EA041662B1 - METHOD FOR PROTEIN-OR TYROSINE KINASE INHIBITION IN CELL USING DIARYL MACROCYCLIC COMPOUND - Google Patents

Description

Cross-reference to related applications

This application claims priority under 35 U.S.C. § 119(e) U.S. Provisional Applications Serial No. 61/931506, filed January 24, 2014, Serial No. 62/049326, Filed September 2014, and Serial No. 62/106301, Filed January 22, 2015, the entire content of which hereby incorporated herein by reference in their entirety.

Technical field

This invention relates to certain diaryl macrocyclic derivatives, pharmaceutical compositions containing them, and methods of using them for the treatment of cancer, pain, neurological diseases, autoimmune diseases and inflammation.

State of the art

Protein kinases are the main regulators of cell growth, proliferation and survival. Genetic and epigenetic changes accumulate in cancer cells, leading to abnormal activation of signal transduction pathways that trigger malignant processes. Manning, G. et al., Science, 2002, 298, 1912-1934. Pharmacological inhibition of such signaling pathways represents a promising intervention for targeted cancer treatment. Sawyers, S., Nature, 2004, 432, 294-297.

MET, together with RON, belongs to a unique subfamily of receptor tyrosine kinases and is predominantly produced in cells of epithelial or endothelial origin. Park, M. et al., Cell, 1986, 45, 895-904. Hepatocyte growth factor (HGF), also known as scattering factor (SF), is the only known naturally occurring high affinity MET ligand and is primarily expressed in cells of mesenchymal origin. Bottaro, D.P. et al., Science, 1991, 251, 802-804. HGF/MET signals control MET-dependent processes of cell proliferation, survival, and migration, which are critical for invasive growth during fetal development and postnatal organ regeneration, and are only fully active in adults for wound healing and tissue regeneration, Trusolino, L. et al., Nature Rev. Mol. Cell biol. 2010, 11, 834-848. The HGF/MET axis is frequently upregulated in many cancers through activating mutation, gene modification, aberrant paracrine, or autocrine ligand production, and is strongly associated with tumorigenesis, invasive growth, and metastasis, Gherardi, E. et al., Nature Rev. Cancer, 2012, 12, 89-103. Additionally, activation of HGF/MET signaling emerges as an important mechanism for resistance to EGFR and BRAF inhibitor treatments through MET amplification and/or upregulation of stromal HGF, Engelman, J.A. et al., Science, 2007, 316, 1039-1043; Wilson, T.R. et al., Nature, 2012, 487, 505-509. With a role in aberrant HGF/MET signaling in human oncogenesis, invasion/metastasis, and resistance, inhibition of the HGF/MET signaling pathway has great potential in cancer therapy.

ALK, together with leukocyte tyrosine kinase (LTK), clusters within the insulin receptor (IR) superfamily of receptor tyrosine kinases. ALK is primarily expressed in the central and peripheral nervous systems, suggesting a possible role in the normal development and function of the nervous system, Pulford, K. et al., Cell Mol. life sci. 2004, 61, 2939. ALK was first discovered as a chimeric protein, NPM (nucleophosmin)-ALK, encoded by a chimeric gene derived from a t(2;5)(p23;q35) chromosome translocation in anaplastic non-small cell lymphoma (ALCL) cell lines. Morris, S.W. et al., Science, 1994, 263, 1281. More than 200 distinct ALK translocation partners have been found in many cancers, including ALCL (60-90% incidence), inflammatory myofibroblastoma tumors (IMT, 50-60%), non-small cell lung carcinomas (NSCLC, 3-7%), colon and rectal cancers (CRC, 0-2.4%), breast cancers (0-2.4%) and other carcinomas. Grande, E. et al., Mol. Cancer Ther. 2011.10, 569-579. ALK chimeric proteins are located in the cytoplasm, and ALK fusion partners participate in the dimerization or oligomerization of chimeric proteins through a turn-turn interaction to generate constitutive activation of the ALK kinase function. Bischof, D. et al., Mol. Cell Biol., 1997, 17, 2312-2325. EML4-ALK, containing portions of the echinoderm microtubulin-associated protein-like e4 (EML4) gene and the ALK gene, first discovered in NSCLC, is highly tumorigenic and has been shown to cause lung adenocarcinoma in transgenic mice. Soda, M. et al., Nature, 2007, 448, 561-566. Oncogenic ALK point mutations occur in both familial and sporadic cases of neuroblastoma. Mosse, Y.P. et al., Nature, 2008, 455, 930-935. ALK is an attractive molecular target for therapeutic intervention in cancer due to its important role in hematopoietic, solid and mesenchymal tumors. Grande, supra.

