colonic obstruction due to lack of normal colonic energy, there is a higher proportion of both hereditary and sporadic loss-of-function RET mutations (Butler Tjaden N., et al., Transl. Res ., 2013, 162: 1-15). Irritable bowel syndrome (IBS) is a common disease affecting 10-20% of people in developed countries and is characterized by abnormal bowel habits, bloating and visceral hypersensitivity (Camilleri, M., N. Engl. J. Med., 2012, 367: 1626-1635). Although the etiology of IBS is unknown, it is thought to be the result of a disruption between the brain and the gastrointestinal tract, a disturbance in the gut microbiome, or an increase in inflammation. The resulting gastrointestinal changes interfere with normal intestinal transit, resulting in diarrhea or constipation. In addition, in many patients with IBS, sensitization of the peripheral nervous system leads to visceral hypersensitivity or allodynia (Keszthelyi, D., Eur. J. Pain, 2012, 16: 1444-1454). See, for example, US Publication No. 2015/0099762.

Accordingly, the present invention provides methods of treating a patient diagnosed with (or identified as having) irritable bowel syndrome (IBS), including diarrhea-predominant, constipation-dominated or intermittent stool disease, functional bloating, functional constipation, functional diarrhea, non-specific functional bowel disorder, functional abdominal pain syndrome, chronic idiopathic constipation, functional disorders of the esophagus, functional gastroduodenal disorders, functional anorectal pain and inflammatory bowel disease, which include administering to a patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form .

Also provided are methods of treating a patient identified or diagnosed as having RET-associated irritable bowel syndrome (IBS) (e.g., a patient identified or diagnosed as having RET-associated irritable bowel syndrome (IBS) with a government-approved, for example, an FDA-approved kit for identifying RET gene dysregulation, RET kinase, or the expression or activity or level of any of them in a patient, or in a biopsy sample obtained from a patient), which comprises administering to said patient a therapeutically effective amount of a compound of formula I- IV or its pharmaceutically acceptable salt, amorphous or polymorphic form.

Also provided herein are methods of treating pain associated with IBS which comprise administering to a patient a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof. In some embodiments, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, is administered in combination with a first other therapeutic agent useful in the treatment of one or more symptoms of IBS.

Also provided are methods of treating irritable bowel syndrome (IBS) in a patient in need thereof, comprising: (a) determining whether the patient has RET-associated IBS (eg, using a regulatory and regulatory agency approved , for example, an FDA-approved kit for identifying RET gene dysregulation, RET kinase, or expression or activity, or the level of any of them in a patient or in a biopsy sample obtained from a patient, or by performing any of the non-limiting examples of assays described herein ); and (b) if the IBS is determined to be RET-associated IBS, administering to said patient a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof.

In some embodiments, the compounds of this invention are useful in the treatment of irritable bowel syndrome (IBS) in combination with one or more additional therapeutic agents or therapies effective in the treatment of irritable bowel syndrome that act by the same or a different mechanism of action. At least one additional therapeutic agent may be administered with a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form, in the same or different dosage forms, by the same or different routes of administration, and on the same or different schedules. administration in accordance with standard pharmaceutical practice known to those skilled in the art.

Non-limiting examples of additional therapeutic agents for the treatment of irritable bowel syndrome (IBS) include probiotics, dietary fiber supplements (eg, psyllium, methylcellulose), antidiarrheals (eg, lopermide), bile acid binding agents (eg, cholestyramine, colestipol, colesevelam) , anticholinergics and antispasmodics (eg, hyoscyamine, dicyclomine), antidepressants (eg, tricyclic antidepressants such as imipramine or notriptyline, or selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine or paroxetine), antibodies (eg , rifaximin), alosetron and lubiprostone.

Accordingly, this document also provides methods for the treatment of irritable bowel syndrome (IBS), comprising administering to a patient in need thereof, a pharmaceutical combination for the treatment of IBS, which contains (a) a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form , (b) an additional therapeutic agent, and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate or sequential use for the treatment of IBS, wherein the amounts of the compound of formula I-IV or its pharmaceutically acceptable salt, amorphous or polymorphic form and additional therapeutic agent together are effective for the treatment of IBS. In one embodiment, the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and the additional therapeutic agent are administered simultaneously as separate dosage forms. In one embodiment, the compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and an additional therapeutic agent are administered as separate dosage forms sequentially, in any order, in a total therapeutically effective amount, for example, in daily or intermediate doses. In one embodiment, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and an additional therapeutic agent are administered simultaneously as a combined dosage form.

This document also provides (i) a pharmaceutical combination for the treatment of irritable bowel syndrome in a patient in need thereof, which contains (a) a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, (b) at least one an additional therapeutic agent (e.g., any of the illustrative additional therapeutic agents described herein for the treatment of irritable bowel syndrome, or known in the art), and (c) optionally at least one pharmaceutically acceptable carrier for simultaneous, separate, or sequential use for the treatment of irritable bowel syndrome, wherein the amounts of a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and an additional therapeutic agent together are effective for the treatment of irritable bowel syndrome; (ii) a pharmaceutical composition containing such a combination; (iii) the use of such a combination for the preparation of a medicament for the treatment of irritable bowel syndrome; and (iv) an industrial package or product containing such a combination as a combined preparation for simultaneous, separate or sequential use; and a method for treating irritable bowel syndrome in a patient in need thereof. In one embodiment, the patient is a human.

The term pharmaceutical combination in this context refers to a pharmaceutical therapy resulting from the mixing or combination of more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients. The term fixed combination means that a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and at least one additional therapeutic agent (e.g., an agent effective for the treatment of irritable bowel syndrome) are administered to a patient simultaneously, as a single composition, or dosage form. The term non-fixed combination means that a compound of formula I-IV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and at least one additional therapeutic agent (for example, an agent effective for the treatment of irritable bowel syndrome) are formulated as separate compositions or dosage forms, so that they can be administered to a patient in need thereof simultaneously, in parallel, or sequentially at various intervening time intervals, such administration providing effective maintenance of two or more compounds in the patient's body. In one embodiment, the compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, and the additional therapeutic agent are formulated as separate unit dosage forms, the individual dosage forms being suitable for sequential or simultaneous administration. This also applies to cocktail therapies, for example, the administration of three or more active ingredients.

In some embodiments, a compound provided herein may be used as an agent for maintenance therapy in a patient undergoing cancer treatment. For example, a compound of formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, may be useful in reducing one or more symptoms associated with treatment with one or more anticancer therapies, such as complications of diarrhea or constipation and/or abdominal pain. pain. See, for example, US Publication No. 2015/0099762 and Hoffman, J.M. et al. Gastroenterology (2012) 142:844-854. Accordingly, a compound or pharmaceutically acceptable salt or composition provided herein may be administered to a patient to manage one or more complications associated with cancer treatment (eg, gastrointestinal complications such as diarrhea, constipation, or abdominal pain) .

In some embodiments, a therapeutically effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, may be administered to a patient undergoing cancer treatment (e.g., a patient experiencing an adverse event associated with cancer treatment, such as an immune adverse event or gastrointestinal complication including diarrhea, constipation and abdominal pain). For example, a compound provided herein, or a pharmaceutically acceptable salt thereof, can be used to treat colitis or IBS associated with the administration of a checkpoint inhibitor; see, for example, Postow, M.A. et al. Journal of Clinical Oncology (2015) 33: 1974-1982. In some such embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, may be formulated to provide low bioavailability and/or targeted delivery to the gastrointestinal tract. See, for example, US Pat. No. 6,531,152.

Also provided is a method for inhibiting RET kinase activity in a cell, comprising contacting said cell with a compound of formula I. In one embodiment, the contacting is carried out in vitro. In one embodiment, the implementation of bringing into contact is carried out in vivo. In one embodiment, the contacting is carried out in vivo, the method comprising administering an effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, in an amorphous or polymorphic form, to a subject having a cell that has RET kinase activity. In some embodiments, the cell is a cancer cell. In one embodiment, the cancer cell is any cancer described herein. In some embodiments, the cancer cell is a RET-associated cancer cell. In some embodiments, the cell is a gastrointestinal tract cell.

Also provided is a method for inhibiting RET kinase activity in a mammalian cell, comprising contacting said cell with a compound of Formula I. In one embodiment, the contacting is carried out in vitro. In one embodiment, the implementation of bringing into contact is carried out in vivo. In one embodiment, the contacting is carried out in vivo, the method comprising administering an effective amount of a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, in an amorphous or polymorphic form, to a mammal having a cell that has RET kinase activity. In some embodiments, the mammalian cell is a mammalian cancer cell. In one embodiment, the mammalian cancer cell is any cancer described herein. In some embodiments, the mammalian cancer cell is a RET-associated cancer cell. In some embodiments, the mammalian cell is a gastrointestinal tract cell.

In this context, the term bringing into contact refers to the association of these fragments in an in vitro system or in an in vivo system. For example, contacting a RET kinase with a compound provided herein includes administering a compound provided herein to an individual or patient, such as a human, having a RET kinase, as well as, for example, administering a compound provided herein to a sample containing a cellular or purified preparation containing RET kinase.

Also provided herein is a method for inhibiting cell proliferation in vitro or in vivo, comprising contacting a cell with an effective amount of a compound of Formula IIV or a pharmaceutically acceptable salt, amorphous or polymorphic form thereof, or a pharmaceutical composition as described herein.

The term "effective amount" means that amount of a compound which, when administered to a patient in need of such treatment, is sufficient to (i) treat a disease or disorder associated with RET kinase, (ii) alleviate, improve, or eliminate one or more symptoms of a particular disease, pathological condition or disorder, or (iii) delaying the onset of one or more symptoms of a particular disease, pathological condition or disorder described herein. The amount of a compound of formula I-IV or a pharmaceutically acceptable salt thereof, amorphous or polymorphic form, which corresponds to such an amount, varies depending on such factors as the specific compound, the state of the disease and its severity, the properties (for example, weight) of the patient in need of treatment , but, nevertheless, can be determined in the usual way by a person skilled in the art.

4. Pharmaceutical compositions and administration.

When used as a pharmaceutical agent, the compound of formula I-IV, including its polymorphic forms and pharmaceutically acceptable salts, can be administered in the form of pharmaceutical compositions. Such compositions may be prepared by methods well known in the art of pharmacy, and may be administered in a variety of ways, depending on whether local or systemic treatment is required and the area to be treated. The introduction can be local (including transdermal, epidermal, ophthalmic and mucous membranes, including intranasal, vagin

- 155 041220 nasal and rectal delivery), pulmonary (for example, by inhalation or insufflation of powders or aerosols, including administration through a nebulizer; intratracheal or intranasal), oral or parenteral. Oral administration may include a once-daily or twice-daily (BID) dosage form. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injections or infusions; or intracranial, eg, intrathecal or intraventricular administration. Parenteral administration may be in the form of a single bolus dose or, for example, via a continuous perfusion dispenser. Pharmaceutical compositions and dosage forms for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Standard pharmaceutical carriers, aqueous, powdered or oily bases, thickeners, and the like may be necessary or desirable.

This document also provides pharmaceutical compositions which contain as an active ingredient a compound of formula I-IV, or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers (auxiliaries). For example, a pharmaceutical composition prepared using a compound of formula I-IV or a pharmaceutically acceptable salt thereof, in amorphous or polymorphic form. In some embodiments, the composition is suitable for topical administration. In preparing the compositions of this invention, the active ingredient is usually mixed with an excipient, diluted with an excipient, or introduced into such a carrier in the form of, for example, a capsule, sachet, paper or other package. If the excipient serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions of this invention may be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or liquid medium), ointments containing, for example , up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile solutions for injection or sterile packaged powders. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is a solid oral dosage form. In some embodiments, the composition is in the form of a tablet or capsule.

This document further provides pharmaceutical compositions comprising a compound of formula I-IV, or a polymorph thereof, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier. Pharmaceutical compositions containing a compound of formula I-IV, or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof, as active ingredient, can be prepared by intimately mixing the compound of formula I-IV, or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof, with a pharmaceutical carrier, according to with conventional pharmaceutical formulation techniques. The carrier may take various forms depending on the desired route of administration (eg, oral, parenteral). In some embodiments, the composition is a solid oral composition.

Suitable pharmaceutically acceptable carriers are well known in the art. A description of these pharmaceutically acceptable carriers is provided in The Handbook of Pharmaceutical Excipients published by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain.

Methods for formulating pharmaceutical compositions are described in many publications such as Pharmaceutical Dosage Forms: Tablets, Second Edition, Revised and Expanded, Volumes 1-3, ed. Lieberman et al; Pharmaceutical Dosage Forms: Parenteral Medications, Volumes 1-2, ed. Avis et al; and Pharmaceutical Dosage Forms: Disperse Systems, vols. 1-2, ed. Lieberman et al; published by Marcel Dekker, Inc.

When preparing compositions in oral dosage forms, any conventional pharmaceutical medium can be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, coloring agents, and the like; for solid oral preparations such as powders, capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrants, and the like. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, acetate sodium, sodium chloride, and the like. Disintegrants include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. Solid oral preparations may also be coated with substances such as sugars or may be enteric coated to alter their main site of absorption. For parenteral administration, the carrier usually consists of sterile water, and other ingredients may be added to increase solubility or preservation. Suspensions or solutions for

- 156 041220 injections can also be obtained using aqueous carriers together with appropriate additives. Pharmaceutical compositions of this invention contain, per unit form, for example, tablet, capsule, powder, injection, full teaspoon, etc., such an amount of the active ingredient as is necessary to deliver the effective dose described herein.

Compositions containing a compound of Formula I-IV, or a polymorphic form or pharmaceutically acceptable salt thereof, may be formulated as unit dosage forms, each dose containing from about 5 to about 1000 mg (1 g), more typically from about 100 to about 500 mg of active ingredient. The term unit dosage form refers to physically separate units suitable as a single dose for humans or other patients, and each unit contains a predetermined amount of the active material (i.e., a compound of formula I-IV or a polymorphic form thereof, or a pharmaceutically acceptable salt thereof ) calculated to provide the desired therapeutic effect, in combination with a suitable pharmaceutical excipient.

In some embodiments, the compositions provided herein contain from about 5 mg to about 50 mg of the active ingredient. Those skilled in the art will appreciate that the compounds or compositions of these embodiments contain about 5 mg to about 10 mg, about 10 mg to about 15 mg, about 15 mg to about 20 mg, about 20 mg to about 25 mg , about 25 mg to about 30 mg, about 30 mg to about 35 mg, about 35 mg to about 40 mg, about 40 mg to about 45 mg, or about 45 mg to about 50 mg of the active ingredient.

In some embodiments, the compositions provided herein contain from about 50 mg to about 500 mg of the active ingredient. Those skilled in the art will appreciate that the compounds or compositions of these embodiments contain about 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg , about 250 mg to about 300 mg, about 350 mg to about 400 mg, or about 450 mg to about 500 mg of the active ingredient. In some embodiments, the compositions provided herein contain about 10 mg, about 20 mg, about 80 mg, or about 160 mg of the active ingredient.

In some embodiments, the compositions provided herein contain from about 500 mg to about 1000 mg of the active ingredient. Those skilled in the art will appreciate that the compounds or compositions of these embodiments contain about 500 mg to about 550 mg, about 550 mg to about 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700 mg , about 700 mg to about 750 mg, about 750 mg to about 800 mg, about 800 mg to about 850 mg, about 850 mg to about 900 mg, or about 900 mg to about 950 mg, or about 950 mg to about 1000 mg of the active ingredient.

The daily dose of a compound of formula I-IV, or a polymorphic form thereof, or a pharmaceutically acceptable salt, may vary over a wide range of 1.0 to 10,000 mg per adult per day or more, or any range in between.

For oral administration, the proposed compositions are preferably presented in the form of tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 10.0, 15.0, 25.0, 50, 0, 100, 150, 160, 200, 250 and 500 mg of the active ingredient for symptomatic dose adjustment for the particular patient to be treated. An effective amount of the drug is typically administered at a dose of about 0.1 mg/kg to about 1000 mg/kg of body weight per day, or any range in between. Preferably, this range is from about 0.5 to about 500 mg/kg of body weight per day, or any range in between. More preferably, from about 1.0 to about 250 mg/kg body weight per day, or any range in between. More preferably, from about 0.1 to about 100 mg/kg of body weight per day, or any range in between. In one example, this range may be from about 0.1 to about 50.0 mg/kg of body weight per day, or any amount or range in between. In another example, this range may be from about 0.1 to about 15.0 mg/kg of body weight per day, or any range in between. In another example, this range may be from about 0.5 to about 7.5 mg/kg of body weight per day, or any amount or range in between. Pharmaceutical compositions containing a compound of formula I-IV, or a polymorphic form thereof, or a pharmaceutically acceptable salt, may be administered 1 to 4 times daily or as a single daily dose.

The active compound may be effective over a wide dosage range and is usually administered in a pharmaceutically effective amount. Optimal doses for administration can be readily determined by one of ordinary skill in the art. Therefore, it should be understood that the amount of the compound actually administered is usually determined by the physician and varies depending on relevant circumstances including the route of administration, the compound actually administered, the efficacy of the drug, the condition being treated, and the severity of the disease state. In addition, factors associated with the particular patient being treated, including patient response, age, weight, diet, time of administration, and severity of the patient's symptoms, may also necessitate dose adjustments.

In some embodiments, the compounds provided herein may be administered in an amount of from about 1 mg/kg to about 100 mg/kg. In some embodiments, a compound provided herein may be administered in an amount of from about 1 mg/kg to about 20 mg/kg, from about

- 157 041220 mg/kg to about 50 mg/kg, from about 10 mg/kg to about 40 mg/kg, from about 15 mg/kg to about 45 mg/kg, from about 20 mg/kg to about 60 mg/kg kg or from about 40 mg/kg to about 70 mg/kg. For example, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg. In some embodiments, such administration may be once daily or twice daily (BID) administration.

In some embodiments, the compounds provided herein may be administered in an amount of about 10 mg twice daily (BID), 20 mg BID, about 40 mg BID, about 60 mg BID, about 80 mg BID, about 120 mg BID, about 160 mg BID and about 240 mg BID. In some embodiments, each dose is administered at least 6 hours after the previous dose. In some embodiments, each dose is administered at least 12 hours after the previous dose.

In some embodiments, a compound of formula I-IV, or a polymorph thereof, or a pharmaceutically acceptable salt, exhibits pH-dependent solubility at reduced pH. Accordingly, patients are also being treated with proton pump inhibitors (PPIs) and/or antacids may be required to adjust the dose of a Formula I-IV compound or polymorph or pharmaceutically acceptable salt thereof (e.g., increasing the dose of a Formula I-IV compound or polymorph thereof, or its pharmaceutically acceptable salt). In some embodiments, the cytochrome P450 (CUP) isoform that metabolizes a compound of Formula I-IV, or a polymorphic form thereof, or a pharmaceutically acceptable salt, is CYP3A4. Accordingly, patients who are also taking agents that inhibit or stimulate CYP3A4 may need to adjust the dose of the compound of formula I-IV or its polymorph, or a pharmaceutically acceptable salt (for example, increase the dose of the compound of formula I-IV or its polymorph, or a pharmaceutically acceptable salt in the case of a CYP3A4 stimulant, or in a dose reduction of a compound of Formula I-IV or a polymorph thereof, or a pharmaceutically acceptable salt in the case of a CYP3A4 inhibitor).

Those of skill in the art will recognize that in vivo and in vitro tests using suitable, known and recognized cell and/or animal models are predictive of the ability of an experimental compound to provide treatment or prevention for a given disorder.

Those skilled in the art will also appreciate that human clinical trials, including first-time human trials, dose adjustment trials, and efficacy trials, in healthy patients and/or in patients suffering from a given disorder, can be conducted in accordance with the methods well known in the clinical and medical field.

5. Sets.

The present invention provides pharmaceutical kits suitable, for example, for the treatment of RET-associated diseases or disorders such as cancer or irritable bowel syndrome (IBS), which contain one or more containers containing a pharmaceutical composition that contains a therapeutically effective amount of a compound of the invention. in this document. Such kits may additionally contain, if necessary, one or more of the various components of conventional pharmaceutical kits, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as is obvious to a person skilled in the art. The kit may also include instructions, both in the form of inserts and in the form of labels, indicating the amount of components to be introduced, instructions for administration and/or instructions for mixing the components.

Examples

The following examples illustrate the invention.

Example 1 Synthesis of a Compound of Formula I

Intermediate connection A1 and A2.

6-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and 4-bromo-6-methoxypyrazolo[1,5-a]pyridine (A2) N=\ N=\ a a

Part A. Preparation of O-(mesitylsulfonyl)hydroxylamine (intermediate R1).

Stage 1. Obtaining tert-butyl-(mesitylsulfonyl)oxycarbamate. To a cooled to 0°C solution of 2,4,6-trimethylbenzene-1-sulfonyl chloride (10.0 g, 45.72 mmol) and tert-butylhydroxycarbamate (6.088 g, 45.72 mmol) in MTBE (100 ml) dropwise TEA (14.46 ml, 48.01 mmol) was added with stirring. The resulting suspension was stirred at 0°C for another 30 min and then warmed to room temperature. The reaction mixture was then diluted with water (100 ml) and adjusted to pH 4 with 1N HCl. HC1( _aq ). The organic layer was dried (Na ₂ SO ₄ ), filtered and concentrated to give the title compound initially as a yellowish oil which upon drying overnight under high vacuum became a white solid (12.89 g, yield 89%).

1 H NMR (CDCl ₃ ): δ 7.66 (br, 1H), 6.98 (s, 2H), 2.67 (s, 6H), 2.32 (s, 3H), 1.31 (s, 9H).

Step 2: Preparation of O-(mesitylsulfonyl)hydroxylamine (intermediate R1). To TFA (117 ml, 1521 mmol) at 0°C was added tert-butyl-(mesitylsulfonyl)oxycarbamate (39.0 g, 124 mmol) slowly over 25 minutes. The reaction mixture was stirred at 0° C. for 1.5 h and then quenched by successively adding crushed ice and water. The resulting thick suspension was vigorously stirred at room temperature for 5 minutes. Without allowing the filter cake to dry out, the solid was collected by gentle suction filtration, then washed with water (4 L) until the filtrate had a pH of 6 (warning: there is a risk of explosion of the dry compound at room temperature). The wet filter cake was then dissolved in dichloromethane (150 ml) and the resulting biphasic solution was separated. The dichloromethane layer was dried over MgSO ₄ for 30 min and then filtered and washed with dichloromethane (420 ml) to give the title compound as a 0.22M solution in dichloromethane.

Part B. Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and 4-bromo-6methoxypyrazolo[1,5-a]pyridine (A2).

Stage 1. Obtaining 1-amino-3-bromo-5-methoxypyridin-1-ium-2,4,6-trimethylbenzenesulfonate. To a solution of O-(mesitylsulfonyl)hydroxylamine (intermediate R1) (26.6 g, 117 mmol) in DCM (570 ml) cooled to 0°C was added 3-bromo-5-methoxypyridine (22.1 g , 117 mmol). The reaction mixture was stirred for 1 h at 0°C, then treated with additional 3-bromo-5-methoxypyridine (250 mg, 1.39 mmol) and stirred for another 2 h at 0°C. The reaction mixture was diluted with Et ₂ O (600 ml), stirred at 0°C for 10 min and then filtered under vacuum, washed with Et ₂ O (3x250 ml). After reducing the volume by about 1/3, more precipitate was obtained from the filtrate, which was collected by filtration. Both filter cakes were dried in vacuo to give the title compound (39.3 g, 83% yield).

1H NMR (CDCl ₃ ) δ 9.25 (br, 1H), 8.99 (m, 1H), 8.74 (m, 1H), 7.46 (m, 1H), 6.83 (s, 2H ), 3.92 (s, 3H), 2.65 (s, 6H), 2.22 (s, 3H).

Stage 2. Preparation of ethyl 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate and ethyl 4-bromo6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate. To a magnetically stirred white suspension of 1-amino-3-bromo-5-methoxypyridin-1-ium-2,4,6-trimethylbenzenesulfonate (33.24 g, 82.42 mmol) in DMF (82 ml) at room temperature TEA (22.98 ml, 164.8 mmol) was added, then ethyl propiolate (16.71 ml, 164.8 mmol) was added dropwise. After vigorous stirring for 2 days, the reaction mixture was slowly quenched by adding it in portions to rapidly stirred ice water (820 ml). The mixture was stirred at room temperature for 10 minutes and then filtered under vacuum. The collected solid was washed with water and air dried to give the title compounds as an orange solid in an isomeric ratio of about 4:1 (by 1H NMR) with the 6-Br isomer as the main isomer (21 g). The wet solid isomeric mixture (about 75% w/w) was used directly in step 3 without further purification. MS (atm. pressure chemical ionization, apci) m/z = 298.9, 300.9 (M+H). The regioisomeric ratio was determined from the MeO chemical shift by 1H NMR (CDCl ₃ ) δ 3.98 (6-Br isomer) versus 3.83 (4-Br isomer).

Stage 3. Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and 4-bromo-6methoxypyrazolo[1,5-a]pyridine (A2). An isomeric mixture of ethyl 6-bromo-4-methoxypyrazolo[1,5-a]pyridine3-carboxylate and ethyl 4-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate from step 2 (15 g, 50.1 mmol) was added to 48% HBr (114 ml) with stirring, then heated at 80°C for 90 minutes, then stirred at room temperature overnight. The resulting suspension was filtered under vacuum and washed with water. The aqueous filtrate and filter cake were treated independently of each other. The filter cake was dissolved in MTBE and filtered under vacuum to remove insoluble impurities. The MTBE filtrate was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to give 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (Intermediate A1) as a beige solid (about 98:2 6-/ 4-Br, 5.08 g). MS (apci) m/z = 226.9, 228.9 (M+H).

1H NMR (CDCl ₃ ) δ 8.26 (m, 1H), 7.82 (d, 1H), 6.61 (m, 1H), 6.43 (m, 1H), 3.94 (s, 3H ).

The original aqueous filtrate of the reaction mixture was independently extracted with EtOAc (2x500 ml). The combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. The crude residue was dissolved in DCM (50 ml) and then filtered to remove insoluble solids. Concentration of the DCM filtrate in vacuo followed by chromatography on silica (0 to 50% EtOAc/hexanes) gave a second batch of 6-bromo-4-methoxypyrazolo[1,5a]pyridine (Intermediate A1) as a white solid (upper spot R _f , 2.06 g) as well as the minor isomer of the title compound 4-bromo-6-methoxypyrazolo[1,5a]pyridine (intermediate A2), also as a white solid (lower spot Rf, 1, 32 g). MS (apci) m/z = 226.9, 228.9 (M+H).

1H NMR (CDCl3) δ 8.02 (m, 1H), 7.85 (d, 1H), 7.17 (d, 1H), 6.55 (m, 1H), 3.80 (s, 3H) .

Intermediate connection A3

- 159 041220

6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde.

POCl ₃ (0.92 ml , 9.909 mmol). The reaction mixture was warmed to room temperature and stirred for 4 h and then diluted with H 2 O (30 ml). The resulting suspension was basified to pH 9-10 with 1 M NaOH( _aq ), then stirred for 1 h and filtered under vacuum, then washed successively with H2O (25 ml) and MTBE (50 ml) to give the title compound (0 .76 g, 90% yield. MS (apci) m/z=256.9 (M+H).

Intermediate connection A4 No. \ jCT

Stage 1. Preparation of (E)-6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime. To a suspension of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde (intermediate A3, 0.76 g, 3.0 mmol) and hydroxylamine hydrochloride (0.31 g, 4.5 mmol) in EtOH (40 ml) was added water (20 ml) and stirred the reaction mixture at 50°C for 4 hours After cooling to room temperature, the reaction mixture was concentrated in vacuo. The residue was suspended in water, then treated with saturated NaHCO ₃ solution ( _aq ) and filtered under vacuum. The solid was washed successively with H ₂ O (25 ml) and MTBE (50 ml) to give the title compound (0.68 g, 84% yield). MS (apci) m/z = 271.9 (M+H).

Stage 2. Obtaining 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile. A solution of (E)-6bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime (17.15 g, 63.50 mmol) in acetic anhydride (707 ml, 7.49 mol) was heated at 120°C during the night. After subsequent distillation to remove acetic anhydride, the resulting residue was dried in vacuo to give the title compound (15.92 g, 99.4% yield).

1H NMR (CDCI3) δ 8.32 (m, 1H), 8.12 (s, 1H), 6.74 (m, 1H), 4.03 (s, 3H).

Intermediate connection A5

3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yltrifluoromethanesulfonate.

Step 1. Preparation of 4-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile. To a solution of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile (intermediate A4, 50 g, 198.4 mmol) and 1-methyl-4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (49.53 g, 238.0 mmol) in dioxane (660 ml) was added 2 M Na ₂ CO ₃ ( _aq ) (297, 5 ml, 595.1 mmol). The reaction mixture was purged with nitrogen for 20 minutes, then Pd(PPh ₃ ) ₄ (4.584 g, 3.967 mmol) was added, then purged with nitrogen for another 5 minutes. The reaction mixture was heated at 80°C for 18 h, then cooled to room temperature and stirred vigorously for 2 h. The suspension was filtered under vacuum, washed successively with H ₂ O (2x300 ml) and MTBE (3x300 ml), then dried in vacuo overnight to give the title compound, which was used in the next step without further purification (52.62 g). MS (apci) m/z = 254.1 (M+H).

Step 2. Preparation of 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3carbonitrile. To a suspension of 4-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3carbonitrile (52.62 g, 207.8 mmol) in DCE (2 L) was added AlCl ₃ (92.86 g, 696.42 mmol) and the reaction mixture was stirred at 80°C for 3 hours. Additional AlCl 3 (2.5 g, 18.75 mmol) was added and the reaction mixture was refluxed for night. After cooling to room temperature, the reaction mixture was diluted with DCE (1 L) and then quenched with portions of H ₂ O (5x500 ml). The mixture was stirred at room temperature for 3 hours, then the resulting suspension was filtered under vacuum and the cake was dried on the filter in a vacuum oven (40°C) to give the title compound, which was used in the next step without further purification (43.69 g) . MS (apci) m/z = 239.9 (M+H).

1H NMR (d ⁶ -DMSO) δ 11.38 (s, 1H), 8.74 (d, 1H), 8.50 (s, 1H), 8.21 (s, 1H), 7.94 (s , 1H), 6.96 (d, 1H), 3.88 (s, 3H).

Step 3. Obtaining 3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-4-160 041220 yltrifluoromethanesulfonate. To a suspension of 4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5a]pyridine-3-carbonitrile (43.69 g, 182.6 mmol) in DMA (365 ml) was added DIEA (63.6 ml, 365.3 mmol) followed by 1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide (71.77 g, 200.9 mmol). The resulting solution was stirred at room temperature for 2 h, and then slowly poured into H ₂ O (4 l). The resulting suspension was stirred for 2 h, then filtered under vacuum. The filter cake was washed with H ₂ O (3x500 ml) and air dried overnight. The filter cake was then dissolved in DCM (1.6 L) and the phases of the resulting biphasic mixture were separated. The organic layer was dried over anhydrous MgSO ₄ , filtered through Celite® and washed with DCM. The combined organic layers were concentrated to give the title compound as 90% pure light brown solid (64.3 g, 95% yield). The purity of the title compound can be further increased to >95% by silica chromatography (0-90% acetone/hexanes). ¹⁹ F NMR (CDCl ₃ ) δ -72.0.

1H NMR (CDCl ₃ ) δ 8.66 (d, 1H), 8.29 (s, 1H), 7.77 (d, 1H), 7.70 (s, 1H), 7.55 (d, 1H ), 4.01 (s, 3H). Intermediate connection A6

tert-Butyl-4-(5-(3-cyano-6-(1-methyl-1Н-πirazol-4-yl)πirazolo[1,5-a]πiridin-4-yl)πiridin-2yl)piperazin-1 -carboxylate.

To a mixture of 3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl-trifluoromethanesulfonate (Intermediate A5; 10.0 g, 26.9 mmol ) and tert-butyl 4-(5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)pyridin-2-yl)piperazin-1-carboxylate (12.6 g, 32 .3 mmol) in dioxane (250 ml) was added a 2 M solution of Na ₂ CO ₃ (in _Odn .) (14.3 g, 135 mmol) and the reaction mixture was purged with nitrogen for 15 minutes, then Pd ₂ (dba) ₃ was added (1.23 g, 1.35 mmol) and X-Phos (2.57 g, 5.39 mmol). The mixture was purged with nitrogen for another 5 minutes and then heated at 80° C. overnight. After cooling to room temperature, the reaction mixture was poured into H ₂ O (1.5 L) and stirred for 2 h. The resulting suspension was filtered and washed successively with H ₂ O (3x200 ml), MTBE (4x100 ml) and hexanes (4x100 ml) to give the title compound as a solid after drying in vacuo overnight (12 g, 92% yield). MS (apci) m/z = 485.2 (M+H).

Intermediate connection A7

6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3 carbonitrile dihydrochloride.

To a solution of tert-butyl-4-(5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4yl)pyridin-2-yl)piperazine -1-carboxylate (A6, 12.0 g, 24.77 mmol) in MeOH (12 ml) and DCM (50 ml) was added HCl (5-6 M in iPrOH, 49.53 ml, 247.7 mmol). After stirring at room temperature for 21 h, the reaction mixture was diluted with MeOH (50 ml) and DCM (50 ml). The suspension was stirred at room temperature until LCMS indicated completion of the reaction. The reaction mixture was filtered, washed with Et ₂ O (5x50 ml) and then dried for 19 h in a vacuum oven at 45° C. to give the title compound (9.97 g, 88% yield). MS (apci) m/z = 385.1 (M+H).

Compound of Formula I

4-(6-(4-((6-Methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4yl)pyrazolo[1 ,5-a]pyridine-3-carbonitrile.

Method 1. Room temperature solution of 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3- carbonitrile dihydrochloride (A7; 0.125 g, 0.273 mmol) in dry DMA (2.5 ml) was treated with TEA (114 μl, 0.820 mmol), Me ₄ N (AcO) ₃ BH (144 mg, 0.547 mmol) and 6-methoxynicotinaldehyde ( 0.0750 g, 0.547 mmol). The resulting mixture was stirred overnight

- 161 041220 at room temperature, then extinguished with water and CHCl ₃ and left to stir for 30 minutes. The resulting biphasic mixture was filtered through a PS sintered filter and the aqueous layer was washed

CHCl ₃ . The organic filtrate was concentrated in vacuo and the C18 residue was purified by reverse phase chromatography (5-90% ACN/water as gradient eluent) to give the title compound (56 mg, 41% yield). MS (apci) m/z = 506.0 (M+H).

Method 2 6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3 α-carbonitrile tetrahydrochloride (A7; 19.0 g) and 6-methoxynicotinaldehyde (7.37 g). DMSO (247 ml) was added followed by triethylamine (15 ml). The yellow suspension was stirred at room temperature for ~2.5 h and then sodium triacetoxyborohydride (15.2 g) was added in one portion and the reaction mixture was heated to 30° C. and stirred overnight. The reaction was considered complete, according to HPLC, and the mixture was cooled in an ice/water bath to 19°C. Water (550 ml) was added slowly keeping the temperature below 30°C. The suspension was stirred for 3.5 hours and then filtered and the filter cake was washed with water (2x285 ml). The solid was dried in a vacuum oven at 45° C. to give the title compound (18.0 g, 99.4%). The crystalline solid was analyzed by RPD to obtain a diffraction pattern corresponding to form A of formula I.

Example 2 Synthesis of compounds of formula II-IV.

Intermediate B1

N=\ ί T

4-bromo-6-hydroxypyrazolo[1,5-α]pyridine-3-carbonitrile.

Stage 1. Obtaining 1-amino-3-bromo-5-methoxypyridin-1-ium-2,4,6-trimethylbenzenesulfonate. To a solution of O-(mesitylsulfonyl)hydroxylamine (intermediate R1, 26.6 g, 117 mmol) in dichloromethane (570 ml) cooled to 0°C was added 3-bromo-5-methoxypyridine (22.1 g, 117 mmol). The reaction mixture was stirred for 1 h at 0°C, then treated with additional 3-bromo-5-methoxypyridine (250 mg, 1.39 mmol) and stirred for another 2 h at 0°C. The reaction mixture was diluted with Et ₂ O (600 ml), stirred at 0°C for 10 min and then filtered under vacuum, and washed with Et ₂ O (3x250 ml). After reducing the volume by about 1/3, more precipitate was obtained from the filtrate, which was collected by filtration. Both filter cakes were dried in vacuo to give the title compound (39.3 g, 83% yield).

¹ H NMR (CDCl ₃ ): δ 9.25 (br, 1H), 8.99 (m, 1H), 8.74 (m, 1H), 7.46 (m, 1H), 6.83 (s , 2H), 3.92 (s, 3H), 2.65 (s, 6H), 2.22 (s, 3H).

Step 2. Preparation of ethyl 6-bromo-4-methoxypyrazolo[1,5-α]pyridine-3-carboxylate and ethyl 4-bromo6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate. To a magnetically stirred white suspension of 1-amino-3-bromo-5-methoxypyridin-1-ium-2,4,6-trimethylbenzenesulfonate (33.24 g, 82.42 mmol) in DMF (82 ml) at room temperature TEA (22.98 ml, 164.8 mmol) was added, then ethyl propiolate (16.71 ml, 164.8 mmol) was added dropwise. After vigorous stirring for 2 days, the reaction mixture was slowly quenched by adding it in portions to rapidly stirred ice water (820 ml). The mixture was stirred at room temperature for 10 minutes and then filtered under vacuum. The collected solid was washed with water and air dried to give the title compounds as an orange solid in an isomeric ratio of about 4:1 (by 1H NMR) with the 6-Br isomer as the main isomer (21 g). The wet solid isomeric mixture (about 75% w/w) was used directly in step 3 without further purification. MS (apci) m/z = 298.9, 300.9 (M+H). The regioisomeric ratio was determined from the MeO chemical shift by 1H NMR (CDCl ₃ ) δ 3.98 (6-Br isomer) versus 3.83 (4-Br isomer).

Step 3. Preparation of 6-bromo-4-methoxy-pyrazolo[1,5-a]pyridine (intermediate B1) and 4-bromo-6-methoxy-pyrazolo[1,5-a]pyridine. An isomeric mixture of ethyl 6-bromo-4-methoxypyrazolo[1,5a]pyridine-3-carboxylate and ethyl 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate from step 2 (15 g, 50.1 mmol) was added to 48% HBr (114 ml) with stirring, then heated at 80°C for 90 minutes, then stirred at room temperature overnight. The resulting suspension was filtered under vacuum and washed with water. The aqueous filtrate and filter cake were treated independently of each other. The filter cake was dissolved in MTBE and filtered under vacuum to remove insoluble impurities. The MTBE filtrate was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to give 6-bromo-4-methoxypyrazolo[1,5-a]pyridine as a beige solid (ca. 98:2 6-/4-Br; 5 .08 g). MS (apci) m/z = 226.9, 228.9 (M+H).

1 H NMR (CDCl ₃ ): δ 8.26 (m, 1H), 7.82 (d, 1H), 6.61 (m, 1H), 6.43 (m, 1H), 3.94 (s, 3H).

The original aqueous filtrate of the reaction mixture was independently extracted with EtOAc (2x500 ml). The combined organic layers were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The crude residue was dissolved in DCM (50 ml) and then filtered to remove insoluble solids.

- 162 041220 substances. Concentration of the filtrate with DCM in vacuo followed by chromatography on silica (0 to 50% EtOAc/hexanes) gave a second batch of 6-bromo-4-methoxypyrazolo[1,5a]pyridine (Intermediate 1) as a white solid (upper spot Rf, 2.06 g) as well as the minor isomer of the title compound 4-bromo-6-methoxypyrazolo[1,5-a]pyridine, also as a white solid (lower spot Rf, 1.32 g). MS (apci) m/z = 226.9, 228.9 (M+H).

1 H NMR (CDCl ₃ ): δ 8.02 (m, 1H), 7.85 (d, 1H), 7.17 (d, 1H), 6.55 (m, 1H), 3.80 (s, 3H).

Stage 4. Obtaining 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde. A solution of 4-bromo-6methoxypyrazolo[1,5-a]pyridine (5.0 g, 22 mmol) in DMF (220 ml) was cooled to 0° C. and then treated slowly with POCl ₃ (6.2 ml, 66 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The reaction mixture was cooled to 0° C., quenched with water (220 ml) and basified with 6 M NaOH( _BODH .) to pH 9-10. The reaction mixture was stirred for 1 h and then filtered under vacuum. The solid was washed successively with water (3x50 ml) and MTBE (3x50 ml). The collected solid was suspended in DCM (500 ml) and washed in an ultrasonic bath for 30 minutes and then filtered under vacuum. The filtrate was saved and the filter cake was dissolved in water (300 ml) and extracted with DCM. The organic extracts as well as the retained DCM filtrate were combined and dried over anhydrous Na ₂ SO ₄ , then filtered and concentrated in vacuo to give the title compound (4.84 g, 86% yield). MS (apci) m/z = 256.9 (M+H).

Stage 5. Obtaining 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime. Water (127 ml) and hydroxylamine hydrochloride were added to a suspension of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde (4.84 g, 19.0 mmol) in EtOH (253 ml) at room temperature. (1.98 g, 28.5 mmol). After stirring at 50°C overnight, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was suspended in water (150 ml) and then slowly quenched with saturated NaHCO ₃ ( _BODH .) solution (30 ml). After stirring for 1 h at room temperature, the suspension was filtered under vacuum and the filter cake was washed successively with H ₂ O (500 ml) and MTBE (100 ml) to give the title compound as a 2:1 E/Z mixture (5 .13 g, quantitative yield), which was used in the next step without further purification. MS (apci) m/z = 271.9 (M+H).

Step 6. Preparation of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile. An E/Z mixture of 4-bromo6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime (4.95 g, 18.33 mmol) in acetic anhydride (172.9 ml, 1833 mmol) was stirred at 140°C in for 25 h and then cooled to room temperature. The resulting suspension was further cooled in an ice bath for 15 minutes and then filtered under vacuum and washed successively with water (200 ml) and MTBE (300 ml) to give the title compound (3.74 g, 81% yield).