Tropomyosin-associated receptor tyrosine kinases (Trks) are a high-affinity receptor for neurotrophins (NTs), a family of nerve growth factor (NGF) proteins. Members of the Trk family are highly expressed in cells of neural origin. Activation of Trks (TrkA, TrkB and TrkC) by their preferred neurotrophins (NGF in TrkA, brain-derived neurotrophic factor [BDNF] and NT4/5 in TrkB and NT3 in TrkC) mediates neuronal survival and differentiation during development. The NT/Trk signaling pathway functions as an endogenous system that protects neurons after biochemical insults, transient ischemia, or physical injury. Thiele, C.J. et

- 1 041662 al., Clin. Cancer Revs. 2009, 15, 5962-5967. However, Trk was originally cloned as an oncogene fused to the tropomyosin gene in the extracellular domain. Activating mutations caused by chromosomal rearrangements or mutations in NTRK1 (TrkA) have been identified in papillary and medullary thyroid carcinoma and more recently in non-small cell lung cancer. Pierotti, M.A. et al., Cancer Lett. 2006, 232, 90-98; Vaishnavi, A. et al., Nat. Med. 2013, 19, 14 69-1472. Because Trks play an important role in pain sensation as well as tumor cell growth and survival signaling, inhibitors of Trk receptor kinases may provide benefits as treatments for pain and cancer.

Receptor tyrosine kinase AXL belongs to the TAM family of proteins and was originally discovered in patients with chronic myeloid leukemia (CML) as an unidentified transforming gene. Verma, A. et al., Mol. Cancer Ther. 2011.10, 1763-1773. The primary ligand for TAM receptors is a growth-stopping specific 6 protein (Gas6). AXL is ubiquitously expressed and has been found in a wide variety of organs and cells, including the hippocampus and cerebellum, monocytes, macrophages, platelets, endothelial cells (EC), heart, skeletal muscle, liver, kidney, and testis. Up-regulation of Gas6/AXL has been reported in many human cancers, including colon, esophagus, thyroid, breast, lung, liver, and astrocytoma-glioblastoma. Increased AXL activation has been observed in both in vitro and in vivo EGFR mutant lung cancer models with acquired erlotinib resistance in the absence of EGFR T790M alteration or MET activation. Zhang, Z. et al., Nat. Genet. 2012, 44, 852-860. Genetic or pharmacological inhibition of AXL restored sensitivity to erlotinib in these tumor models. Increased expression of AXL and in some cases its ligand Gas6 has been found in EGFR mutant lung cancers derived from individuals with acquired resistance to tyrosine kinase inhibitors. Therefore, AXL is a promising therapeutic target for patients with EGFR mutant lung cancer who have become resistant to EGFR inhibitors.

Crizotinib (PF-02341066) is a tyrosine kinase drug targeting MET/ALK/ROS1/RON with moderate activity against TRKs and AXL. Cui, J.J. et al., J. Med. Chem. 2011, 54, 6342-6363. It has been approved for the treatment of certain patients with late stage (locally advanced or metastatic) NSCLC expressing an abnormal ALK fusion gene identified by a companion diagnostic test (Vysis ALK Break Apart FISH Probe kit). Like imatinib and other kinase inhibitor drugs, resistance invariably develops over time with crizotinib treatment. Resistance mechanisms include ALK gene amplification, secondary ALK mutations, and abnormal activation of other kinases including KIT and EGFR. Katayama, R. et al., Sci. Transl. Med. 2012, 4, 120ral7. Based on the clinical success of second generation ABL inhibitors for the treatment of imatinib resistance in CML patients, a second generation of ALK inhibitors is emerging. These drugs are aimed at treating crizotinib-resistant NSCLC patients with more potent inhibition against both wild-type and mutant ALK proteins. Gridelli, C. et al., Cancer Treat Rev. 2014, 40, 300-306.