1 H NMR (d ⁶ -DMSO): δ 8.70 (s, 1H), 8.60 (s, 1H), 7.78 (s, 1H), 3.83 (s, 3H).

Step 7 Preparation of 4-bromo-6-hydroxypyrazolo[1,5-a]pyridine-3-carbonitrile: Suspension of 4bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile (50.0 g, 198 4 mmol) in DCE (500 ml) was treated with AlCl ₃ (79.34 g, 595.1 mmol). In an atmosphere of N ₂ ( _Γ ), the resulting mixture was stirred for 19 h at 76°C, then cooled to room temperature. Using THF (1750 ml) as wash solvent, the reaction mixture was poured into a mechanically stirred suspension of sodium sulfate decahydrate (10 eq., 639 g) in THF (1000 ml). After stirring overnight at room temperature, the resulting suspension was filtered and the solid was washed with additional THF (2x250 ml). The filtrate was concentrated in vacuo and the resulting solid was dried under high vacuum for 3 days to give the title compound (46.18 g, 98% yield) of sufficient purity for further use.

¹ H NMR (d ⁶ -DMSO): δ 10.48 (s, 1H), 8.58 (s, 1H), 8.38 (d, 1H), 7.64 (3, 1H).

Intermediate B2

4-bromo-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile.

A mixture of 4-bromo-6-hydroxypyrazolo[1,5-a]pyridine3-carbonitrile (intermediate B1; 10.0 g, 42.0 mmol) and K ₂ CO ₃ ( _mb ) (17 .4 g, 126 mmol) in DMF (50 ml) with 2,2-dimethyloxirane (36.9 ml, 420 mmol). After sealing the flask, the reaction mixture was stirred for 12 h at 60°C, then for 12 h at 85°C. The mixture was left to cool to room temperature. The room temperature mixture was poured into water (400 ml) then stirred for 1 hour at room temperature. The resulting suspension was filtered under vacuum and the filter cake was washed with water. The solid was collected and dried in vacuo to give the title compound pure (11 g, 84% yield).

- 163 041220

Intermediate B3

4-(6-fluoropyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile.

4-Bromo-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile mixture (Intermediate B2; 10.0 g, 32.2 mmol), 2-fluoro-5 -(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)pyridine (10.8 g, 48.4 mmol) and Pd(PPh ₃ ) ₄ (1.12 g, 0.967 mmol) in dioxane (200 ml) was treated with a 2 M solution of Na ₂ CO ₃ ( _B o _DH .) (64.5 ml, 129 mmol). The resulting mixture was purged with Ar _(g) , then stirred for 12 h at 85°C in an atmosphere of N ₂ ( _Γ ). After cooling to room temperature, the resulting mixture was poured into cold water (1.5 L). The pH of the mixture was adjusted to about pH 6 by adding 10% citric acid. After stirring for 1 hour at room temperature, the resulting suspension was filtered under vacuum. The solid was collected and dried in vacuo to give pure title compound (10 g, 95% yield).

Intermediate B4

4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-а]pyridine- 3-carbonitrile dihydrochloride.

Stage 1. Obtaining tert-butyl-3-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5a]pyridin-4-yl)pyridin-2-yl)-3, 6-diazabicyclo[3.1.1]heptane-6-carboxylate. 4-(6-Fluoropyridin3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile mixture (Intermediate B3; 1.70 g, 8.55 mmol) , tert-butyl ester of 3,6-diazabicyclo[3.1.1]heptane-6-carboxylic acid (1.70 g, 8.55 mmol) and K ₂ CO ₃ ( _tv ) (7.88 g, 57.0 mmol ) in DMSO (7 ml) was stirred for 12 h at 90°C. The resulting thick suspension was diluted with an additional amount of DMSO (2 mL) and stirred for 12 h at 90°С. The mixture was cooled to room temperature and diluted with water (100 ml). The aqueous mixture was washed with DCM. The combined organic extracts were dried over anhydrous MgSO ₄ ( _TV ), filtered and concentrated in vacuo. The crude residue was purified by silica chromatography (30-80% EtOAc/hexanes as gradient eluent) to give the pure title compound (2.87 g, 100% yield). MS (apci) m/z = 505.2 (M+H).

Stage 2. Preparation of 4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2methylpropoxy)pyrazolo[1,5-a] pyridine-3-carbonitrile dihydrochloride.

Solution of tert-butyl-3-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridin-4yl)pyridin-2-yl)-3,6-diazabicyclo [3.1.1]heptane-6-carboxylate (see step 1; 3.05 g, 6.04 mmol) in DCM (20 ml) was treated with 4N. HCl solution in dioxanes (15.1 ml, 60.4 mmol). The resulting mixture was stirred for 12 hours at room temperature and then concentrated in vacuo. The crude residue was diluted with DCM and toluene and then sonicated, then concentrated in vacuo to give the title compound as the dihydrochloride salt (2.44 g, quant.). MS (apci) m/z = 405.2 (M+H).

Compound of Formula II

6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6diazabicyclo[3.1.1]heptan-3-yl)pyridine-3 -yl)pyrazolo[1,5-a]pyridine-3-carbonitrile.

To a mixture of 4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2methylpropoxy)pyrazolo[1,5-a]pyridine- 3-carbonitrile dihydrochloride (Intermediate B4, 6.0 g, 12.6 mmol) in DCM (30 mL) was added triethylamine (5.3 mL, 37.7 mmol) followed by 6 methoxynicotinaldehyde (2.59 g, 18.9 mmol) and then in one portion of sodium triacetoxyborohydride (5.33 g, 25.1 mmol) with stirring. Additional DCM (30 ml) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into water (200 ml) and extracted with DCM (200 ml). After phase separation, the organic layer was washed with water (2x200 ml). The combined aqueous washings were re-extracted into DCM (200 ml). Volume

- 164 041220 dinyne organic extracts were washed with brine (250 ml), passed through a fritted phase separator and treated with activated charcoal (Darco G-60, 6 g). After stirring at room temperature for 2 h, the mixture was filtered under vacuum, washed with DCM (3x10 ml). The filtrate was treated with 3-(trimethoxysilyl)propane-1-thiol (11 g, 7.4 mmol) and stirred on a magnetic stirrer overnight. The mixture was filtered under vacuum and concentrated on a rotary evaporator to about 200 ml. To the DCM solution obtained above, heptane (150 ml) was added in portions with stirring until a cloudy solution was obtained. After 90 minutes of stirring, the white suspension was filtered under vacuum, washed with heptane (200 ml) to give the title product as a fine crystalline white powder (3.0 g, 59%).

Recrystallization of the compound of formula II in DMSO/water was carried out as follows. The reaction flask was charged with the compound of formula II (10.1 g) and DMSO (110 ml). The mixture was heated to 50°C until complete dissolution of the solid. The mixture was cooled to 25°C and filtered through a fine filter. DMSO (10 ml) was loaded through the filter as a wash solution. The resulting solution was heated to 45° C. and water (5 ml) was added slowly. The mixture was stirred for 30 minutes and a seed layer was obtained. Water (25 ml) was added over 1 hour and the slurry was allowed to mature at 45° C. for another 1 hour. Then the suspension was left to cool to 25°C and stirred for 2 h. The suspension was filtered and the filter cake was washed with water (20 mlx3), MeOH (20 mlx2), and MTBE (20 mlx2). The filter cake was dried at room temperature in a vacuum oven to give 9.35 g (74%) of the title compound. MS (apci) m/z = 526.2 (M+H).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 8.64 (d, 1H, J=2.3 Hz), 8.55 (s, 1H), 8.38 (d, 1H, J=2 .3 Hz), 8.04 (d, 1H, J=2.3 Hz), 7.80 (dd, 1H, J=8.6, 2.3 Hz), 7.64 (dd, 1H, J = 8.6, 2.3 Hz), 7.27 (d, 1H, J=2.0 Hz), 6.76 (d, 1H, J=8.6 Hz), 6.73 (d, 1H , J=8.2 Hz), 4.67 (s, 1H), 3.85 (s, 2H), 3.79 (s, 3H), 3.72 (d, 2H, J=12.5 Hz ), 3.64 (d, 2H, J=5.9 Hz), 3.51 (bd, 2H), 3.47 (s, 2H), 2.47 (m, 1H), 1.55 (d , 1H), 1.20 (s, 6H).

Compound of Formula III

O

6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3yl)pyridin-3-yl)pyrazolo[1 ,5-a]pyridine-3-carbonitrile.

To a mixture of 4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2methylpropoxy)pyrazolo[1,5-a]pyridine- 3-carbonitrile dihydrochloride (intermediate B4, 4.50 g, 9.43 mmol) in DMSO (63 ml) was added 6-methoxynicotinic acid (1.73 g, 11.3 mmol) followed by Hunig's base (5.25 ml , 30.2 mmol) and HATU (4.30 g, 11.3 mmol). The mixture was stirred at room temperature for 1 h, then poured into a mixture of water (500 ml) and saturated NaHCO ₃ solution (50 ml), then stirred for another 4 h. The suspension was vacuum filtered and the collected solid was first triturated with MTBE (100 ml), then purified by flash chromatography on silica (1-15% MeOH in DCM with 1% NH4OH) to give the title product. The resulting product was recrystallized by dissolving in a minimum amount of DCM, then slowly adding MTBE until a suspension formed. After filtration followed by drying under high vacuum at 40-45° C. for 2 days, the title product was obtained as a crystalline white powder (4.0 g, 78.6%).

Further recrystallization of the title product in CH ₃ CN/water was carried out as follows. A mixture of the title product (37 g) in CH3CN (222 ml) was heated to reflux to give a clear solution. Water (333 ml) was then slowly added at the same temperature, with stirring. After the addition, heating was stopped and the mixture was allowed to cool to room temperature and stirred overnight. The solid was collected by vacuum filtration and dried under high vacuum at 40-45° C. overnight to give the title product as a crystalline white powder (24 g, 65%).

1H NMR (DMSO-d ⁶ ) δ 8.60-8.65 (d, 1H), 8.53 (s, 1H), 8.49-8.51 (m, 1H), 8.28-8, 31 (d, 1H), 7.91-7.95 (m, 1H), 7.73-7.78 (m, 1H), 7.23-7.25 (m, 1H), 6.81- 6.85 (m, 1H), 6.65-6.69 (d, 1H), 4.84-4.94 (shm, 1H), 4.66 (s, 1H), 4.51-4, 63 (shm, 1H), 4.04-4.20 (shm, 1H), 3.88 (s, 3H), 3.83 (s, 2H), 3.60-3.63 (m, 2H) , 3.42-3.53 (shm, 1H), 2.75-2.85 (m, 1H), 1.63-1.69 (m, 1H), 1.18 (s, 6H). MS (apci) m/z = 540.2 (M+H).

- 165 041220

Compound of formula IV

6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin3-yl)pyrazolo[1,5-a]pyridine -3-carbonitrile.

To a suspension of 4-(6-fluoropyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3carbonitrile (intermediate B3, 90 mg, 0.276 mmol) in DMSO ( 2 ml) DIEA (193 μl, 1.10 mmol) was added followed by 4-(pyridin-2-ylmethyl)piperidin-4-ol hydrochloride (69 mg, 0.303 mmol). The reaction mixture was stirred at 90°C for 60 h, then directly purified using C18 reverse phase chromatography (using 5-95% acetonitrile in water with 0.1% TFA as a gradient eluent). Fractions containing the desired product were combined, partially concentrated in vacuo to remove ACN, then partitioned between saturated NaHCO ₃ solution ( _vogn .) and DCM. The biphasic mixture was extracted with additional DCM (2x). The combined organic extracts were dried over anhydrous MgSO ₄ ( _TV ), filtered and concentrated in vacuo. The residue was sonicated in Et ₂ O (2 ml) and then concentrated in vacuo to give the title compound (48 mg, 35% yield). MS (apci) m/z = 499.2 (M+H).

Additional recrystallization and isolation of form A. The compound of formula IV (10 g) was added to the reaction vessel, DMSO (50 ml) was added and the mixture was stirred. After 10 minutes the compound of formula IV had completely dissolved and then water (4x5 ml, 20 ml total) was added dropwise. After stirring for 10 minutes, a thin suspension formed. Additional water (3x5 ml, total 15 ml) was added and after stirring for 30-60 min the suspension was filtered. The filter cake was washed with water (10 ml), MTBE (50 ml) and then the solid was dried at room temperature under vacuum to give 9.2 g of the compound of formula IV. The crystalline solid was analyzed by DSC and RPD to obtain the corresponding endotherm and form A diffraction pattern for the compound of formula IV.

Example 3 Selection of polymorphic salts for a compound of formula I.

A. Solubility Tests for the Free Base of a Compound of Formula I.

The solubility of the free base of the compound of formula I was tested in 13 solvents at 25 and 60°C. Solubility in mixed solvent systems such as DCM/EtOH, DMSO/EtOH, DMSO/water, THF/EtOH and THF/water was also determined.

Solubility tests were carried out as follows. 10-20 mg of free base was stirred in 1 ml of solvent overnight at room temperature; the solubility was recorded at 25°C. Then the samples were heated to 60°C. If the solid dissolved on heating, additional free base was added in portions of 3-5 mg. The results of the solubility test are presented in table. 5-10 and in Fig. 1A-1E.

Table 5

Total solubility of the free base of the compound of formula I

Solvent Solubility at 25 °C (mg/ml) Does the solid dissolve at 60°C? EtOAc 0.36 No iPAc 0.25 No Meon 0.07 No EtOH 0.04 No toluene 0.35 No acetone 0.44 No IPA 0 No IEC 0.69 No THF 2.59 Yes (10.2-13.0 mg/ml) ACN 0.33 No DMA 12.9 >40 mg/ml dhe 4.6 Didn't study DXM 15.5 N/A water 0.07 N/A 0.1 MHC1 1.5 Didn't study

- 166 041220

The free base showed solubility of 2.59 mg/ml, 12.9 mg/ml and 15.5 mg/ml in THF,

DMA and DXM, respectively. For most solvent systems studied, the free base did not dissolve after heating to 60°C. 10-13 mg of the free base dissolved in THF at 60° C., although the solution turned milky at 13 mg. The DMA solubility study at 60° C. was terminated after the addition of a total of 40 mg of the free base.

Table 6

Solubility of the free base of the compound of formula I in DCM/EtOH at room temperature

% DCM in EtOH Solubility (mg/ml) 0 0.05 10 0.06 20 0.26 thirty 0.87 40 2.53 50 7.42 60 16.6 70 22.7 80 29.0 90 34.2 100 17.3

The solubility of the free base in DCM/EtOH mixtures is shown in FIG. 1A and in table. 6 above.

Table 7

Solubility of the free base of the compound of formula I in DMSO/EtOH at room temperature

% DMSO in EtOH Solubility (mg/ml) 0 0.05 10 0.09 20 0.18 thirty 0.39 40 0.61 50 1D4 60 1.80 70 2.57 80 4.28 90 4.44 100 6.96

The solubility of the free base in DMSO/EtOH mixtures is shown in FIG. 1B and table. 7 above.

- 167 041220

Table 8

Solubility of the free base of the compound of formula I in DMSO/H2O at room temperature

% DMSO in H ₂ O Solubility (mg/ml) 0 0 10 0.09 20 0.08 thirty 0.09 40 0.10 50 0.09 60 0.10 70 0.15 80 0.35 90 1.52 100 6.96

The solubility of the free base in DMSO/H2O mixtures is shown in FIG. 1C and in table. 8 above.

Table 9

Solubility of the free base of the compound of formula I in THF/EtOH at room temperature

% THF in EtOH Solubility (mg/ml) 10 0.29 20 0.34 thirty 0.55 40 0.82 50 1.38 60 1.97 70 2.51 80 3.09 90 3.18 100 2.59

The solubility of the free base in THF/EtOH mixtures is shown in FIG. 1D and in table. 9 above.

Table 10

Solubility of the free base of the compound of formula I in THF/H2O at room temperature

% THF in EtOH Solubility (mg/ml) 10 0.15 20 0.13 thirty 0.35 40 1.36 50 2.50 60 3.76 70 6.17 80 6.65 90 6.56 100 2.59

The solubility of the free base in THF/H ₂ O mixtures is shown in FIG. 1E and in table. 10 above.

Studies were carried out to determine the conditions for the possible recrystallization of free os

- 168 041220 new compounds of formula I, as shown in table. eleven.

Table 11

Attempts to recrystallize the free base of a compound of formula I

Scale (g) Solvent (vol.)// Anti-solvent (vol.) Temperature (°C) Time (h) LTF (mg/ml) Notes 0.05 DMSO (18)// EtOH 200 proof (27) from 70 °C to room, t-ry 16 1.20 The free base was dissolved in DMSO at 70°C. EtOH was added slowly. A solid formed slowly 1 hour after the start of cooling. Refrigerate during the night. Filtered. 78.6% were isolated. 0.10 DMSO (18)// EtOH 200 proof (25) from 70 °C to room, t-ry 16 1.20 The free base was dissolved in DMSO at 70°C. EtOH was added slowly until cloudy. At 70 °C a solid formed rapidly. Refrigerate during the night. Filtered. Allocated 83.6%. 0.10 DMSO (18)// EtOH 200 proof (18) from 70 °C to room, t-ry 16 2.26 The free base was dissolved in DMSO at 70°C. EtOH was added slowly until cloudy. Refrigerate during the night. Filtered. Allocated 80.5%.

5.0 DMSO (20)// EtOH 200 proof (20) from 70 °C to room, t-ry 16 1.6 The free base was dissolved in DMSO at 70°C. EtOH was added slowly until cloudy. Refrigerate during the night. Filtered. Allocated 88%. 0.12 DMSO (18)//EUN 200 proof (33) from 70 °C to room, t-ry 16 1.00 The free base was dissolved in DMSO at 70°C. EtOH was added slowly until cloudy. Refrigerate during the night. Filtered. Allocated 85%. 0.10 DMSO (18)//EUN 200 proof (28) from 70 °C to room, t-ry 16 1.03 The free base was dissolved in DMSO at 70°C. Cooled to 40°C, the solution became cloudy, EtOH was added slowly. A solid formed immediately. Allocated 78.2%. 0.10 DMSO (18)//EUN 190 proof (28) from 70 °C to room, t-ry 72 0.70 The free base was dissolved in DMSO at 70°C. EtOH was added slowly until cloudy. Refrigerate during the night. Not filtered.

- 169 041220

0.10 90% DCM/EtOH (25)// EtOH 200 proof (19) from 40 °C to room, t-ry 16 2.38 The free base was dissolved in 90% DCM in EtOH at 40°C. EtOH was added slowly until cloudy. The solid decomposed slowly at 40 °C. Refrigerate during the night. 0.10 66% DCM/EtOH (30)// EtOH 200 proof (20) from 40 °C to room, t-ry 16 3.70 The free base was dissolved in 66% DCM in EtOH at 40°C. EtOH was added slowly, the solution remained a solution. Refrigerate during the night. In the morning a solid was observed. 1.0 DMSO (6)//H2O (24) from 100 °C to room, t-ry 24 1.1 The free base was dissolved in DMSO at 100°C. H ₂ O was added slowly to form a slurry. Heated for 10 hours at 100°C, then cooled overnight to room temperature. t-ry. Washed with 20 volumes of H ₂ O. Dry in an oven. Exit - TBD. 1.0 DMSO (10)//EtOH 200 proof (20) from 100 °C to room, t-ry 24 0.9 The free base was dissolved in DMSO at 100°C. Cooled down to 75°C. Slowly added EtOH to obtain a suspension. Stirred in for 10 hours at 75 °C, then cooled overnight to room. t-ry. Filtered. Washed with 20 volumes of H ₂ O. Dry in an oven. Exit - TBD. 1.0 EtOH 200 proof (20) from 75 °C to room, t-ry 24 0.6 The free base was suspended in EtOH at 75°C. Heated for 24 hours at 75°C. Cooled to room temperature, t-ry and filtered. Washed with 20 volumes of H ₂ O. Dry in an oven. Exit - TBD.

The free base of the compound of formula I was found to dissolve in 18 volumes of DMSO at 70°C and in 6 volumes of DMSO at 100°C. EtOH was added to the DMSO solution at 70°C; the solution turned cloudy after ~18 volumes of EtOH (1:1 DMSO/EtOH). An additional amount of EtOH led to the destruction of the solid at 70°C.

B. Characterization of the free base of the compound of formula I.

The compound of formula I, prepared as described in Example 1 above, was analyzed by polarized light microscopy (MPS), RPD, DSC, TGA, DSP, FT-IR and 1H NMR and identified as form A (Fig. 2A-2G ). The X-ray powder diffraction pattern is shown in Fig. 2A. The peaks in the X-ray powder diffraction pattern are shown below in

- 170 041220 tab. 12. An overlay of X-ray powder diffraction patterns from different batches of the free base of the compound of formula I is shown in FIG. 2B. A small endothermic event was observed on DSC starting at about 187°C, followed by an endothermic event starting at about 224°C (FIG. 2C). The isothermal (25°C) EAF curve is shown in FIG. 2D. TGA analysis showed a weight loss of 1.1% from the start of heating to about 238°C (FIG. 2E). The Fourier IR spectrum is shown in Fig. 2F. The NMR spectrum (in DMSO-d ⁶ ) is shown in FIG. 2g.