By modulating multiple targets in a group of structurally related tyrosine kinases MET, ALK, AXL, and TRK, the compounds described herein target crizotinib resistance, EGFR inhibitor drug resistance, and other primary indications with abnormal cell signaling due to MET, ALK, AXL, and/or or TRK mutations and gene amplification. The compounds described herein are inhibitors of MET, wild and mutant ALKs, AXL and TRKs and will be useful in the treatment of cancer patients with abnormal signals from any one or more of the MET, ALK, AXL or TRKs.

The Janus kinase family (JAKs) includes JAK1, JAK2, JAK3, and TYK2, and they are cytoplastic tyrosine kinases required for physiological signaling of cytokines and growth factors. Quintas-Cardama, A. et al., Nat. Rev. drug discov. 2011, 10(2), 127-40; Pesu, M. el al., Immunol. Rev. 2008, 223, 132-142; Murray, P. J., J. Immunol. 2007, 178(5), 2623-2329. JAKs are activated by ligand-induced oligomerization, resulting in the activation of a downstream transcriptional signaling pathway called STAT (signal transducers and transcription activators). Phosphorylated STATs dimerize and translocate to the nucleus to trigger the expression of specific genes involved in proliferation, apoptosis, and differentiation that are essential for hematopoiesis, inflammation, and immune response. Murray, supra.

Mouse knockout studies implied the main roles of JAK-STAT signals, with some overlap. JAK1 plays a critical role in signaling various pro-inflammatory cytokines such as IL-1, IL-4, IL-6 and tumor necrosis factor alpha (TNFa). Muller, M. et al., Nature, 1993, 366(6451), 129-135. JAK2 functions for hematopoietic growth factor signals such as Epo, IL-3, IL-5, GM-CSF, thrombopoietin somatotropin and prolactin-mediated signals. Neubauer, H. et al., Cell, 1998 93(3), 397-409. JAK3 is involved in mediating immune responses and TYK2 is associated with JAK2 or JAK3 for signaling cytokines such as IL-12. Nosaka, T. et al., Science, 1995, 270(5237), 800-802; Vainchenker, W. et al., Semin. cell. dev. Biol. 2008, 19(4), 385-393.

Aberrant regulation of JAK/STAT pathways has been implicated in many human pathologies.

- 2 041662 diseases, including cancer (JAK2) and rheumatoid arthritis (JAK1, JAK3). The JAK2 recovery-of-function mutation (JAK2V617F) has been found in many patients with MPN. Levine, R.L. et al., Cancer Cell, 2005, 7(4), 387-397; Kralovics, R. et al., N. Engl. J. Med. 2005, 253(17), 1779-1790; James, C. et al., Nature, 2005, 434(7037), 1144-1148; Baxter, E.J. et al., Lancet, 2005, 365(9464), 1054-1061. A mutation in the JH2 pseudokinase domain of JAK2 results in substantially kinase activity. Cells containing the JAK2V617F mutation acquire a cytokine-independent ability to grow and often turn into a tumor, providing a reliable basis for the development of JAK inhibitors as a targeted therapy.

Many JAK inhibitors have shown significant benefit in clinical trials for splenomegaly and disease-related constitutional symptoms in patients with myelofibrosis, including the first FDA-approved inhibitor ruxolitinib in 2011. Quintas-Cardama, supra Sonbol, M.B. et al., Ther. Adv. Hematol. 2013, 4(1), 15-35; La Fave, L.M. et al., Trends Pharmacol. sci. 2012, 33(11), 574-582. Recently collected clinical data related to treatment with ruxolitinib, indicating that JAK inhibitors work in both wild-type and mutated JAK2 cases. Verstovsek, S. et al., N. Engl. J. Med. 2012, 366(9), 799-807; Quintas-Cardama, A. et al., Blood, 2010, 115(15), 3109-3117. The discovery of selective JAK2 inhibitors for JAK1/3 remains an unsolved problem. In addition, hyperactivation of JAK2/signal transducers and transcriptional activators 3 (JAK2/STAT3) is responsible for abnormal dendritic cell differentiation, leading to abnormal dendritic cell differentiation and accumulation of immunosuppressive myeloid cells in cancer (Nefedova, Y. et al., Cancer Revs. 2005;65(20):9525-35). In Pten-null senescent tumors, activation of the Jak2/Stat3 pathway establishes an immunosuppressive tumor microenvironment that promotes tumor growth and resistance to chemotherapeutic agents (Toso, A. et al., Cell Reports 2014, 9, 75-89). Thus, pharmacological inhibition of the JAK2/STAT3 pathway may be an important new therapeutic strategy to enhance antitumor activity through the regulation of antitumor immunity.