Table 12

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24.12 3.69 5.20 5.60 24.57 3.62 30.30 33.00 24.94 3.57 1.80 2.00 25.32 3.51 5.00 5.40 25.60 3.48 2.50 2.70 26.45 3.37 8.00 8.70 26.65 3.34 8.90 9.60 27.17 3.28 2.50 2.70 27.72 3.22 15.70 17.10 28.25 3.16 1.30 1.40 28.56 3.12 3.80 4.10 29.31 3.04 1.60 1.70 29.64 3.01 3.40 3.70 30.16 2.96 0.80 0.80 30.75 2.91 3.50 3.80 31.24 2.86 2.00 2.20 31.72 2.82 1.00 1.10 32.19 2.78 0.50 0.60 33.16 2.70 1.30 1.40 34.05 2.63 2.30 2.50 34.40 2.60 1.10 1.20 35.02 2.56 0.80 0.90 35.44 2.53 1.40 1.50

C. Initial selection of salts.

An initial selection of salts was made for the compound of formula I using counterions of inorganic and organic acids and DCM or DMA as solvent (solubility of the free base of the compound of formula I in DCM = 15.5 mg/ml; solubility of the free base of the compound of formula I in DMA = 12, 9 mg/ml). Acid and solvent combinations are shown in Table 1 below. 13.

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Table 13

The results of the primary selection of salts

Acid dissolve Tel Appearance Source ³ After 13 hours After heating to 40 °C for 4.5 h. After heating to 40°C and cooling overnight After evaporation After dilution with MTBE Maleic acid ¹ DXM Firm with a pink tint Firm with a faint pink tinge N/A N/A N/A N/A L-Dairy acid DXM Transparent, no solids Transparent, without hard water, with a pink tint N/A N/A Brown, sticky, May-like substance when AI dries N/A Acetic acid DXM Transparent, no solids Transparent, without hard water, with a yellow tint N/A N/A Beige solid when dry AI N/A Adipine acid DXM A little cloudy, a little "solid" on the walls of the flask A little cloudy, hard to tell if there is solid A little cloudy, hard to tell if there is solid Slightly cloudy, small particles float in the solution N/A N/A Fumaric acid DXM Turbid Cloudy, fine solid particles Cloudy, fine solid particles Cloudy, fine solid particles N/A N/A D- Glutamic acid DXM Turbid A little cloudy, hard to tell if there is solid Cloudy, milky Cloudy, milky N/A N/A D-malic acid ¹ DXM Slightly pink solids Slightly pink solids N/A N/A N/A N/A

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L-malic acid ¹ DXM White solid particles Solid particles, white (with a pink tint) N/A N/A N/A N/A MSOH DXM White solid particles Clear, brown solid on walls Transparent, milky particles on the walls (loss of solvent on a loose lid) Clear milky solid on the walls N/A N/A pTsOH. _H2O DXM Cloudy, milky Cloudy, milky Cloudy, milky Cloudy, milky N/A N/A Benzenesulfo new acid DXM Transparent, with a light yellow tint Clear, light pink, slightly solid A little solid on the walls, milky solution A little solid on the walls, milky solution N/A N/A Benzoic acid DXM Transparent, with a light yellow tint Transparent, light yellow N/A N/A Beige solid when dry AI N/A Malonic acid DXM Transparent, with a light yellow tint White, cloudy, some solid on the walls of the flask A little solid on the walls, small particles A little solid on the walls, small particles N/A N/A HBg/N ₂ O DXM Turbid, pink "sticky" luck on the walls of the flask Cloudy, milky, pink "sticky" luck on the walls of the flask Cloudy, milky, pink solid on the sides of the flask Cloudy, milky, pink solid on the sides of the flask N/A N/A

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Amber acid DXM Transparent, with a light yellow tint Very small particles Fine particles, some solid matter on the walls Very small particles N/A n/a L-tartaric acid ² DXM Cloudy, small particles Cloudy, can't tell if solids are being filtered out Solid particles with a pink tint Solid particles with a pink tint N/A n/a HC1 / H ₂ O DXM Turbid Cloudy, milky Brown "sticky" solid on walls, milky Brown "sticky" solid on the walls, milky n/a n/a

Sulfuric acid DXM Clear, some solid stuck to the sides of the flask Clear, some solid stuck to the sides of the flask Solid on the walls of the flask, some solid floating Clear solution/white "solid" on the sides of the flask n/a N/A Phosphoric acid DXM Transparent, with a yellow tint Transparent, with a yellow tint Clear yellow solution, brown solid adhered to flask Clear yellow solution, brown solid adhered to flask n/a n/a Citric acid ² DXM Cloudy, small particles Particulate White colors N/A N/A n/a n/a propionic acid DXM Transparent Transparent, with a yellow tint N/A N/A Beige solid when dry AI n/a D-tartaric acid ² DXM Cloudy, some solid matter Particulate White colors n/a n/a N/A n/a

- 175 041220

L- Glutamic acid DXM Solid particles float in solution A little cloudy, hard to tell if there is solid A little cloudy, hard to tell if there is solid Cloudy, milky N/A n/a Maleic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion L-Lactic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion Acetic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion Adipine acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion Fumaric acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion D- Glutamic acid DMA Turbid cloudy emulsion cloudy emulsion cloudy emulsion n/a Cloudy emulsion D-malic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion L-malic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion Methansulfo new acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion pTsOH.H ₂ O DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion Benzenesulfo new acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion Benzoic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint n/a Cloudy emulsion

- 176 041220

Malonic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion NVg/NgO DMA Solid particles dirty white filtered white solid slow filtration N/A N/A N/A N/A succinic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion L-tartaric acid3 DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Clear solution propionic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion Sulfuric acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion

Phosphoric acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion Lemon acid DMA Clear solution Clear solution Clear solution Clear solution N/A Cloudy emulsion HC1 / H ₂ O DMA White solid particles Filtered off white solid N/A N/A N/A N/A propionic acid DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion D-tartaric acid ² DMA Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint Transparent, with a light yellow tint N/A Cloudy emulsion L- Glutamic acid DMA cloudy emulsion cloudy emulsion cloudy emulsion cloudy emulsion N/A Cloudy emulsion HC1/ dioxane DMA emulsion i Emulsion emulsion i Emulsion N/A emulsion i

¹ Acids that gave solids after 13 hours that could be filtered.

² After heating and cooling, the solid was filtered off.

³ Observations for experiments in DCM were recorded after all experiments were set up (~1 h after the first flask).

Samples that yielded a solid after 13 hours at room temperature were filtered and analyzed. Samples that did not form a solid after stirring overnight and were clear solutions were evaporated to dryness (DCM reactions) or MTBE added (DMA reactions). The resulting solids were isolated and analyzed. Samples that yielded unfilterable solids were subjected to heat cycling; the solid (if any) was isolated by filtration after heating and cooling. The appearance of the solids listed above in the table. 13.

Filtered and analyzed the solids formed from the following acids: maleic acid, D-malic acid, L-malic acid, citric acid, L-tartaric acid, D-tartaric acid, HBr and HCl. The solids obtained after evaporating the DCM were also analyzed for the following acids: acetic acid, benzoic acid and propionic acid.

In the initial selection, eleven salts were received for further analysis (MPS, DSC, etc.). Salts were prepared from the following acids: maleic acid, acetic acid, D-malic acid, L-malic acid, citric acid, L-tartaric acid, D-tartaric acid, benzoic acid, propionic acid, HCl and HBr.

An additional screening of salts of sulfuric and phosphoric acids was also carried out, as shown in Table 14 below.

Table 14

Screening Conditions for Salts of Sulfuric and Phosphoric Acids

Scale Solvent (vol.) Acid (eq.) Temperature Time (h) Notes 20 mg THF (100) H ₂ SO ₄ / H ₂ O (1.1) 25°C 24 Did not become a solution. Milky solution overnight, a solid adhered to the walls of the flask. 20 mg EtOH (100) H ₂ SO ₄ / H ₂ O (1.1) 25°C 24 When the acid was added, a thick substance formed, turned into a solid gel. Milk solution overnight. 20 mg 1:1 DCM/EtOH (SO) H ₂ SO ₄ / H ₂ O (1.1) 25°C 24 When the acid was added, the solid mostly dissolved. Sticky substance on the walls. Milk solution overnight.

20 mg 4:1 DCM/EtOH (SO) H ₂ SO ₄ / H ₂ O (1.1) 25°C 24 The free base was completely dissolved before the addition of the acid. Acid formed almost immediately. Filtered solid overnight. 20 mg THF (100) H ₃ RO ₄ / H ₂ O (1.1) 25°C 24 It did not become a solution, first a thick substance formed. Milk solution overnight. 20 mg EtOH (100) H ₃ RO ₄ / H ₂ O (1.1) 25°C 24 It did not become a solution, a thick substance formed. Milk solution overnight. 20 mg 1:1 DCM/EtOH (SO) H ₃ RO ₄ / H ₂ O (1.1) 25°C 24 The sticky substance on the walls of the flask became thicker over time. Milk solution overnight. 20 mg 4:1 DCM/EtOH (SO) H ₃ RO ₄ / H ₂ O (1.1) 25°C 24 The free base was completely dissolved before the addition of the acid. Acid formed almost immediately. Gel overnight.

The free base of the compound of formula I was found to be soluble in 4:1 DCM/EtOH, showing limited solubility in THF, EtOH and 1:1 DCM/EtOH. The conditions listed above in table. 14 do not provide a filterable phosphoric acid salt. The use of sulfuric acid in 4:1 DCM/EtOH provided a filterable solid after stirring overnight.

- 178 041220

Solids derived from the acids listed in table. 15 were analyzed by MPS and

DSC; appearance and melting point of these substances, determined using DSC, are presented in table. 15. DSC curves are shown in FIG. 3-14.

Table 15

Appearance and melting point of isolated salts

Acid Appearance of the solid after filtration or evaporation of the solvent Melting point (DSC) N/A free base 229.23°C Maleic acid Grey/light brown solid 226.84°C D-malic acid Grey/light brown solid 212.69°C L-malic acid Grey/light brown solid 213.52°C Lemon acid Grey/light brown solid 197.53°C L-tartaric acid Grey/light brown solid 222.66°C D-tartaric acid Grey/light brown solid 219.24°C Acetic acid Beige solid 229.06°C Benzoic acid Beige solid 229.16°C propionic acid Beige solid 228.77°C NVg Beige solid 225.25°C HC1 Off-white solid 241.25°C _H2SO4 Off-white solid 285.95°C

The DSC thermogram for the maleic acid salt identified during the initial salt screening is shown in FIG. 3. An endothermic event was observed on DSC with an onset at about 224°C. A small endothermic event was observed on DSC with an onset at about 208°C.

The DSC thermogram for the acetic acid salt identified during the initial salt sampling is shown in FIG. 4. An endothermic event was observed on DSC with onset at about 227°C.

The DSC thermogram for the D-malic acid salt identified during the initial salt screening is shown in FIG. 5. An endothermic event was observed on DSC with an onset at about 211°C.

The DSC thermogram for the benzoic acid salt identified during the initial salt screening is shown in FIG. 6. An endothermic event was observed on DSC with an onset at about 228°C.

The DSC thermogram for the L-tartaric acid salt identified during the initial salt screening is shown in FIG. 7. An endothermic event was observed on DSC with an onset at about 216°C.

The DSC thermogram for the citric acid salt identified during the initial salt sampling is shown in FIG. 8. An endothermic event was observed on DSC with an onset at about 190°C.

The DSC thermogram for the propionic acid salt identified during the initial salt screening is shown in FIG. 9. An endothermic event was observed on DSC with an onset at about 226°C. An endothermic event was observed on DSC with an onset at about 148°C.

The DSC thermogram for the D-tartaric acid salt identified during the initial salt screening is shown in FIG. 10. An endothermic event was observed on DSC with an onset at about 210°C. A small endothermic event was observed on DSC with onset at about 159°C.

The DSC thermogram for the L-malic acid salt identified during the initial salt screening is shown in FIG. 11. An endothermic event was observed on DSC with an onset at about 211°C.

The DSC thermogram for the sulfuric acid salt identified during the initial salt sampling is shown in FIG. 12. An endothermic event was observed on DSC with an onset at about 277°C. A small endothermic event was observed on DSC with an onset at about 35°C.

C. Scaling up and optimizing selected salts.

Scaled the production volume of salts of hydrochloric acid (HCl) and hydrobromic acid (HBr) obtained during the primary selection of salts, as shown in table. 16 for further analysis.

Table 16

Scaling up production of HCl and HBr salts

Scale b (d) Acid a (eq.) Volume m DMA Max, temperature a (°C) Volume of anti-solvent (MTBE) ripening time Isolated solid 0.2 g 36% HC1 (1.1) 75 28°C 125 Overnight with stirring 152 mg, 71% yield. Found 6.2% C1 (expected value 6.5%) 0.2 g 48% HBg (1.1) 50 40°C 150 During the night without stirring 215 mg, 86% yield. Found 12% Br (expected value 13.5%)

The DSC thermogram for the hydrochloric acid salt prepared at ~200 mg scale is shown in FIG. 13. An endothermic event was observed on DSC with an onset at about 233°C.

The DSC thermogram for the hydrobromic acid salt obtained on a ~200 mg scale is shown in FIG. 14. An endothermic event was observed on DSC with an onset at about 195°C.

Alternative conditions for the formation of the HCl salt were studied. For example, the volume of DMA was reduced to 50 volumes, and the reaction mixture was heated to 40°C, as shown in table. 17 (line 1). At this temperature, HCl was added; then the resulting mixture was cooled to room temperature. The DSC thermogram of the hydrochloric acid salt prepared in this manner is shown in FIG. 15A. An endothermic event was observed on DSC with an onset at about 230°C.

An HCl salt was also prepared using a 1:1 DCM/EtOH mixed solvent system in 50 volumes at 30°C. The DSC thermogram of the hydrochloric acid salt prepared in this manner is shown in FIG. 15V. An endothermic event was observed on DSC with onset at about 224°C.

Table 17

Alternative Conditions for the Preparation of the HCl Salt

Scale (g) Acid (eq.) DMA Volume Max. temperature (°C) Volume anti-solvent (MTBE/vol.) ripening time Isolated solid 0.2 g 36% HC1 (1.1) 75 28°C 125 Overnight with stirring 152 mg, 71% yield. Found 6.2% C1(expected value 6.5%) 0.2 g 48% HBg (1.1) 50 40°C 150 Through the night without stirring 215 mg, 86% yield. Found 12% Br (expected value 13.5%)

Four salts (salts with HCl, HBr, L- and D-malic acid) and the free base of the compound of formula I were prepared on a 2 g scale (Table 18). Four salts were analyzed and described by MPS, DSC, TGA, DSP, RPD, FTIR and 'H NMR (FIGS. 16A-16F, 17A-17D and 18A-18H).

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Table 18

Scaling up production of HCl, HBr, L-malic and D-malic acid salts

Scale b (d) Solvent (vol.) Acid (eq.) Time(h) Pace Melting point (DSC) Actual Yield (%) Notes 2.0 DMA (50) HC1 (1.1) Zch. from 40 °C to room, t-ry 240.61°C 1.88 g (87.8%) HC1 was added at 40°C. Filtered and. White solid.

2.0 3.6:1 DHM/JUN (46) HC1 (1.1) 20 o'clock 25°C 237.99°C 1.93 g (90.1%) Chlorine analysis showed 1 eq. HC1 2.0 3.6:1 DHM/JUN (46) NVg (1.1) 20 o'clock 25°C 237.91°C 2.26 g (97.4%) Bromine analysis showed 1 eq. NVg 2.0 3.6:1 DHM/JUN (46) LAlic acid (0.97) 20 o'clock 25°C 208.31°C 1.64 g 66.8% 2.0 3.6:1 DHM/JUN (46) DAlic acid (0.97) 20 o'clock 25°C 208.25°C 1.60 g 65.2% 2.2 3.6:1 DHM/JUN (46) LAlic acid (0.97) 20 o'clock 25°C N/A 2.11 g 75.9% 2.5 3.6:1 DHM/JUN (46) DAlic acid (0.97) 20 o'clock 25°C N/A 2.41 g 76.2%

Analysis of chlorine and bromine confirmed the formation of the corresponding mono-HX salts. Salts of L- and D-malic acid were obtained with a yield of 75-78%; the HCl salt was obtained in 90% yield.

For the HCl salt (DMA), an endothermic event was observed on DSC with onset at about 233°C (Fig. 16A); a small exothermic event was observed on DSC with an onset at about 253°C. For the HCl salt (EtOH/DCM), an endothermic event was observed on DSC with an onset at about 223°C; a small endothermic event was observed on DSC with an onset at about 71°C (Fig. 16D). Isothermal (25° C.) DSP analysis showed that the HCl salt (DMA) is hygroscopic (FIG. 16B). At a relative humidity of 80%, an increase in weight of about 5% was observed. A certain hysteresis was observed. When removed from the chamber, the HCl salt sample remained a free-flowing powder; no spreading was observed due to the absorption of moisture from the air. TGA analysis of the HCl salt (DMA) showed a weight loss of 7.4% from the start of heating to about 255°C (FIG. 16C). TGA analysis of the HCl salt (EtOH/DCM, mixed solvent system) showed a weight loss of 8.2% from the start of heating to about 255°C (FIG. 16D). An overlay of the X-ray powder diffraction patterns of the HCl salt (prepared in DMA, from different batches, before/after EAF) and the HCl salt (EtOH/DCM) is shown in FIG. 16E. ^{The 1H} NMR spectrum of the HCl salt (prepared in EtOH/DCM) in DMSO-66 is shown in FIG. 16F.

For the HBr salt, an endothermic event was observed on DSC with an onset at about 215°C; a small endothermic event was observed on DSC with onset at about 34°C (FIG. 17A). Salt TGA analysis

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HBr showed a weight loss of about 10.3% from the start of heating to about 255°C (FIG. 17A). The X-ray powder diffraction pattern of the HBr salt is shown in FIG. 17V. Fourier IR spectrum

The HBr salt is shown in FIG. 17C. The 1H NMR spectrum of the HBr salt in DMSO-d6 is shown in FIG. 17D.

For the L-malic acid salt, an endothermic event was observed on DSC with an onset at about 205° C. (FIG. 18A). For the D-malic acid salt, an endothermic event was observed on DSC with an onset at about 206° C. (FIG. 18B). TGA analysis of the L-malic acid salt showed a weight loss of about 17.7% from the start of heating to about 253°C (FIG. 18A). TGA analysis of the D-malic acid salt showed a weight loss of about 18.4% from the start of heating to about 250°C (FIG. 18B). Isothermal (25° C.) DSP analysis showed that both L-malic and D-malic acid salts were hygroscopic (FIGS. 18C and 18D). At 80% RH, a weight increase of about 2.8% was observed. A certain hysteresis was observed. When removed from the chamber, samples of salts with L-malic and D-malic acid remained a free-flowing powder; no spreading was observed due to the absorption of moisture from the air. An overlay of X-ray powder diffraction patterns of L- and D-malic acid salts is shown in FIG. 18E. An overlay of the FTIR spectra of L- and D-malic acid salts is shown in FIG. 18F. The 1H NMR spectra of L- and D-malic acid salts in DMSO-d ⁶ are shown in FIG. 18G-18H respectively.

Two batches of L-malic acid salt were tested in DSC, TGA and RPD analyses. For the L-malic acid salt (batch A), an endothermic event was observed on DSC with an onset at about 210° C. (FIG. 18I). TGA analysis of L-malic acid salt (lot A) showed a weight loss of about 18.0% from the start of heating to about 250°C (Fig. 18J). For the L-malic acid salt (batch B), an endothermic event was observed on DSC with an onset at about 217° C. (FIG. 18K). TGA analysis of L-malic acid salt (batch B) showed a weight loss of about 17.7% from the start of heating to about 250°C (FIG. 18L). An overlay of the X-ray powder diffraction patterns of the L-malic acid salts (lots A and B) and the free base of the compound of formula I is shown in FIG. 18M.

The solubility of the HCl, HBr, L-malic acid and D-malic acid salts of the compound of formula I was studied in various solvent systems.

The HCl salt solubility test was carried out as follows. About 10-20 mg of HCl salt was stirred in 1 ml of solvent. In each solvent system, some solids were observed after 2 hours. Then the samples were heated to 65°C for 3 h, after which the appearance was recorded. The resulting mixture was then allowed to cool to room temperature overnight. The results of the solubility test are presented in table. 19.

Table 19 Solubility of HCl salt

Solvent Appearance at 25°C after 2 hours Does the solid dissolve at 65°C? Solubility at 25 °C (mg/ml) Water Solid particles No 0.24 Meon Solid particles No 0.58 EtOH Solid particles No 0.12 IPA Solid particles No 0 EtOAc Solid particles No 0 iPAc Solid particles No 0 THF Solid particles No 0 ACN Solid particles No 0 Toluene Solid particles No 0 DXM Solid particles n/a 0.10 Acetone Solid particles N/A 0 IEC Solid particles No 0 MTBE Solid particles No 0 2-methyl-THF Solid particles No 0 EDC Solid particles No 0 dmso Solid particles Close to 18.7 mg/ml 5.94 0DMNS1 Solid particles Didn't study 2.18

The HCl salt was found to be insoluble in IPA, EtOAc, iPAc, THF, ACN, toluene, acetone, MEK, MTBE, 2-methyl-THF and DCE. Solubility <1 mg/ml in water, MeOH, EtOH and DCM was observed. A solubility of about 6 mg/ml in DMSO was observed.

The HBr salt solubility test was carried out as follows. About 10-20 mg of HBr salt was stirred in 1 ml of solvent. In each solvent system, some solids were observed after 2 hours. Then the samples were heated to 65°C for 3 h, after which the appearance was recorded. The resulting mixture was then allowed to cool to room temperature overnight. The results of the solubility test are presented in table. 20.