ROS1 kinase is a receptor tyrosine kinase with an unknown ligand. The normal functions of the human ROS1 kinase have not been fully understood. However, ROS1 kinase has been reported to undergo genetic rearrangement to create constitutively active chimeric proteins in a variety of human cancers, including glioblastoma, non-small cell lung cancer (NSCLC), cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastic tumor, angiosarcoma and epithelioid hemangioendothelioma (Davies, K.D. et al., Clin. Cancer Revs. 2013, 19(15):4040-4045). ROS1-targeted chimeric proteins with crizotinib have shown promising clinical efficacy in NSCLC patients whose tumors are positive for ROS1 genetic abnormalities (Shaw, A.T. et al., N. Engl. J. Med. 2014, 371(21): 1963-1971). Acquired resistance mutations have been observed in patients treated with crizotinib (Awad, M. M. et al., N. Engl. J. Med. 2013, 368(25):2396-2401). There is an urgent need to develop a second generation of ROS1 inhibitors to overcome ROS1 resistance to crizotinib.

There remains a need for small molecule inhibitors of these multiple protein or tyrosine kinase targets with desirable pharmaceutical properties. Certain diaryl macrocyclic compounds have been found in the context of this invention to have such a favorable activity profile.

The essence of the invention

In one aspect, the invention relates to a method for inhibiting in a cell a protein or tyrosine kinase selected from MET, ALK, ROS1, AXL, TRK and/or JAK, comprising contacting the cell with an effective amount of a compound of the formula

or a pharmaceutically acceptable salt thereof, wherein the contact is in vitro, ex vivo or in vivo.

For brevity, descriptions of publications cited in this specification, including patents, are hereby incorporated herein by reference.

Description of graphic materials

Fig. 1 illustrates an x-ray powder diffraction pattern of 11-fluoro-14-methyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-1][1,4,8,10]benzoxatriazacyclotridecine free base crystalline polymorph form 1 -4(5H)-it (example 20).

Fig. 2 shows a differential scanning calorimetry thermogram of the crystalline polymorph form 1 of the free base 11-fluoro-14-methyl-6,7,13,14-tetrahydro-1,15thienopyrazolo[4,3-N1,4,8,10]benzoxatriazacyclotridecin-4( 5H)-she (example 20).

- 3 041662

Detailed description of the invention

Before the present invention is described further, it should be understood that the present invention is not limited to the specific embodiments described, as these may, of course, vary. In addition, it should be understood that the terminology used herein for the purposes of describing particular embodiments of the invention is not intended to be limiting, as the scope of this invention will only be limited by the appended claims.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which this invention pertains. All patents, applications, published applications, and other publications cited in this document are incorporated herein by reference in their entirety. If a definition set forth in this section conflicts with or is otherwise inconsistent with a definition set forth in a patent, patent application, or other publication that is incorporated herein by reference, then the definition set forth in this section shall take precedence over the definition which is incorporated into this document by reference.

As used herein and in the appended claims, the singular forms include the plural unless the context clearly dictates otherwise. It should also be noted that claims may be drafted in such a way as to exclude any optional element. Thus, this statement is intended to serve as a foregoing basis for the use of such exclusive terminology as exclusively, only, etc., in connection with the enumeration of the elements of a claim or for the use of a negative limitation.

As used herein, the terms including and containing are used in their open, non-limiting sense.

To provide a more concise description, some quantitative expressions given in this document are defined by the term approximately. It is understood that whether or not the term is used explicitly approximately, each amount specified herein is intended to indicate an actual set value and is also intended to indicate an approximation to such given value as would reasonably be inferred from ordinary skill in the art. techniques, including equivalents and approximations due to experimental conditions and/or measurement conditions for such a given value. Whenever yield is given as a percentage, such yield refers to the mass of the object for which the yield is given relative to the maximum amount of the same object that can be obtained under specific stoichiometric conditions. Concentrations that are expressed as a percentage refer to mass ratios unless otherwise indicated.

Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing in accordance with this invention, the preferred methods and materials are described below. All publications cited in this document are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which these publications are cited.

Unless otherwise indicated, the methods and techniques of embodiments of this invention are generally performed in accordance with conventional methods well known in the art and as described in various general and more specific references that are cited and discussed in the present description. See, for example, Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University PRevss, 2002, p. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001.