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Table 20 Solubility of the HBr salt

Solvent Appearance at 25°C after 2 hours Does the solid dissolve at 65°C? Solubility at 25 °C (mg/ml) water Solid particles No 0 Meon Solid particles No 0 EtOH Solid particles No 0 IPA Solid particles No 0 EtOAc Solid particles No 0 iPAc Solid particles No 0 THF Solid particles No 0 ACN Solid particles No 0 DXM Solid particles No 0 Acetone Solid particles No 0 IEC Solid particles No 0 MTBE Solid particles No 0 2-methyl-THF Solid particles No 0 dhe Solid particles No 0 DMSO Solid particles No 1.95

The HBr salt exhibited low solubility in the solvent systems studied; the solid did not dissolve when heated to 65°C. The solubility of 1.95 mg/ml in DMSO was observed.

The L-malic acid salt solubility test was carried out as follows. About 10-20 mg of L-malic acid salt was stirred in 1 ml of solvent at room temperature overnight. The results of the solubility test are presented in table. 21.

Table 21

Solubility of L-malic acid salt

Solvent Lot 1: Solubility at 25°C (mg/mL) Lot 2: Solubility at 25°C (mg/mL) water 0.26 0.23 Meon 0.52 0.58 EtOH 0.16 0.15 IPA 0 0 EtOAc 0.20 1.58 iPAc 0 0.33 THF 2.53 5.42 ACN 0.28 0.85 Toluene 0.29 0.62 DXM 0.24 5.50 Acetone 0.61 1.57 IEC 0.51 2.31 MTBE 0 0 2-methyl-THF 0.61 1.21 Dhe 0.13 4.97 DMSO 14.2 15.4 0.1 MHC1 2.30 Didn't study

The solubility test was carried out on two batches of the L-malic acid salt. Observed a certain variability in solubility between batches. A solubility of 14-15 mg/ml in DMSO was observed.

A second solubility test to determine the solubility of the L-malic acid salt at elevated temperatures was performed as follows. About 10-20 mg of L-malate salt was stirred in 1 ml of solvent at room temperature for 30 minutes. The samples were heated to 60°C for 3.0 h; solubility was recorded. Then the samples were heated to 70°C for about 3.0 h; solubility was recorded. The results of the second dissolution test are presented in table. 22.

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Table 22

Solubility of L-malic acid salt at elevated temperatures Solvent Does the solid dissolve Does the solid dissolve

substance at 60 °C? substance at 70 °C? water No No Meon No N/A EtOH No No IPA No No EtOAc No No iPAc No No THF at 65 °C almost 12.3 mg/ml N/A ACN No No Toluene No No DXM N/A N/A Acetone No N/A IEC No No MTBE No No 2-methyl-THF No No dhe No No DMSO Yes (>36 mg/ml) Yes (>56 mg/ml) 0.1 MHC1 Yes (>13.4 mg/ml) N/A

The D-malic acid salt solubility test was carried out as follows. About 10-20 mg of D-malic acid salt was stirred in 1 ml of solvent overnight. For testing the solubility at elevated temperatures, about 10-20 mg of D-malic acid salt was stirred in 1 ml of solvent at room temperature for 30 minutes. The samples were heated to 60°C for 3.0 h; solubility was recorded. Then the samples were heated to 70°C for about 3.0 h; solubility was recorded. The results of the solubility test are presented in table. 23.

Table 23

Solubility of D-malate salt

Solvent Solubility at 25 °C (mg/ml) Does the solid dissolve at 60°C? Does the solid dissolve at 70°C? water 0.35 No No Meon 0.49 No N/A EtOH 0.14 No No IPA 0 No No EtOAc 0.21 No No iPAc 0.12 No No THF 2.65 At 65 °C ~10 mg/ml N/A ACN 0.23 No No Toluene 0 No No DXM 0.17 N/A N/A Acetone 0.65 No n/a IEC 0.61 No No MTBE 0 No No 2-methyl-THF 0.62 No No Dhe 0.14 No No DMSO 16.7 Yes (>40 mg/ml) Yes (>56 mg/ml) 0.1 MHC1 2.90 Yes (>15 mg/ml) N/A

The production scale of the L-malic acid salt and the HCl salt was further scaled up. The L-malic acid salt was obtained in 77.7% yield and the HCl salt was obtained in 91% yield. Then the conditions for obtaining a salt of L-malic acid was optimized to increase the output, as shown in table. 24.

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Table 24

Scale up production of HCl salt and L-malic acid salt

Scale (G) Solvent (vol.) Acid (eq.) Time (h) Pace. Actual Yield (%) Notes 10 3.6:1 DCM/EtOH (46) LAlic acid (0.97) 20 o'clock 25°C 9.83 g (77.7%) 10 3.6:1 DCM/EtOH (46) HC1 (1.1) 20 o'clock 25°C 10.05 g (91.4%) 2.0 -3.3:1 DCM/EtOH (31) LAlic acid (0.97) 20 o'clock 25°C 2.01 g (79.4%) The free base was dissolved at room temperature, tre in 30 vol. 4:1 DCM/EtOH. L-malic acid was added as a solution in EtOH (1 vol.). 1.5 -6:1 DCM/EtOH (26 vol) L- Malic acid (1.05) 20 o'clock 25°C 1.64 g (86.2%) The free base was dissolved at room temperature, tre in 30 vol. 4:1 DCM/EtOH. L-malic acid was added as a solution in EtOH (1 vol.). 8.0 -6:1 DCM/EtOH (26 vol) L- Malic acid (1.05) 20 o'clock 25°C 8.70 g (86.0%) The free base was dissolved at room temperature, tre in 25 vol. 9:1 DCM/EtOH. L-malic acid was added as a solution in EtOH (1 vol.).

8.0 -6:1 DCM/EtOH (26 vol) L- Malic acid (1.05) 20 o'clock 25°C 8.86 g (87.5%) The free base was dissolved at room temperature, tre in 25 vol. 9:1 DCM/EtOH. L-malic acid was added as a solution in EtOH (1 vol.). The suspension was then cooled to 0°C for 1.5 hours, then filtered.

The solvent volume was reduced to 30 volumes using a 4:1 DCM/EtOH ratio. Addition of L-malic acid (0.97 eq.) in 1 volume of EtOH increased the total solvent volume to 31 volumes and the solvent ratio was about 3.3:1 DCM/EtOH. These conditions ensured an increase in the yield of L-malate salt to 79.4%.

The solvent system DCM/EtOH 9:1 was reduced to 25 volumes. L-malic acid (1.05 eq.) was dissolved in 1 volume of EtOH, resulting in a total amount of solvent of 26 volumes, and the final ratio of solvents was about 6:1 DCM/EtOH. These conditions ensured an increase in the yield of L-malate salt to 86.2%. Malic acid salt was obtained with a yield of 86% when scaling these conditions up to 8 grams. The second reaction on the scale of 8 grams was carried out under the same conditions, but the suspension was cooled to 0°C for 1.5 h before filtering. In this reaction, a yield of 87.5% was obtained.

D. Stability screening for free base and selected salts.

The stability of the free base of formula I and the HCL salt, the L-malic acid salt and the D-malic acid salt was checked by weekly HPLC analysis for 4 weeks. Samples were stored at 40° C. and 75% RH. The results of the stability test are presented in table. 25.

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Example 4 Selection of Polymorphic Salts for Compound of Formula II A. Instruments and Methods for Performing the Analysis.

The analysis tools and methods used to screen for the polymorphs described in Examples 2 and 3 below were as follows.

X-ray powder diffraction (RPD).

RPD analysis was performed on a Panalytical X'pert Pro instrument, scanning samples from 3 to 35° 2θ. The material was carefully ground and loaded into a multiwell plate with Kapton or Mylar polymer film to hold the sample. The multiwell plate was then inserted into a Panalytical diffractometer operating in transmission mode and analyzed using the following experimental conditions.

Source Data Origin: RPD Dimension (*.XRDML).

Scan axis: gonio.

Home position [°2θ]: 3.0066.

End position [°2θ]: 34.9866.

Step size [°2θ]: 0.0130.

Scan step time [s]: 18.8700.

Scan type: continuous.

PSD Mode: Scan PSD Length [°2θ]: 3.35.

Divergence [°2θ]: 0.0000.

Type of divergence slot: fixed.

Divergence gap size [°]: 1.0000.

Measurement temperature [°C]: 25.00.

Anode material: Cu K-alpha1 [A]: 1.54060.

K-alpha2 [A]: 1.54443 K-beta [A]: 1.39225.

Ratio K-A2 / K-A1: 0.50000.

Generator settings: 40 mA, 40 kV.

Goniometer radius [mm]: 240.00.

Diet, focus diverg. gap [mm]: 91.00.

Incident beam monochromator: No.

Rotation: No.

Microscopy in polarized light (MPS).

The presence of crystallinity (double refraction) was determined using an Olympus BX50 polarizing microscope equipped with a Motic camera and video capture software (Motic Images Plus 2.0). All images were recorded with a 20x objective unless otherwise noted.

Thermogravimetric analysis (TGA).

About 5 mg of material was weighed into an open aluminum crucible and loaded into a combined thermogravimetric/differential thermal analyzer (TG/DTA) and kept at room temperature. The sample was then heated at a rate of 10° C./min from 20° C. to 300° C., and during this time the change in the mass of the sample was recorded, as well as any differential thermal events (DTA). Nitrogen was used as purge gas at a flow rate of ³⁰⁰ cc/min.

Differential Scanning Calorimetry (DSC).

About 5 mg of the substance was weighed into an aluminum DSC crucible and sealed with a perforated aluminum lid. Then the crucible with the sample was placed in a Seiko DSC6200 instrument (equipped with a cooling device), cooled and kept at 20°C. After reaching a stable heat flux response, the sample and the control sample were heated at the scan rate

10°C/min. and read the response heat flux.

Nuclear magnetic resonance (NMR).

NMR experiments were performed on a Bruker AVIIIHD spectrometer equipped with a DCH cryoprobe operating at 500.12 MHz for protons. The experiments were carried out in deuterated solvents, and each sample was obtained at a concentration of about 10 mm.

Dynamic vapor sorption (DSP).

About 10 mg of sample was placed on a mesh vapor sorption balance and mounted on a DVS-1 dynamic vapor sorption balance manufactured by Surface Measurement Systems. The sample was profiled from 40-90% relative humidity (RH) in 10% increments, holding the sample at each stage until a stable weight was reached (99.5% stage completion). After completion of the sorption cycle, the sample was dried using the same procedure to 0% RH, and then a second sorption cycle was performed to 40% RH. The mass change during the sorption/desorption cycles was plotted to determine the hygroscopic nature of the sample. RPD analysis was then carried out on the remaining solid.

Gravimetric vapor sorption (GSP).

Approximately 10-20 mg of sample was placed on a mesh vapor sorption balance and mounted on a Hiden Analytical IGASorp moisture sorption analyzer. The sample was profiled from 40-90% RH in 10% increments, holding the sample at each stage until a stable weight was reached (98% stage completion). After completion of the sorption cycle, the sample was dried using the same procedure to 0% RH, and finally returned to its original state of 40% RH. The mass change during the sorption/desorption cycles was plotted to determine the hygroscopic nature of the sample.

High performance liquid chromatography with ultraviolet detection (HPLC-UV)

Device. HPLC with UV detector.

Column: ACE Excel 3 Super C18, column 75 mm x 4.6 mm.

Column temperature: 40°C.

Automatic sampler temperature: 25°C.

UV wavelength: 235 nm.

Sample Injection Volume: 5.0 Limits of Quantification (QL).

Flow rate: 1.0 ml/min.

Mobile phase A: 0.1% TFA in water.

Mobile phase B: 0.1% TFA in acetonitrile.

Gradient program:

Time (minutes) Solvent B (%) 0.00 10 15.00 90 15.10 10 20.00 10

Charged aerosol detection (CAP).

Device. HPLC with charged aerosol detection.

Column: Thermo Acclaim P2 50x2.1 mm, 3.0 QL.

Temperature: 30°C.

Automatic sampler temperature: room temperature.

Sample injection volume: 10 QL.

Flow rate: 0.5 ml/min.

Mobile phase A: deionized water.

Mobile phase B: 10 mM ammonium formate buffer pH 3.65.

Gradient program:

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Time (minutes) Solvent B (%) 0.00 35 2 35 12 65 15.3 65 18.6 40 19.3 35 23 35

B. Initial characteristic.

The compound of formula II, prepared as described in Example 2 above, was analyzed by RPD, TG/DTA, DSC, DSP, MPS, ¹ H NMR and HPLC and identified as Form 1. The material had a high degree of crystallinity, according to RPD ( Fig. 62A). MPS analysis showed agglomerates of small needle-like crystals. On the TG/DTA curve (FIG. 62C), a small endotherm was observed starting at about 189°C, followed by a sharp endotherm starting at about 199°C associated with a melt transition. Thermal decomposition followed this event. The initial endotherm is possibly related to the transition between solid phases, followed by melting of the more stable solid form. DSC showed a small endotherm beginning at about 184°C followed by a sharp endotherm beginning at about 199°C (FIG. 62B). The material was moderately hygroscopic as measured by DSP (FIGS. 62D and 62E) with a weight gain of about 3.6% at 90% RH. After DSP, no changes in this shape were observed. The purity of the material, according to HPLC, was 99.2%. ¹ H NMR in 66-DMSO indicated trace amounts of DCM (0.04 eq.) and MeOH (0.06 eq.) in the material (FIG. 62F).

C. Primary selection of salts.

The primary selection of salts was carried out for the compounds of formula II, using the acidic counterions and solvent systems presented below in table. 26 and 27.

Table 26

Proven Acids

Hydrochloric acid Sulfuric acid 1,2- Etand isul phokis ota Methansulfonic acid Naphthalene-2-sulfonic acid Benzenesulfonic acid 2- Hydroxyethanesulfonic acid L-Aspartic Acid Maleic acid ethanesulfonic acid L-Glutamic acid L-tartaric acid Lemon acid D-Glucuronic acid L-malic acid D-Gluconic acid L-Lactic acid L-Ascorbic acid succinic acid Oxalic acid Phosphoric acid l-T oluenesulfonic acid Fumaric acid hippuric acid Benzoic acid

Table 27 Solvent systems Solvent system

THF/water (1%)

1,4-Dioxane/water (10%)

MeCN/water (20%)

Acetone/water (10%)

IPA/water (10%)

EUN/water (10%)

Salt screening was performed as follows. A basic aqueous acid solution was added to

- 188 041220 about 30 mg of the compound of formula II in each of the solvent systems listed above in the table. 27.

If stock solutions could not be obtained, the acid was added neat to a solution of the compound of formula II. The masses and volumes of acids used to obtain basic solutions are presented in Table 1 below. 28.

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Table 28

Stock solutions of counterions for primary screening of salts

Acid Acid base solution Known suitable solvent Amount of acid added per vial/l bottle Other Amount of acid Solvent volume (ml) hydrochloric acid 83.5 µl 1 Water 57.1 µl Sulfuric acid 56.1 µl 1 Water 57.1 µl 1,2 Ethandisulfonic acid 234.2 mg 1 EtOH 57.1 µl added 1 equiv. HCI P- Toluene sulfonic acid 190.2 mg 1 Water 57.1 µl Methansulfonic acid 64.9 µl 1 Water 57.1 µl

Naphthalene-2sulfonic acid 255.8 mg 1 EtOH 57.1 µl added 1 equiv. HCI Benzenesulfonic acid 168.3 mg 1 Water 57.1 µl Oxalic acid 90.0 mg 1 Water 57.1 µl 2- Hydroxyethanesulfo new acid 148.1 mg 1 Water 57.1 µl added 1 equiv. HCI L-Aspartic Acid N/A N/A N/A 7.7 mg Added undiluted Maleic acid 116.1 mg 1 Water 57.1 µl Phosphoric acid 98.0 mg 1 Water 57.1 µl ethanesulfonic acid 81.6 1 Water 57.1 µl L-Glutamic acid N/A N/A N/A 8.5 mg Added undiluted L-tartaric acid 150.1 mg 1 Water 57.1 µl Fumaric acid N/A n/a N/A 6.5 mg Added undiluted Lemon acid 192.1 mg 1 Water 57.1 µl D-Glucuronic acid 194.1 mg 1 Water 57.1 µl L-malic acid 134.1 mg 1 Water 57.1 µl hippuric acid N/A n/a N/A 10.3 mg Added undiluted D-Gluconic acid 319.0 µl 1 Water 57.1 µl

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L-Lactic acid 76.0 µl 1 Water 57.1 µl L-Ascorbic acid 176.1 mg 1 Water 57.1 µl Benzoic acid 122.1 mg 1 IPA 57.1 µl succinic acid 118.1 mg 1 Meon 57.1 µl

The samples were then subjected to periodic temperature changes from room temperature (room temperature) to 40° C. in 4-hour cycles for 24 hours. Each mixture was filtered and the isolated solids (wet) analyzed by RPD for crystallinity and shape. Samples in which no solid was observed were stored without a lid to allow the solvent to evaporate. The remaining samples were placed in an oven at 40°C and RH 75% overnight. The samples were then re-analyzed by RPD to determine changes in shape or crystallinity. Observations and results, including various forms of diffractograms observed during screening, are presented below in table. 29-53 and in FIG. from 24A-24B to 47A-47B.

Table 29

Observations for hydrochloric acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension Suspension I Suspension I Suspension I Suspension I After temperature cycling white solid Pale orange solid white solid white solid white solid Pale yellow solid RPD after temperature cycling Form 2 Form 2 Form 2 Form 2 Form 2 Form 2 RPD after stability test Form 2 Form 2 Form 2 Form 2 Form 2 Form 2

The diffractograms after temperature cycling and after the stability test for hydrochloric acid are shown in FIG. 24A and 24B, respectively.

Table 30

Sulfuric Acid Observations and RPD Results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension Suspension I Suspension I Suspension I After temperature cycling yellow resinous substance Pale yellow solution Colorless solution Pale yellow solution White solid in yellow solution Pale yellow solution RPD after temperature cycling Solid Free Solid Free Solid Free Solid Free Form 3 Solid Free RPD after stability test and Solid Free Solid Free Solid Free Solid Free Form 5 Solid Free

The diffractograms after temperature cycling and after the stability test for sulfuric acid are shown in FIG. 25A and 25B respectively.

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Table 31

Observations for 1,2-ethanedisulfonic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Up to temperature cycling Suspension I Suspension I Suspension Suspension I Suspension Suspension I After temperature cycling Pale yellow solution Pale yellow solution Colorless solution Pale yellow solution Colorless solution Pale yellow solution RPD after temperature cycling Solid Free Solid Free Solid Free Solid Free Solid Free Solid Free RPD after stability test and Solid Free Solid Free Solid Free Solid Free Solid Free Solid Free

Table 32

Observations for p-toluenesulfonic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension Suspension Suspension Suspension Suspension Suspension After temperature cycling yellow solution yellow solution white solid White solid in yellow solution White solid in yellow solution White solid in yellow solution RPD after temperature cycling Weak diffraction Solid Free Solid Free Form 4 Amorphous Amorphous RPD after stability test Form 4 Solid Free Solid Free Form 4 Form 4 Amorphous

The diffractograms after temperature cycling and after stability testing for p-toluenesulfonic acid are shown in FIG. 26A and 26B, respectively.

Table 33

Observations for methanesulfonic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension I Suspension Suspension I Suspension After temperature cycling yellow solution yellow solution yellow solution white solid white solid white solid RPD after temperature cycling Solid Free Solid Free Solid Free Amorphous Form 2 Amorphous RPD after stability test and Solid Free Solid Free Solid Free Amorphous Form 2 Amorphous

- 192 041220

The diffractograms after temperature cycling and after stability testing for methanesulfonic acid are shown in FIG. 27A and 27B, respectively.

Table 34

Observations for naphthalene-2-sulfonic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EUN/water (10%) Before temperature cycling Suspension Suspension Suspension Suspension Suspension Suspension After temperature cycling yellow solution yellow solution Pale yellow solution white solid White solid in yellow solution White solid in yellow solution RPD after temperature cycling Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2 RPD after stability test Form 2/ Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2/ Form 5 Form 2

Diffractograms after temperature cycling and after stability testing for naphthalene-2-sulfonic acid are shown in FIG. 28A and 28B, respectively.

Table 35

Observations for benzenesulfonic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension I Suspension Suspension I Suspension I After temperature cycling Pale yellow solution white solid Pale yellow solution white solid white solid white solid RPD after temperature cycling Solid Free Solid Free Solid Free Weak diffraction Form 2 Form 2 RPD after stability test Solid Free Solid Free Solid Free Weak diffraction Form 2 Form 2

The diffractograms after temperature cycling and after stability testing for benzenesulfonic acid are shown in FIG. 29A and 29B, respectively.

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Table 36

Observations for oxalic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EUN/water (10%) Before temperature cycling Suspension I Suspension I Suspension I Suspension Suspension Suspension After temperature cycling Pale yellow solid White solid in yellow solution white solid white solid white solid white solid RPD after temperature cycling Solid Free Form 6 Solid Free Amorphous Amorphous Amorphous RPD after stability test and Solid Free Form 6 Solid Free Form 2/ Form 6 Form 6 Form 6

The diffractograms after temperature cycling and after the stability test for oxalic acid are shown in FIG. 30A and 30B, respectively.

Table 37

Observations for 2-hydroxyethanesulfonic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPAZ water (10%) EUN/water (10%) Up to temperature cycling Suspension Suspension I Suspension Suspension Suspension I Suspension I After temperature cycling white solid white solid white solid white solid white solid white solid RPD after temperature cycling Form 1 (free base) / Form 2 Form 7 Form 1 (free base) Form 1 (free base) Form 2/ Form 7 Form 7 RPD after stability test and Form 1 (free base) / Form 2 Form 7 Form 1 (free base) / Form 8 Form 1 (free base) / Form 8 Form 2/ Form 7 Form 7

The diffractograms after temperature cycling and after stability testing for 2-hydroxyethanesulfonic acid are shown in FIG. 31A and 31B, respectively.

- 194 041220

Table 38

Observations for L-aspartic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Up to cyclic temperature changes Suspension I Suspension Suspension Suspension Suspension I Suspension After cyclic temperature changes yellow solid Pale yellow solution Colorless solution white solid white solid white solid RPD after cyclic temperature changes Form 8 Form 1 (free base) / Form 8 Form 1 (free base) / Form 8 Form 1 (free base) / Form 8 Form 8 Form 1 (free base) / Form 8 RPD after stability test and Form 8 Form 1 (free base) / Form 8 Form 8 Form 14 Form 8 Form 1 (free base) / Form 8

The diffractograms after temperature cycling and after stability testing for L-aspartic acid are shown in FIG. 32A and 32B, respectively.

Table 39

Maleic Acid Observations and RPD Results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension Suspension Suspension I Suspension I Suspension I After temperature cycling yellow solid Colorless solution Colorless solution white solid white solid white solid RPD after temperature cycling Weak diffraction Solid Free Solid Free Amorphous Amorphous Amorphous RPD after stability test and Form 15 Solid Free Solid Free Amorphous Amorphous Amorphous

The diffractograms after temperature cycling and after the stability test for maleic acid are shown in FIG. 33A and 33B, respectively.

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Table 40

Phosphoric Acid Observations and RPD Results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EUN/water (10%) Before temperature cycling Suspension Suspension I Suspension I Suspension I Suspension I Suspension After temperature cycling yellow resinous substance yellow solution Pale yellow resinous substance Pale yellow resinous substance white solid yellow resinous substance RPD after temperature cycling Amorphous Solid Free Solid Free Form 9 Form 10 Weak diffraction RPD after stability test and Form 10 Solid Free Solid Free Form 9/ Form 10 Form 10 Form 10

The diffractograms after temperature cycling and after the stability test for phosphoric acid are shown in FIG. 34A and 34B, respectively.

Table 41

Observations for ethanesulfonic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EUN/water (10%) Before temperature cycling Suspension I Suspension I Suspension I Suspension I Suspension Suspension I After temperature cycling yellow solution yellow resinous substance Pale yellow resinous substance Pale yellow resinous substance white solid yellow solution RPD after temperature cycling Solid Free Solid Free Solid Free Solid Free Amorphous Solid Free RPD after stability test Solid Free Solid Free Solid Free Solid Free Amorphous Solid Free

The diffractograms after temperature cycling and after stability testing for ethanesulfonic acid are shown in FIG. 35A and 35B, respectively.

- 196 041220

Table 42

Observations for L-Glutamic Acid and RPD Results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Up to temperature cycling Suspension I Suspension I Suspension Suspension Suspension Suspension After temperature cycling White solid in yellow solution white solid white solid white solid white solid white solid RPD after temperature cycling Form 8 Form 7 Form 1 (free base) Form 1 (free base) Form 1 (free base) / Form 2 Form 1 (free base) / Form 2 RPD after stability test and Form 8 Form 7 Form 1 (free base) Form 1 (free base) Form 1 (free base) / Form 2 Form 1 (free base)

Diffractograms after temperature cycling and after stability testing for L-glutamic acid are shown in FIG. 36A and 36B, respectively.

Table 43

Observations for L-tartaric acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EUN/ water (10%) Before temperature cycling Suspension Suspension Suspension I Suspension Suspension I Suspension After temperature cycling White solid in yellow solution white solid white solid white solid white solid white solid RPD after temperature cycling Weak diffraction Weak diffraction Solid Free Weak diffraction Form 11 Weak diffraction RPD after stability test and Weak diffraction Weak diffraction Solid Free Weak diffraction Form 11 Weak diffraction

The diffractograms after temperature cycling and after the stability test for L-tartaric acid are shown in FIG. 37A and 37B, respectively.

- 197 041220

Table 44

Fumaric Acid Observations and RPD Results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension Suspension I Suspension I Suspension I After temperature cycling Pale yellow solid white solid Colorless solution white solid white solid white solid RPD after temperature cycling Form 12 Solid Free Solid Free Solid Free Form 8 Form 8 RPD after stability test and Form 12 Solid Free Solid Free Solid Free Form 8 Form 8

The diffractograms after temperature cycling and after the stability test for fumaric acid are shown in FIG. 38A and 38B, respectively.

Table 45

Observations for citric acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EUN/water (10%) Up to temperature cycling Suspension Suspension I Suspension Suspension Suspension Suspension I After temperature cycling yellow solid Pale yellow solution Colorless solution Pale yellow resinous substance white solid white solid RPD after temperature cycling Weak diffraction Solid Free Solid Free Weak diffraction Form 1 (free base) Solid Free RPD after stability test and Weak diffraction Solid Free Solid Free Weak diffraction Form 1 (free base) Solid Free

The diffractograms after temperature cycling and after the stability test for citric acid are shown in FIG. 39A and 39B, respectively.

- 198 041220

Table 46

Observations for D-Glucuronic Acid and RPD Results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension I Suspension I Suspension Suspension I Suspension I Suspension After temperature cycling Pale yellow solution Pale yellow solution Pale yellow solution white solid white solid Colorless solution RPD after temperature cycling Solid Free Solid Free Form 1 (free base) Solid Free Form 8 Form 8 RPD after stability test and Solid Free Solid Free Form 1 (free base) Solid Free Form 8 Form 1 (free base)

Diffractograms after temperature cycling and after stability testing for D-glucuronic acid are shown in FIG. 40A and 40B respectively.

Table 47

Observations for L-malic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension Suspension I Suspension I Suspension Suspension I Suspension After temperature cycling yellow solid Pale yellow solution white solid white solid white solid white solid RPD after temperature cycling Amorphous Solid Free Solid Free Form 1 (free base) Form 8 Form 8 RPD after stability test and Amorphous Solid Free Solid Free Form 1 (free base) Form 8 Form 1 (free base)

The diffractograms after temperature cycling and after the stability test for L-malic acid are shown in FIG. 41A and 41B, respectively.

- 199 041220

Table 48

Observations for hippuric acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Up to cyclic temperature changes Suspension I Suspension Suspension Suspension Suspension Suspension After cyclic temperature changes White solid in yellow solution white solid white solid white solid white solid white solid RPD after cyclic temperature changes Form 8 Form 1 (free base) / Form 8 Form 1 (free base) Form 1 (free base) Form 8 Form 8 RPD after stability test and Form 8 Form 1 (free base) / Form 8 Form 1 (free base) Form 1 (free base) Form 1 (free base) / Form 8 Form 1 (free base) / Form 8

The diffractograms after temperature cycling and after the stability test for hippuric acid are shown in FIG. 42A and 42B, respectively.

Table 49

Observations for D-gluconic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Up to temperature cycling Suspension I Suspension I Suspension Suspension Suspension Suspension After temperature cycling yellow solid white solid white solid white solid white solid white solid RPD after temperature cycling Form 13 Form 7 Form 1 (free base) Form 1 (free base) Form 8 Form 1 (free base) / Form 8 RPD after stability test and Form 13 Form 7 Form 1 (free base) Form 1 (free base) Form 1 (free base) / Form 8 Form 1 (free base) / Form 8

Diffractograms after temperature cycling and after stability testing for D-gluconic acid are shown in FIG. 43A and 43B, respectively.

- 200 041220

Table 50

Observations for L-Lactic Acid and RPD Results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EUN/ water (10%) Up to temperature cycling Suspension I Suspension Suspension Suspension Suspension I Suspension After temperature cycling Pale yellow solid white solid white solid white solid white solid white solid RPD after temperature cycling Form 13 Form 1 (free base) Form 1 (free base) Form 1 (free base) Form 8 Form 8 RPD after stability test and Form 13 Form 1 (free base) Form 1 (free base) Form 1 (free base) Form 8 Form 1 (free base) / Form 8

The diffractograms after temperature cycling and after the stability test for L-lactic acid are shown in FIG. 44A and 44B, respectively.

Table 51

Observations for L-ascorbic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EUN/ water (10%) Before temperature cycling Suspension I Suspension Suspension Suspension Suspension Suspension After temperature cycling yellow solution White solid in yellow solution orange solid orange solid yellow solid yellow solid RPD after temperature cycling Solid Free Form 1 (free base) Form 1 (free base) Form 1 (free base) Form 8 Form 1 (free base) / Form 8 RPD after stability test and Solid Free Form 1 (free base) / Form 2 Form 1 (free base) Form 1 (free base) / Form 2 Form 1 (free base) / Form 8 Form 1 (free base) / Form 8

The diffractograms after temperature cycling and after stability testing for L-ascorbic acid are shown in FIG. 45A and 45B, respectively.

Table 52

Observations for benzoic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone/ water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension Suspension Suspension Suspension I Suspension Suspension

- 201 041220

After temperature cycling white solid white solid white solid white solid white solid white solid RPD after temperature cycling Form 8 Form 1 (free base) / Form 8 Form 1 (free base) / Form 8 Form 1 (free base) Form 8 Form 8 RPD after stability test and Form 1 (free base) / Form 8 Form 1 (free base) / Form 8 Form 1 (free base) / Form 8 Form 1 (free base) Form 1 (free base) / Form 8 Form 1 (free base) / Form 8

The diffractograms after temperature cycling and after the stability test for benzoic acid are shown in FIG. 46A and 46B, respectively.

Table 53

Observations for succinic acid and RPD results

Solvent THF/ water (1%) Dioxane / water (10%) MeCN/ water (20%) Acetone / water (10%) IPA/ water (10%) EtOH/ water (10%) Before temperature cycling Suspension Suspension Suspension I Suspension Suspension Suspension After temperature cycling White solid in yellow solution White solid in yellow solution white solid white solid white solid white solid RPD after temperature cycling Form 1 (free base) / Form 8 Form 1 (free base) / Form 8 Form 8 Form 1 (free base) Form 8 Weak diffraction RPD after stability test and Form 1 (free base) / Form 8 Form 1 (free base) Form 8 Form 1 (free base) Form 1 (free base) Form 1 (free base)

The diffractograms after temperature cycling and after the stability test for succinic acid are shown in FIG. 47A and 47B, respectively.

Based on the results of the RPD primary selection of salts, identified primary suitable salts, presented below in table. 54.

Table 54

Primary suitable salts, according to RPD

1 Sulfate made in IPA/water (10%) 2 Tosylate prepared in acetone/water (10%) 3 Naphthalene-2-sulfonate obtained in a mixture THF/water (10%) 4 Oxalate prepared in 1,4-dioxane/water (10%) 5 Oxalate obtained by evaporation from a mixture of THF/water 6 Phosphate made in acetone/water (10%) 7 Phosphate made in IPA/water (10%) 8 Tartrate made in IPA/water (10%) 9 Fumarate prepared in THF/water

Each salt was thermally analyzed by TG/DTA to identify possible salt forms. Eight of the nine suitable salts have been found to be solvates or hydrates.

- 202 041220

The following results are obtained.

Sulfate (IPA/water (10%)): observed weight loss from the start of heating. No obvious thermal transitions were observed. Observed thermal decomposition around 257°C. The TG/DTA curve of the sulfate salt is shown in FIG. 48.

Tosylate (acetone/water (10%)): a weight loss of about 6% from the start of heating was observed, followed by a second weight loss of 3% due to solvent loss. A broad endotherm was observed starting at about 28°C, followed by a broad exotherm starting at about 158°C. Observed thermal decomposition around 192°C. The TG/DTA curve of the tosylate salt is shown in FIG. 49.

Naphthalene-2-sulfonate (THF/water (10%)): weight loss was observed from the start of heating. No obvious thermal transitions were observed. The TG/DTA curve of the naphthalene-2-sulfonate salt is shown in FIG. 50.

Oxalate (1,4-dioxane/water (10%)): a weight loss of about 0.6% from the start of heating was observed. A broad endotherm was observed starting at 194° C., followed by thermal decomposition. The TG/DTA curve of the oxalate salt is shown in FIG. 51.

Oxalate (evaporation from THF/water): A broad endotherm was observed starting at about 31° C., with a corresponding weight loss of about 6.2%. A broad endotherm was observed with an onset of about 100° C., with a corresponding weight loss of about 1.6%. Observed thermal decomposition at about 150°C. DSC analysis showed a wide endotherm with an onset around 27°C. The TG/DTA curve of the oxalate salt is shown in FIG. 52.

Phosphate (acetone/water (10%)): a weight loss of about 3% from the start of heating was observed. The TG/DTA curve of the phosphate salt is shown in FIG. 53.

Phosphate (IPA/water (10%)): a weight loss of about 2.5% from the start of heating was observed. A small endotherm was observed with an onset around 162°C. Observed thermal decomposition after 200°C. The TG/DTA curve of the phosphate salt is shown in FIG. 54.

Tartrate (IPA/water (10%)): a weight loss of about 3.7% from the start of heating was observed. A slight endotherm was observed with an onset around 173° C., followed by thermal decomposition. The TG/DTA curve of the tartrate salt is shown in FIG. 55.

Fumarate (THF/water): a weight loss of 5.2% was observed starting at 128° C., corresponding to a slight endotherm at the same temperature. A second small endotherm was observed with an onset around 190° C., with a corresponding weight loss of 2.2%. A sharp melting endotherm was observed starting at 202° C. associated with the melting of form 2 (free base). The TG/DTA curve of the fumarate salt is shown in FIG. 56.

D. Secondary selection of salts.

The phosphate salt obtained in IPA/water (10%) in the primary salt screening was scaled up and further characterized. The scaled method is described below. 300 mg of a compound of formula II was suspended in 3 ml of IPA/water (10%) (10 ml/g), then 1.0 eq. 1M stock solution of phosphoric acid. The mixture was shaken and temperature cycled in 4 hour increments at 40° C. overnight. The resulting solid was filtered and washed with IPA.

The phosphate salt produced on a larger scale was crystalline according to RPD and MPS. The RPD peaks of the phosphate salt are presented in Table 1 below. 5 and in FIG. 23A. The diffraction diffraction pattern corresponds to the data obtained during the primary selection. In thermal analysis, the melting point was set at about 167°C (FIG. 23C). A weight loss of about 1.3% from the start of heating was observed. A second weight loss of about 1.2% was observed starting at about 167°C. The material was very hygroscopic as measured by HSP (FIGS. 23D and 23E), with a mass increase of about 14%, but no change in shape or crystallinity as determined by post-HSP analysis. A broad water peak was observed on the 1H NMR spectrum, indicating salt formation (FIG. 23F). The purity of the salt, according to HPLC, was 97.7%. Impact-activated dissociation (CAD) analysis showed that the salt has a ratio of 1.4:1, PO ₄ :free base.

- 203 041220

Table 55

Phosphate Salt RPD Peaks

Position [°2-theta] SHPV on the left [°2Θ] Area [imp*°20] distance d [A] Height [imp.] Rel. INT. [%] 3.61 0.10 151.75 24.51 1503.25 100.00 6.20 0.15 87.25 14.26 576.19 38.33 7.19 0.15 57.90 12.30 382.36 25.44 8.91 0.15 35.23 9.93 232.63 15.48 9.54 0.10 40.79 9.27 404.11 26.88 10.31 0.15 69.31 8.58 457.72 30.45 12.50 0.10 41.74 7.08 413.47 27.51 13.00 0.15 39.81 6.81 262.90 17.49 14.61 0.10 44.88 6.06 444.57 29.57 15.12 0.10 35.26 5.86 349.27 23.23 15.89 0.15 133.60 5.58 882.27 58.69 16.72 0.10 101.45 5.30 1004.92 66.85 17.84 0.10 92.25 4.97 913.84 60.79 18.22 0.10 108.09 4.87 1070.70 71.23 19.13 0.10 48.49 4.64 480.33 31.95 20.28 0.13 108.57 4.38 860.39 57.24 20.92 0.10 56.42 4.25 558.88 37.18 22.15 0.18 82.62 4.01 467.65 31.11 23.05 0.10 39.45 3.86 390.79 26.00 23.88 0.10 41.78 3.73 413.88 27.53 24.21 0.13 39.71 3.68 314.69 20.93 24.61 0.15 30.06 3.62 198.52 13.21 25.10 0.13 37.82 3.55 299.73 19.94 25.52 0.13 75.71 3.49 599.97 39.91 27.04 0.10 26.96 3.30 267.03 17.76 27.30 0.26 78.12 3.27 309.55 20.59 28.52 0.18 61.69 3.13 349.18 23.23 29.21 0.15 16.56 3.06 109.36 7.28 30.07 0.20 21.19 2.97 104.97 6.98 31.22 0.15 18.15 2.87 119.87 7.97 34.03 0.20 8.71 2.63 43.12 2.87

Portions of the phosphate salt form were weighed into 3 glass tubes to study the stability of the salt. Each tube was stored for 7 days under the following conditions: 1) room temperature and lighting; 40°C and 75% RH; or 3) 80°C. After 7 days, each sample was analyzed by RPD to determine crystallinity and shape. The material was stable under stress conditions with no change in shape or crystallinity observed after a 7 day stability test under any conditions tested. The material, which was stored at room temperature, was 98.03% pure. The material, which was stored at 40°C/75% RH, was 97.44% pure. The material, which was stored at 80° C., was 64.96% pure. One major impurity was identified, most likely a degradation product.

The thermodynamic solubility of the phosphate salt was studied at pH 1, pH 4.5 and pH 6.8. below opi

- 204 041220 san method for studying thermodynamic solubility. About 10 mg of the phosphate salt was weighed into a 2 ml glass tube. 2 ml of the required buffer solution was added to the tube and the resulting suspension was stirred at room temperature overnight. Each solution was centrifuged and the resulting solid was collected. There was not enough solid collected to analyze the phosphate salts from pH 1 and pH 4.5 buffers. The phosphate salt from pH 6.8 buffer was analyzed by RPD and shown to be Form 1. The mother liquor from each experiment was analyzed by HPLC to determine the solubility of each salt at each pH. The results are presented below in table. 56.

Table 56 Thermodynamic solubility

Salt buffer pH Concentration (mg/ml) Results of the RAP Phosphate 1 4.1 Not enough solid 4.5 0.97 Not enough solid 6.8 <0.02 Form 1

Example 5 Screening for Polymorphs of a Formula II Compound A. Assay Tools and Methods.

The assay tools and methods used to screen for polymorphs described in Example 5 are described in Example 4 above.

B. Solubility screening in solvents.

Solubility screening in solvents was performed in 32 solvent systems as follows. About 10 mg of a compound of formula II was placed in each of 32 tubes and 5 volume aliquots of the appropriate solvent system were added to the appropriate tubes. Before each addition, the dissolution of the mixture was checked. In the absence of visible dissolution, the mixture was heated to about 40°C and checked again. This procedure was repeated until visible dissolution or until the addition of 100 volumes of solvent. The undissolved material from the dissolution test was isolated and analyzed by RPD. The solvent systems and the approximate solubility of the compound of formula II in each solvent system tested are shown in Table 1 below. 57, where (P) means partial solubility at 100 volumes.

Table 57

List of Solubility Screening Solvents

Solvent Approximate solubility (mg/ml) 1 1,4-Dioxane <5 (P) 2 1-Butanol <5 3 1-Propanol <5 4 Acetone <5 5 Acetone: water (50%) <5 6 Anizol <5 (P) 7 CHCh <5 (P) 8 Cyclohexane <5

- 205 041220

9 Cyclohexanone <5 (P) 10 DXM <5 (P) eleven tert-Butyl methyl ether N/A 12 DMSO 41 13 ethanol <5 14 EtOAc <5 15 Heptane N/A 16 IPA <5 17 Isopropyl acetate N/A 18 Isopropyl ether N/A 19 MeCN N/A 20 MeCN: water (20%) 5 21 IEC <5 22 MeOAc <5 23 Meon N/A 24 2-Ethoxyethanol <5 (P) 25 2-Methyl-THF <5 (P) 26 MIBC <5 27 Nitromethane <5 (P) 28 NMP 17 29 THF <5 (P) thirty THF:water (1%) 48 31 Toluene N/A 32 Water <5

This substance was found to have only low solubility in most solvent systems tested, with moderate solubility observed in only 3 solvent systems (DMSO, NMP and THF/water (1%)).

The resulting solids were filtered and analyzed by RPD to determine shape and crystallinity. The results are presented below in table. 58. In FIG. 57 shows the XRD patterns of the resulting solids.

- 206 041220

Table 58

Results of RPD of obtained solids

Solvent Form 1 1,4-Dioxane Not enough solid 2 1-Butanol Form 7 3 1-Propanol Form 8 4 Acetone Amorphous 5 Acetone: water (50%) Amorphous 6 Anizol Not enough solid 7 SNS1z Form 1/ Form 13 8 Cyclohexane Form 1 9 Cyclohexanone Not enough solid 10 DXM Not enough solid eleven tert-Butyl methyl ether Form 1 12 DMSO Amorphous 13 ethanol Form 8 14 EtOAc Weak diffraction 15 Heptane Form 1 16 IPA Form 2 17 Isopropyl acetate Form 1 18 Isopropyl ether Form 1 19 MeCN Form 1 20 MeCM:water (20%) Form 2/ Form 13 21 IEC Not enough solid 22 MeOAc Form 7 23 Meon Form 1 24 2-Ethoxyethanol Not enough solid 25 2-Methyl-THF Form 7 26 MIBC Form 1 27 Nitromethane Form 1 28 NMP Not enough solid 29 THF Form 13 thirty THF:water (1%) Form 8 31 Toluene Form 1 32 Water Form 2/ Form 13

C. Primary polymorphic screening.

Primary polymorphic screening was performed using the solvent systems presented in Table 207 041220 below. 59, which were selected according to the solvent solubility screening described above.

Table 59

List of primary polymorphic screening solvents

Solvent 1 Acetone 2 Acetone/water (50%) 3 Anizol 4 1-Butanol 5 2-butanone 6 Chloroform 7 Cyclohexane 8 Cyclohexanone 9 dichloromethane 10 1,4-Dioxane eleven ethanol 12 ethyl acetate 13 Dimethyl sulfoxide 14 MeCN/water (20%) 15 Methyl acetate 16 2-Ethoxyethanol 17 Nitromethane 18 Methyl isobutyl ketone 19 2-Methyl-THF 20 2-Propanol 21 1-Propanol 22 Tetrahyd rofuran 23 L/-Methylpyrrolidone 24 THF/water (1%) 25 Water

1. Experiments with temperature cycling (ripening).

Suspensions for temperature cycling (maturing) experiments were prepared using the solvent systems from Table 1. 59 above as follows. About 40 mg of a crystalline compound of formula II was suspended in an appropriate solvent system. In the case of complete dissolution, an additional amount of the substance was added until a suspension was obtained. The resulting suspensions were subjected to successive 4-hour heating-cooling cycles from 40°C to room temperature for 72 hours.

After temperature cycling, each mixture was filtered and the isolated solids (wet) analyzed by RPD for crystallinity and shape. The samples were then stored at 40° C. and 75% RH overnight, and re-analyzed by RPD to determine possible changes in shape or crystallinity. The results are presented below in table. 60 and in FIG. 58A-58D.

- 208 041220

Table 60

Experiments with temperature cycling (ripening)

Solvent Observations After temperature cycling After stability tests 1,4-Dioxane Solid Weak diffraction Form 1 1-Butanol Solid Weak diffraction Form 1 1-Propanol Solid Form 8 Form 1 Acetone Solid Weak diffraction Form 1 Acetone: water (50%) Solid Form 1 Form 1 Anizol Solid Form 1 Form 1 SNS1 ₃ Solid Amorphous Form 1 Cyclohexane Solid Form 1 Form 1 Cyclohexanone Solid Weak diffraction Form 1 DXM Solid Amorphous Form 1 DMSO Solid Form 1 Form 1 ethanol Solid Weak diffraction Form 1 EtOAc Solid Form 1 Form 1 IPA Solid Form 8 Form 1 MeCM:water (20%) Solid Weak diffraction Form 1 IEC Solid Weak diffraction Form 1 MeOAc Solid Weak Form 1 diffraction 2-Ethoxyethanol Solid Weak diffraction Form 1 2-Methyl-THF Solid Weak diffraction Form 1 MIBC Solid Form 1 Form 1 Nitromethane Solid Weak diffraction Form 1 NMP Not enough solid Not enough solid Not enough solid THF Solid Weak diffraction Form 1 THF: water (1%) Solid Weak diffraction Form 1/ Form 8 Water Solid Form 1 Form 1

The supernatant from each experiment was divided equally into 3 tubes for evaporation, quenching and antisolvent addition experiments (described below).

2. Evaporation experiments.

For the evaporation experiments, a portion of the supernatant samples from the suspensions containing the compound of formula II were transferred to test tubes. Then the tubes were opened and left to evaporate at room temperature. The isolated material was analyzed by RPD without pre-drying. The results are presented below in table. 61 and in FIG. 59.

- 209 041220

Table 61

Evaporation experiments

Solvent Solid Observations Form 1,4-Dioxane Yes Pale yellow solid Form 17 1-Butanol No N/A N/A 1-Propanol No N/A N/A Acetone Yes white solid Form 1/ Form 8 Acetone: water (50%) Yes White needle crystals N/A Anizol Yes white solid N/A SNS1 ₃ Yes yellow solid Form 1/ Form 8 Cyclohexane No N/A N/A Cyclohexanone No N/A N/A DXM Yes white solid Form 1 DMSO Yes white solid Form 1 ethanol Yes white solid N/A EtOAc Yes white solid Amorphous IPA No N/A N/A MEANS: water (20%) Yes White needle crystals Form 1 IEC Yes white solid Amorphous MeOAc Yes white solid Form 1 2-Ethoxyethanol Yes white solid Form 1 2-Methyl-THF Yes white solid N/A MIBC No N/A N/A Nitromethane Yes Pale yellow solid Form 1 NMP No N/A N/A THF Yes yellow solid Form 1/ Form 8 THF: water (1%) No N/A N/A Water No N/A N/A

3. Rapid cooling experiments.

The quench experiments were carried out at 5°C and -20°C. Part of the samples of the supernatant solution from the suspensions containing the compound of formula II, was transferred into test tubes and left to cool at 5°C in the refrigerator. After obtaining a sufficient amount of material (as a result of precipitation), the substance was analyzed without prior drying, using RPD. Samples that did not precipitate were transferred to a freezer and allowed to cool at -20°C. After obtaining a sufficient amount of material, the substance was analyzed without prior drying, using RPD. The results of quench experiments carried out at 5° C. are presented in Table 1 below. 62 and in FIG. 60. In the samples that were stored at -20°C, no solid was observed after 10 days.

Table 62

Quench experiments (5°C)

Solvent Solid Temperature Observations Form 1,4-Dioxane Yes 2°C white solid Amorphous 1-Butanol No N/A N/A N/A 1-Propanol No N/A N/A N/A Acetone Yes 2°C white solid Form 1/ Form 8 Acetone: water (50%) No N/A Anizol Yes 2°C white solid Form 18 SNS1 ₃ No N/A N/A N/A Cyclohexane No N/A N/A N/A Cyclohexanone Yes 2°C white solid Amorphous DXM Yes 2°C white solid N/A DMSO N/A N/A N/A N/A ethanol No N/A N/A N/A EtOAc Yes 2°C white solid Amorphous IPA No N/A N/A N/A MeCM:water (20%) Yes 2°C White needle crystals Form 1 IEC Yes 2°C white solid Amorphous MeOAc Yes 2°C white solid Amorphous 2-Ethoxyethanol Yes 2°C white solid Amorphous 2-Methyl-THF No N/A N/A N/A MIBC No N/A N/A N/A Nitromethane Yes 2°C White needle crystals Amorphous NMP No N/A N/A N/A THF No N/A N/A N/A THF: water (1%) No N/A N/A N/A Water No N/A N/A N/A

4. Experiments with the addition of an antisolvent.

Antisolvent addition experiments were carried out by transferring a portion of the supernatant samples from the suspensions containing the compound of formula II into test tubes and gradually adding the antisolvent to the saturated solutions. After obtaining a sufficient amount of material, the substance was analyzed without prior drying, using RPD. The solvents used in the experiments with the addition of anti-solvent, are presented below in table. 63. The results of experiments with the addition of anti-solvent are presented below in table. 64 and in FIG. 61.

- 211 041220

Table 63

Solvents and antisolvents for antisolvent experiments

Sample Solvent Anti solvent 1 Acetone MTBE 2 Acetone/water (50%) MTBE 3 Anizol MTBE 4 1-Butanol MTBE 5 2-butanone MTBE 6 Chloroform MTBE 7 Cyclohexane MTBE 8 Cyclohexanone Heptane 9 dichloromethane MTBE 10 1,4-Dioxane DIPE eleven ethanol DIPE 12 ethyl acetate MTBE 13 Dimethyl sulfoxide N/A 14 MeCN/water (20%) MTBE 15 Methyl acetate MTBE 16 2-Ethoxyethanol Water 17 Nitromethane MTBE 18 Methyl isobutyl ketone DIPE 19 2-Methyl-THF Water 20 2-Propanol MTBE 21 1-Propanol MTBE 22 Tetrahydrofuran MTBE

- 212 041220

Table 64

Experiments with the addition of an anti-solvent

Solvent Antisolvent Antisolvent volume solids about IA observed Form 1,4-Dioxane DIPE 0.5 Yes white solid Form 1 1-Butanol MTBE 1 Yes white solid n/a 1-Propanol MTBE 1.5 No n/a N/A Acetone MTBE 1 Yes white solid Form 1/ Form 8 Acetone: water (50%) MTBE 1 Yes White needle crystals n/a Anizol Water 1 Yes white solid n/a SNS1 ₃ MTBE 1.5 Yes white solid Form 1/ Form 8 Cyclohexane MTBE 1 No n/a n/a Cyclohexane he Heptane 1 Yes white solid Amorphous DXM MTBE 1 Yes white solid n/a DMSO n/a n/a No n/a n/a ethanol DIPE 1 No n/a n/a EtOAc MTBE 1 No n/a N/A IPA MTBE 1 No n/a N/A MEANS: water (20%) MTBE 1 No n/a N/A IEC MTBE 1 Yes white solid N/A MeOAc MTBE 1 Yes white solid Amorphous 2Ethoxyethano l Water 1 Yes white solid Amorphous 2-Methyl-THF Water 1 No n/a n/a MIBC DIPE 1 No n/a n/a Nitromethane MTBE 1 No n/a n/a NMP Water 1 Yes Yellow needle crystals Form 1 THF MTBE 1 Yes white solid Amorphous THF:water (1%) MTBE 1 No n/a n/a Water MTBE 1 No n/a n/a

D. Secondary polymorphic screening.

During the initial screening of polymorphs in solvents, four forms were identified: form 1, form 2, form 7, and form 8. RPD after GSP, 'H NMR spectroscopy, HPLCV and MPS.

A seven-day stability test was also carried out as follows. A portion of each polymorph was weighed into 3 glass tubes. Each tube was stored for 7 days under the following conditions: 1) room temperature and lighting; 40°C and 75% RH; or 3) 80°C. After 7 days, each sample was analyzed by RPD to determine crystallinity and shape. Additional studies have shown that Form 1 is the most stable form of the compound of formula II, identified during polymorphic screening. Forms 2 and 7 were found to be hydrated forms of the compound of formula II which dehydrated to Form 1 on heating. Form 8 was found to be a solvate with IPA which desolvated to Form 1 on heating. No change in appearance was observed after 7 days for none of the samples, and no change in shape, according to the RPD, after 7 days for any of the samples, although the sample of form 8, which was stored at 40 ° C / 75% RH, appeared to have a higher content of amorphous substances.

1. Form 1.

Form 1 is the most stable form of the compound of formula II identified during polymorphic screening. Form 1 was prepared on a 300 mg scale as follows. About 500 mg of the free base of the compound of formula II was weighed into a 20 ml scintillation tube. Ethyl acetate (4 ml) was added to the tube and the resulting suspension was subjected to temperature cycling from 40°C to room temperature for 72 hours. The sample was filtered and the collected material was dried overnight under vacuum at 40°C.

The RPD after drying (40° C. under vacuum) of the material showed Form 1. The RPD Form 1 peaks are shown in Table 1 below. 65 and in FIG. 19A. The substance was crystalline. According to the MPS data, agglomerates of particles with strong birefringence were observed. A small endothermic event was observed, according to DSC data (FIG. 19B), with an onset around 190°C. A melting transition was observed with an onset around 203°C. A small endothermic event was observed, according to TG/DTA (FIG. 19C), with an onset around 190° C. with a corresponding weight loss of 0.4%. A melting transition was observed with an onset around 204° C., with a corresponding weight loss of 0.2%. After the melting transition, decomposition was observed. The material was moderately hygroscopic as measured by GSP (FIGS. 19D and 19E) with a weight gain of 2.6% at 90% RH. After analysis of the GSP, no change in shape was observed, according to the RPD. According to ¹ H NMR (Fig. 19F), a trace amount of solvent was observed. The obtained spectra were consistent with the spectra of the obtained material (compounds of formula II).

- 214 041220

Table 65

Peaks RPD form 1

Position [°2-theta] FWHM left [°2θ] Area [imp*°20] distance d [A] Height [imp.] Rel. INT. [%] 4.95 0.19 24.33 17.84 97.48 6.05 8.23 0.19 61.56 10.74 246.62 15.32 9.75 0.19 82.45 9.07 330.34 20.52 12.77 0.19 41.33 6.93 165.59 10.28 13.80 0.19 35.96 6.41 144.06 8.95 14.77 0.19 46.81 5.99 187.52 11.65 15.51 0.19 79.80 5.71 319.71 19.86 16.53 0.12 267.93 5.36 1610.16 100.00 17.11 0.19 44.95 5.18 180.08 11.18 17.82 0.12 93.39 4.97 561.24 34.86 18.86 0.16 220.48 4.70 1060.02 65.83 22.34 0.19 29.93 3.98 119.89 7.45 22.81 0.19 29.57 3.89 118.47 7.36 23.75 0.19 145.46 3.74 582.78 36.19 24.99 0.12 52.20 3.56 313.71 19.48 25.32 0.12 99.61 3.52 598.63 37.18 25.59 0.19 132.87 3.48 532.35 33.06 25.96 0.19 158.21 3.43 633.84 39.36 26.56 0.12 46.49 3.35 279.36 17.35 27.07 0.37 96.42 3.29 193.14 12.00 28.28 0.12 55.67 3.15 334.57 20.78 28.86 0.19 64.48 3.09 258.35 16.04

2. Form 2.

Form 2 was isolated from form 7 and considered to be the hydrated form of the compound of formula II, which dehydrates to form 1 upon heating. Form 2 was prepared on a 300 mg scale as follows. About 500 mg of the free base of the compound of formula II was weighed into a 20 ml scintillation tube. Ethanol/water (4 ml, 10%) was added to the tube and the resulting suspension was temperature cycled from 40° C. to room temperature for 72 hours. The sample was filtered and the collected material was dried overnight on the filter.

The analysis of the RPD of the substance of the suspension (obtained on an enlarged scale) was consistent with the data of the material obtained on a small scale, observed in the primary screening of salts - the diffraction pattern corresponded to the diffraction pattern of form 7. The analysis of the RPD, carried out after drying (on the filter layer) of the substance, showed a form 1. Peaks RAP form 2 are presented below in table. 66 and in FIG. 20A. The substance was crystalline. According to the MPS data, agglomerates of particles with strong birefringence were observed. A small endothermic event was observed, according to DSC data (FIG. 20B) with an onset around 192°C. A melting transition of form 1 was observed with an onset around 204°C. According to TG/DTA data (FIG. 20C), a weight loss of 0.7% from the start of heating was observed. A small endothermic event was observed with an onset around 194° C., with a corresponding weight loss of 0.2%. Observed the transition between solid phases and the transition of the melting of form 1 with an onset of about 205°C. After the melting transition, decomposition was observed. The material was moderately hygroscopic as measured by GSP (FIGS. 20D and 20E) with a 2% weight gain at 90% RH. After analysis of the GSP, no change in shape was observed, according to the RPD. ¹ H NMR (not shown) showed a trace amount of solvent. The obtained spectra were consistent with the spectra of the obtained material (compounds of formula II).

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Table 66

Peaks RPD form 2

Position [°2-theta] SHPV on the left [°20] Area [imp*°20] distance d[A] Height [name.] Rel. INT. [%] 6.19 0.15 92.45 14.28 610.55 9.20 8.91 0.10 34.21 9.93 338.85 5.11 10.29 0.15 65.01 8.60 429.31 6.47 13.43 0.41 18.88 6.59 46.76 0.70 13.87 0.15 11.28 6.38 74.48 1.12 14.68 0.15 76.40 6.03 504.57 7.60 15.12 0.10 194.68 5.86 1928.53 29.06 15.87 0.15 16.00 5.58 105.68 1.59 16.50 0.15 76.75 5.37 506.86 7.64 16.79 0.10 39.53 5.28 391.55 5.90 17.14 0.10 28.80 5.17 285.27 4.30 17.42 0.13 43.66 5.09 346.03 5.21 17.82 0.10 669.86 4.98 6635.61 100.00 18.14 0.10 64.77 4.89 641.58 9.67 18.72 0.15 11.62 4.74 76.73 1.16 19.09 0.15 11.11 4.65 73.40 1.11 20.38 0.10 80.18 4.36 794.26 11.97 21.08 0.15 100.83 4.21 665.90 10.04 22.10 0.15 85.85 4.02 566.98 8.54 22.81 0.10 44.29 3.90 438.70 6.61 23.37 0.10 62.75 3.81 621.59 9.37 24.20 0.10 182.81 3.68 1810.90 27.29 24.61 0.10 61.83 3.62 612.45 9.23 25.00 0.13 45.50 3.56 360.61 5.43 25.48 0.10 27.06 3.50 268.10 4.04 26.14 0.10 10.42 3.41 103.22 1.56 27.21 0.10 49.21 3.28 487.46 7.35 27.40 0.10 29.76 3.26 294.85 4.44 27.97 0.13 22.74 3.19 180.20 2.72 29.03 0.15 32.60 3.08 215.30 3.24 29.36 0.13 49.11 3.04 389.19 5.87 29.63 0.13 56.71 3.01 449.38 6.77 29.98 0.13 44.37 2.98 351.62 5.30 30.50 0.10 13.45 2.93 133.23 2.01 31.20 0.10 58.94 2.87 583.89 8.80 31.66 0.10 19.31 2.83 191.32 2.88 32.22 0.13 43.31 2.78 343.20 5.17 32.61 0.20 9.60 2.75 47.53 0.72 34.09 0.13 20.42 2.63 161.81 2.44 34.46 0.10 25.96 2.60 257.14 3.88

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3. Form 7.

Form 7 was considered the hydrated form of the compound of formula II, which dehydrates to form 1 upon heating. Form 7 was prepared on a 300 mg scale as follows. About 500 mg of the free base of the compound of formula II was weighed into a 20 ml scintillation tube. 4 ml of 1,4-dioxane/water (10%) was added to the tube and the resulting suspension was temperature cycled from 40° C. to room temperature for 72 hours. The sample was filtered and the collected material was dried overnight on the filter.

The RPD analysis of the substance of the suspension was consistent with the data for the substance obtained on a small scale, identified in the primary screening of salts. RPD after drying (on the filter layer) of the substance showed form 1.

RPD peaks.

Form 7 are presented below in table. 67 and in FIG. 21A. The substance was crystalline. According to the MPS data, agglomerates of particles with strong birefringence were observed. An endotherm was observed, according to DSC data (Fig. 21B), with an onset around 147°C. A small endotherm was observed with an onset around 196°C associated with the observed transition to Form 1. A melting transition of Form 1 was observed with an onset around 205°C. According to TG/DTA (FIG. 21C), an endotherm was observed with an onset of about 147° C. with a corresponding weight loss of about 7% due to solvent loss. The weight loss corresponded to about 2 equivalents of water present. A small endotherm was observed with an onset of about 196°C associated with the observed transition to Form 1. A melting transition of Form 1 was observed with an onset of about 206°C. The material was slightly hygroscopic as measured by GSP (FIGS. 21D and 21E), with a 0.8% increase in mass at 90% RH, and no change in shape was observed by RPD analysis after GSP. According to 1H NMR (Fig. 21F), a trace amount of solvent was observed.

Table 67

Peaks RPD form 7

Position [°2-theta] FWHM left [°20] Area [imp*°29] distance d[A] Height [imp.] Rel. INT. [%] 4.88 0.15 124.64 18.10 823.10 14.40 6.19 0.15 19.85 14.28 131.10 2.29 8.59 0.15 142.39 10.30 940.35 16.45 9.87 0.15 176.65 8.96 1166.58 20.41 10.31 0.15 28.54 8.58 188.50 3.30 11.62 0.10 34.57 7.62 342.41 5.99 12.58 0.10 36.56 7.03 362.18 6.34 14.14 0.15 140.39 6.27 927.13 16.22 14.84 0.15 143.38 5.97 946.87 16.57 15.77 0.26 452.10 5.62 1791.38 31.34 16.58 0.15 865.48 5.35 5715.58 100.00 17.26 0.10 199.94 5.14 1980.57 34.65 18.04 0.10 290.40 4.92 2876.67 50.33 18.97 0.10 237.60 4.68 2353.62 41.18 19.34 0.15 399.14 4.59 2635.93 46.12 19.91 0.10 334.71 4.46 3315.58 58.01 21.35 0.10 180.69 4.16 1789.93 31.32 21.84 0.10 138.81 4.07 1375.00 24.06 23.34 0.10 258.86 3.81 2564.23 44.86 24.28 0.13 108.82 3.67 862.39 15.09 25.12 0.10 217.06 3.55 2150.16 37.62 25.73 0.10 158.86 3.46 1573.65 27.53

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4. Form 8.

Form 8 was identified as an isopropyl alcohol (IPA) solvate which desolvates to form 1 on heating. Form 8 was prepared on a 300 mg scale as follows. About 500 mg of the free base of the compound of formula II was weighed into a 20 ml scintillation tube. 4 ml of IPA was added to the tube and the resulting suspension was subjected to temperature cycling from 40°C to room temperature for 72 hours. The sample was filtered and the collected material was dried overnight under vacuum at 40°C.

The RPD analysis of the substance of the suspension was consistent with the data for the substance obtained on a small scale, observed in the primary screening of polymorphs and salts. RPD analysis after drying (40° C. under vacuum) of the material showed Form 8. The RPD Form 8 peaks are shown in Table 1 below. 68 and in FIG. 22A. The substance was crystalline. According to the MPS data, agglomerates of particles with strong birefringence were observed. An endotherm was observed, according to DSC data (Fig. 22B), with an onset around 168°C. A small endotherm was observed with an onset of about 190°C associated with the observed transition to form 1. A melting transition of form 1 was observed with an onset of about 203°C. According to TG/DTA (FIG. 22C), an endotherm was observed with an onset of about 165° C. with a corresponding weight loss of about 4% due to solvent loss. The weight loss corresponded to about 0.5 equivalents of IPA present in the sample. A small endotherm was observed with an onset of about 191°C associated with the observed transition to form 1. A melting transition of form 1 was observed with an onset of about 205°C. The material was moderately hygroscopic as measured by GSP (FIGS. 22D and 22E) with a 2.6% weight gain at 90% RH. Filed RPD after analysis of GSP, observed an increased content of amorphous matter. According to 1H NMR (Fig. 22F), 0.5 eq. IPA.

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Table 68

Peaks RPD form 8

Position [°2-theta] FWHM left [°2θ] Area [imp*°20] distance d[A] Height [imp.] Rel. INT. [%] 6.19 0.15 92.45 14.28 610.55 9.20 8.91 0.10 34.21 9.93 338.85 5.11 10.29 0.15 65.01 8.60 429.31 6.47 13.43 0.41 18.88 6.59 46.76 0.70 13.87 0.15 11.28 6.38 74.48 1.12 14.68 0.15 76.40 6.03 504.57 7.60 15.12 0.10 194.68 5.86 1928.53 29.06 15.87 0.15 16.00 5.58 105.68 1.59 16.50 0.15 76.75 5.37 506.86 7.64 16.79 0.10 39.53 5.28 391.55 5.90 17.14 0.10 28.80 5.17 285.27 4.30 17.42 0.13 43.66 5.09 346.03 5.21 17.82 0.10 669.86 4.98 6635.61 100.00 18.14 0.10 64.77 4.89 641.58 9.67 18.72 0.15 11.62 4.74 76.73 1.16 19.09 0.15 11.11 4.65 73.40 1.11 20.38 0.10 80.18 4.36 794.26 11.97 21.08 0.15 100.83 4.21 665.90 10.04 22.10 0.15 85.85 4.02 566.98 8.54 22.81 0.10 44.29 3.90 438.70 6.61 23.37 0.10 62.75 3.81 621.59 9.37 24.20 0.10 182.81 3.68 1810.90 27.29 24.61 0.10 61.83 3.62 612.45 9.23 25.00 0.13 45.50 3.56 360.61 5.43

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25.48 0.10 27.06 3.50 268.10 4.04 26.14 0.10 10.42 3.41 103.22 1.56 27.21 0.10 49.21 3.28 487.46 7.35 27.40 0.10 29.76 3.26 294.85 4.44 27.97 0.13 22.74 3.19 180.20 2.72 29.03 0.15 32.60 3.08 215.30 3.24 29.36 0.13 49.11 3.04 389.19 5.87 29.63 0.13 56.71 3.01 449.38 6.77 29.98 0.13 44.37 2.98 351.62 5.30 30.50 0.10 13.45 2.93 133.23 2.01 31.20 0.10 58.94 2.87 583.89 8.80 31.66 0.10 19.31 2.83 191.32 2.88 32.22 0.13 43.31 2.78 343.20 5.17 32.61 0.20 9.60 2.75 47.53 0.72 34.09 0.13 20.42 2.63 161.81 2.44 34.46 0.10 25.96 2.60 257.14 3.88

Example 6 A polymorph of a compound of formula III.

The compound of formula III, prepared as described in Example 2, was analyzed by XRD, DSC and 1H NMR and found to be Form A. The X-ray powder diffraction pattern of Form A is shown in FIG. 63A. Form A X-ray powder diffraction peaks are shown in Table 1. 69. According to DSC data, an endothermic event was observed with an onset around 83°C (FIG. 63B).

Table 69

Peaks RPD form A of the compound of formula III

2-Theta Intensity 2-Theta Intensity 2.02 4930 13.42 3244 2.74 2840 14 1121 3.12 2219 14.5 1495 4.24 2468 15.24 3953 4.66 5782 15.86 2721 5.1 1247 16.68 1776 6.24 1061 17.3 9498 6.76 3997 17.72 1523 7.16 905 18.76 5838 7.74 827 19.2 9152 8.7 811 20.24 3605 9.22 1144 20.96 1217 9.34 1419 21.92 2319 9.72 815 22.1 1894 10.2 776 23.2 1306 10.56 755 23.86 5921 11.5 740 24.1 2517 11.86 715 25.24 1657 12.66 1246 25.48 2717 13.24 1713 25.7 1941

Example 7 Polymorph of the compound of formula IV.

A. Initial characteristic.

The compound of formula IV, obtained as described in example 2, was analyzed using

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XRD, DSC and 1H NMR, and found to be Form A. The X-ray powder diffraction pattern of Form A is shown in FIG. 64A. Form A X-ray powder diffraction peaks are shown in Table 1. 70. According to DSC data, an endothermic event was observed with an onset around 149°C (FIG. 64B).

Table 70

Peaks RPD form A of the compound of formula IV

2-Theta d(A) Height N% 8.32 10.62 373.20 100.00 9.05 9.77 8.90 2.40 10.11 8.74 0.90 0.20 10.65 8.30 1.20 0.30 11.49 7.70 30.90 8.30 12.25 7.22 27.50 7.40 13.03 6.79 24.40 6.50 13.51 6.55 3.30 0.90 14.08 6.29 0.70 0.20 14.38 6.16 1.40 0.40 14.68 6.03 4.30 1.20 15.60 5.68 3.00 0.80 16.27 5.44 265.60 71.20 16.64 5.32 94.40 25.30 16.89 5.25 5.00 1.40 17.57 5.04 24.40 6.50 18.08 4.90 42.00 11.30 18.61 4.76 39.00 10.50 18.85 4.70 41.00 11.00 19.15 4.63 23.80 6.40

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19.35 4.58 92.10 24.70 19.80 4.48 3.60 1.00 20.04 4.43 49.20 13.20 20.28 4.37 30.50 8.20 20.45 4.34 59.60 16.00 20.90 4.25 0.30 0.10 21.59 4.11 68.30 18.30 21.92 4.05 120.80 32.40 22.69 3.92 13.90 3.70 23.01 3.86 4.10 1.10 23.34 3.81 6.80 1.80 23.81 3.73 11.40 3.10 24.41 3.64 26.50 7.10 25.19 3.53 11.20 3.00 25.71 3.46 7.30 2.00 26.06 3.42 17.40 4.70 27.04 3.29 12.40 3.30 28.21 3.16 3.40 0.90 28.78 3.10 1.10 0.30 29.56 3.02 1.20 0.30 30.41 2.94 17.90 4.80 31.04 2.88 3.00 0.80 31.87 2.81 8.40 2.20 32.10 2.79 21.40 5.70 33.12 2.70 1.30 0.40

33.61 2.66 3.60 1.00 34.16 2.62 5.80 1.50 34.81 2.58 4.10 1.10 35.11 2.55 6.20 1.60 36.76 2.44 2.40 0.60 37.00 2.43 2.40 0.60 37.42 2.40 1.80 0.50 37.84 2.38 2.10 0.60 39.36 2.29 2.00 0.50

B. Solubility screening.

The solubility of the compounds of formula IV (free base) was tested in various mixtures of solvents, as shown in table. 71.

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Table 71

Solubility of the compound of formula IV (free base) at about 25°C

Solvent mg/ml Acetone 42.9 Acetone: water (1:1) 45.8 Acetone: heptane (1:1) 6.1 Butanol 16.6 MTBE 5.5 ethanol 5.2 EUN: water (1: 1) 51 EYN: water (90:10) 55 ethyl acetate 21.0 Heptane 0.0 IEC 30.7 IPA 7.0 ACN 21.2 Chloroform >59 Chloroform: heptane (1:1) P,7 DXM >70 p-dioxane >62 Hexane 0.0 methanol >74 THF >37 THF:heptane (30:70) 0.6 Water 0.007

C. Polymorphic screening.

Additional polymorphic screening was performed using crystallization techniques such as cooling and solvent/anti-solvent precipitation. The results are presented in table. 72.

Table 72

Polymorphic Screening of a Compound of Formula IV

Solvent Crystallization technology Results of the RAP Acetone Suspended at ~25°C for ~3 days Form A Acetone/water Vapor diffusion at room temperature, t-re Form A Acetone:water (1:1) Suspended at ~25°C for ~3 days Form A Aceto n/heptane Vapor diffusion at room temperature, t-re Form A Acetone:heptane (2:1) API was dissolved at ~55 °C, slowly cooled to room temperature without stirring Form A Acetone: heptane (1:1) Suspended at ~25°C for ~3 days Form A CAN Suspended at ~25°C for ~3 days Form A 2-Butanol Suspended at ~25°C for ~3 days Form A 2-Butanol Suspended at ~5°C for ~3 days Form A

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Chloroform Slow evaporation at room temperature -- Chloroform/heptane Vapor diffusion at room temperature, t-re Form A Chloroform:heptane (1:1) Suspended at ~25°C for -3 days Form A DXM Slow evaporation at room temperature — p-dioxane Rapid evaporation at room, t- Re EtOH Suspended at ~25°C for -3 days Form A EUN: water (1:1) Suspended at ~25°C for -3 days Form A EYN: water (90:10) Suspended at ~25°C for -3 days Form B EtOAc Suspended at ~25°C for -3 days Form A EtOAc Suspended at -5°C for -3 days Form A EtOAc API was dissolved at ~70°C, slowly cooled to room temperature Form A Heptane Suspended at ~25°C for -3 days Form A Hexane Suspended at ~25°C for -3 days Form A IPA Suspended at ~25°C for -3 days Form A IPA API was dissolved at ~70°C, slowly cooled to room temperature without stirring Form A Meon Slow evaporation at room temperature —

MeOH/water Vapor diffusion at room temperature, oily substance -- IEC Suspended at ~25°C for -3 days, not enough solids MTBE Suspended at -25°C for -3 days Form A THF Slow evaporation at room temperature -- THF: heptane (30:70) Suspended at -25°C for -3 days Form A THFteptane (1:4) Precipitation using solvent/anti-solvent, observed the formation of a precipitate and an oily substance, left to stir at room temperature for -1 day Form A TNB:water (1:4) Precipitation with solvent/anti-solvent, precipitate and small amount of oil observed, stirred at -5°C, oil persisted THF/heptane Vapor diffusion at room temperature, t-re Form A Water Suspended at -25°C for -3 days Form A

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Form B. A new RPD diffraction pattern of the substance was observed from a suspension in ethanol/water (90:10) as shown in Table 1 above. 72. A substance with a new diffraction pattern was designated form B.

Form B was prepared on a ~65 mg scale by suspending a formulation containing Form A and 1 ml of 9:1 EtOH:H ₂ O at 25° C. for ~36 h. 8000 rpm, five minutes) and then dried in a vacuum oven with N ₂ flow at room temperature overnight (~18 h), followed by RPD and DSC analysis.

Form B was further analyzed by RPD and DSC. The RPD data is shown in Fig. 65A and in table. 73. The DSC thermogram of Form B is shown in FIG. 65V. An endothermic event was observed by DSC with an onset of about 162°C, which is about 12°C higher than Form A. An overlay of X-ray powder diffraction patterns of polymorphs A and B of the compound of formula IV is shown in FIG. 66.

Table 73

Form B RPD Peaks of a Compound of Formula IV

2-Theta d(A) Height N% 4.85 18.19 20.60 5.30 7.45 11.86 390.20 100.00 7.69 11.48 24.90 6.40 9.70 9.11 127.30 32.60 9.94 8.89 88.80 22.80 13.74 6.44 142.80 36.60 14.54 6.09 94.00 24.10 14.89 5.94 1.90 0.50 15.43 5.74 19.40 5.00 15.76 5.62 3.30 0.90 16.31 5.43 20.70 5.30 16.89 5.24 149.00 38.20 17.15 5.17 14.30 3.70 17.39 5.09 10.60 2.70 17.79 4.98 10.80 2.80 18.08 4.90 25.60 6.60 18.48 4.80 20.60 5.30 19.44 4.56 126.00 32.30 19.90 4.46 127.60 32.70 20.80 4.27 2.50 0.60 21.31 4.17 100.00 25.60 21.31 4.17 100.00 25.60 21.44 4.14 62.10 15.90 22.21 4.00 2.60 0.70

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22.41 3.96 15.80 4.00 22.80 3.90 27.70 7.10 23.05 3.86 9.80 2.50 23.53 3.78 9.50 2.40 24.25 3.67 23.30 6.00 24.50 3.63 27.00 6.90 24.91 3.57 10.00 2.60 25.35 3.51 3.80 1.00 25.71 3.46 0.90 0.20 26.31 3.39 15.70 4.00 26.74 3.33 2.60 0.70 26.94 3.31 4.20 1.10 27.39 3.25 31.00 7.90 28.24 3.16 13.10 3.40 28.92 3.08 9.20 2.40 29.56 3.02 0.70 0.20 30.04 2.97 3.70 0.90 31.12 2.87 2.10 0.50 31.76 2.82 5.70 1.50 31.99 2.80 2.10 0.50 32.27 2.77 1.00 0.30 32.98 2.71 2.80 0.70 33.61 2.66 10.20 2.60 34.19 2.62 2.00 0.50 34.68 2.58 1.20 0.30

35.11 2.55 1.70 0.40 35.66 2.52 1.70 0.40 35.84 2.50 2.80 0.70 36.32 2.47 1.30 0.30 38.05 2.36 2.90 0.70 39.00 2.31 1.80 0.50 39.21 2.30 3.00 0.80

Example 8: RET Enzyme Assay.

Compounds of formulas I-IV were tested for their ability to inhibit wild-type RET kinase and V804M mutant RET kinase using CisBio's HTRF® KinEASE™-TK assay technology. Briefly, the N-terminal cytoplasmic domain of human recombinant GST-tagged RET (aa 658-end) from Eurofins (0.25 nM RET; catalog number 14-570M) or the N-terminal cytoplasmic domain of V804M mutant human recombinant RET with GST α-label (aa 658-end) from Millipore (0.25 nM enzyme; cat. no. 14760) was incubated with 250 nM biotin TK substrate (CisBio, cat. no. 62TKOPEC) and 1 mM ATP along with test compound in buffer , consisting of 25 mm HEPES with pH 7.4, 10 mm MgCl ₂ , 0.01% Triton X-100 and 2% DMSO in a volume of 8 μl. The compound was typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30 minute incubation at 22° C., the reaction was quenched by the addition of 8 μl of a quench solution containing 31.25 nM Sa-XL665 and 1X TK-ab-cryptate in HTRF detection buffer (all from CisBio, catalog number 62TKOPEC). After 1 hour incubation at 22° C., the progress of the reaction was determined using a PerkinElmer EnVision multimodal plate reader by dual HTRF wavelength detection, and the percentage of control (ROC) was calculated using the logarithmic emissivity. 100 ROC was determined without using experimental compound, and 0 ROC was determined using pre-quenched control reactions. ROS values were fitted to a 4-parameter logistic curve and IC ₅₀ was determined as the inhibitor concentration at which ROS is 50 for the fit curve. It has been established that the

The definition of formula I has IC ₅₀ values of 4.5 nM and 18.9 nM for inhibition of wild-type RET enzyme and V804M mutant RET kinase, respectively. The compound of formula II was found to have IC ₅₀ values of 14.0 nM and 24.1 nM for inhibition of wild-type RET enzyme and V804M mutant RET kinase, respectively. The compound of formula III was found to have IC ₅₀ values of 29.9 nM and 59.0 nM for inhibition of wild-type RET enzyme and V804M mutant RET kinase, respectively. The compound of formula IV was found to have IC ₅₀ values of 17.0 nM and 121.7 nM for inhibition of wild-type RET enzyme and V804M mutant RET kinase, respectively.

Example 9 Cellular RET Assay.

The efficacy of the RET kinase inhibitory compound in cells was determined on HEK293 cells expressing the Kif5b-RET fusion protein. Briefly, HEK-293 cells expressing the Kif5b-RET fusion protein were plated at 50,000 cells per well in poly-D-lysine coated 96-well plates the day before the assay. Cells were incubated for 1 hour with test compound in DMEM (Dulbecco's Modified Eagle's Medium) at a final DMSO concentration of 0.5%. The compound of formula I-IV was usually prepared in 3-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After 1 hour, the medium was removed, the cells were fixed with 3.8% formaldehyde for 20 minutes, washed with PBS and permeabilized for 10 minutes with 100% methanol. Plates were then washed with PBS-0.05% Tween20 and blocked with LI-COR blocking solution (LI-COR, catalog # 927-40000) for 1 hour. Plates were washed with PBS-0.05% Tween20, then incubated with anti- phospho-RET (Tyr1062) (Santa Cruz, cat. no. sc-20252-R) and anti-GAPDH antibody (Millipore, cat. no. MAB374) for 2 hours. The plates were washed with PBS-0.05% Tween20 and incubated with secondary anti-rabbit 680 (Molecular Probes, cat. no. A21109) and anti-mouse 800 (LI-COR, cat. no. 926-32210) antibodies for 1 hour. All antibodies were diluted with LI-COR blocking solution containing 0.05% Tween. The plates were washed with PBS-0.05% Tween20, 100 μl of PBS was added to each well, and the plates were read on a LI-COR Aerius fluorescent plate reader. Phospho-RET signal was normalized to GAPDH signal. 100 ROC (percentage of control) was determined without using experimental compounds, and 0 ROC was determined using 1 μM control inhibitor. ROS values were fitted to a 4-parameter logistic curve. The IC ₅₀ value is the point where the curve intersects 50 ROS. The compound of formula I was found to have an IC ₅₀ value of 4.0 nM for RET kinase inhibition in HEK-293 cells expressing the Kif5b-RET fusion protein. The compound of formula II was found to have an IC ₅₀ value of 4.2 nM for RET kinase inhibition in HEK-293 cells expressing the Kif5b-RET fusion protein. The compound of formula III was found to have an IC ₅₀ value of 10.9 nM for RET kinase inhibition in HEK-293 cells expressing the Kif5b-RET fusion protein. The compound of formula IV was found to have an IC ₅₀ value of 16.5 nM for RET kinase inhibition in HEK-293 cells expressing the Kif5b-RET fusion protein.

Claims (9)

CLAIM 1. A crystalline form of a compound of formula II having the formula wherein said crystalline form is form 1 characterized by an X-ray powder diffraction (XRD) diffraction pattern containing peaks at °2θ values: a) 16.5±0.2, 18.9±0.2 and 26.0±0.2; b) 16.5±0.2, 18.9±0.2, 23.8±0.2, 25.3±0.2 and 26.0±0.2; c) 16.5±0.2, 17.8±0.2, 18.9±0.2, 23.8±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2 and 28.3±0.2; or d) 9.8±0.2, 16.5±0.2, 17.8±0.2, 18.9±0.2, 23.8±0.2, 25.0±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2 and 28.3±0.2. 2. The crystalline form of a compound of formula II having the formula wherein said crystalline form is form 2 characterized by an X-ray powder diffraction (XRD) diffraction pattern containing peaks at °2θ values: a) 15.1±0.2, 17.8±0 .2 and 24.2±0.2; b) 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2 and 24.2±0.2; - 227 041220 c) 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2 and 24.6±0.2; or d) 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2 and 31.2±0.2. 3. The crystalline form of the compound of formula II having the formula where the specified crystalline form is form 7, characterized by an X-ray powder diffraction (XRD) diffraction pattern containing peaks at values of °2θ: a) 16.6±0.2, 18.0±0.2 and 19.9±0.2; b) 16.6±0.2, 18.0±0.2, 19.3±0.2, 19.9±0.2 and 23.3±0.2; c) 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.9±0.2, 23.3±0.2 and 25.1±0.2; or d) 15.8±0.2, 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.91±0.2, 21.4±0.2, 23.3±0.2 and 25.1±0.2. 4. The crystalline form of the compound of formula II having the formula N=\ N^zAcN And νΑ>| where the specified crystalline form is form 8 solvate with isopropyl alcohol, characterized by an X-ray powder diffraction (XRD) diffraction pattern containing peaks at values of °2θ: a) 15.1±0.2, 17.8±0.2 and 24.2±0.2; b) 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2 and 24.2±0.2; c) 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2 and 24.6±0.2; or d) 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2 and 31.2±0.2. 5. Solid oral pharmaceutical composition containing a pharmaceutically acceptable carrier and a crystalline form according to any one of claims 1-4. 6. The pharmaceutical composition of claim 5 wherein said crystalline form of the compound of formula II is Form 1 and said composition contains less than about 15% by weight of other forms of the compound. 7. The pharmaceutical composition of claim 5 wherein said crystalline form of the compound of formula II is Form 2 and said composition contains less than about 15% by weight of other forms of the compound. 8. The pharmaceutical composition of claim 5 wherein said crystalline form of the compound of formula II is Form 7 and said composition contains less than about 15% by weight of other forms of the compound. 9. The pharmaceutical composition of claim 5 wherein said crystalline form of the compound of formula II is Form 8 and said composition contains less than about 15% by weight of other forms of the compound.