The chemical nomenclature for the compounds described herein was generally derived using the commercially available ACD/Name 2014 program (ACD/Labs) or ChemBioDraw Ultra 13.0 (Perkin Elmer).

It should be understood that certain features of the invention, which for clarity are described in the context of separate embodiments of the invention, can also be presented in combination in one embodiment of the invention. Conversely, various features of the invention, which for brevity are described in the context of one embodiment of the invention, may also be presented individually or in any suitable subcombination. All combinations of embodiments of the invention relating to chemical groups represented by variables are specifically covered by the present invention and are described herein as if each and all combinations were described individually and explicitly to the extent that such combinations cover compounds , which are stable compounds (ie compounds that can be isolated, characterized and tested for biological activity). Additionally, all subcombinations of chemical groups listed in the embodiments of the invention describing such variables are also specifically covered by the present invention and are described herein as follows,

- 4 041662 as if each and all such subcombinations of chemical groups were described separately and explicitly in this document.

Chemical definitions

Any formula depicted herein is intended to represent a compound of that structural formula, as well as certain variations or forms. For example, a formula given herein is intended to include the racemic form, or one or more enantiomeric, diastereomeric, or geometric isomers, or mixtures thereof. In addition, any formula given in this description, is intended to refer also to the hydrate, solvate, or polymorphic form of such a compound, or mixtures thereof.

Any formula provided herein is also intended to represent the unlabeled forms as well as the isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Examples of isotopes that may be included in the compounds of this invention include the isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine such as ² H, ³ H, ^PS , ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ³⁶ Cl, and ¹²⁵ I, respectively. Such isotopically labeled compounds are useful in metabolic studies (preferably with ¹⁴ C), reaction kinetics studies (e.g. with ² H or ³ H), detection or imaging techniques [such as positional emission tomography (PET) or single photon emission computed tomography (SPECT)] including tissue distribution assays of drugs or substrates, or radioactive treatment of patients. In addition, substitution with heavier isotopes such as deuterium (ie ² H) may provide certain therapeutic advantages as a result of higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements. The isotopically labeled compounds of this invention and their prodrugs can generally be prepared by following the procedures described in the Schemes or in the Examples and Preparations described below by replacing a readily available isotopically labeled reagent with a non-isotopically labeled reagent.

The present invention also includes pharmaceutically acceptable salts of the compound and pharmaceutical compositions containing such salts, and methods of using such salts.

A pharmaceutically acceptable salt means a free acid or base salt of a compound provided herein that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. See, in general, S.M. Berge, et al., Pharmaceutical Salts, J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are pharmacologically effective and suitable for contact with the tissues of the subject without undue toxicity, irritation or allergic reaction. The compound described herein may have a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or more than one of each type, and accordingly react with a variety of inorganic or organic bases and inorganic and organic acids to form a pharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monoacid phosphates, diacid phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates. , propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butene-1,4-dioates, hexine-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates , propylsulfonates, besilates, xylosulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates and mandelates. For lists of other suitable pharmaceutically acceptable salts, see REmington's Pharmaceutical Sciences, ^17th Edition, Mack Publishing Company, Easton, Pa., 1985.

For a compound of the invention which contains a basic nitrogen, a pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, and the like, or an organic acid such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranosidyl acid such as glucuronic acid or galacturonic acid, alpha hydroxy acid such as mandelic acid, citric acid slots, or tartaric acid, an amino acid such as aspartic acid or glutamic acid, an aromatic acid such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid, or cinnamic acid, a sulfonic acid such as lauryl sulfonic acid

- 5 041662 acid, p-toluenesulfonic acid, methanesulfonic acid or ethanesulfonic acid, or any compatible mixture of acids, such as those exemplified herein, and any other acid and mixture thereof that are considered equivalent or acceptable substitutes, taking into account the usual level of knowledge in this technology.

Pharmaceutical compositions

For therapeutic purposes, pharmaceutical compositions containing a compound described herein may additionally contain one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance that is non-toxic and otherwise biologically suitable for administration to a subject. Such excipients facilitate the administration of the compounds described herein and are compatible with the active ingredient. Examples of pharmaceutically acceptable excipients include stabilizers, lubricants, surfactants, diluents, antioxidants, binders, coloring agents, fillers, emulsifiers, or flavor modifying agents. In preferred embodiments of the invention, the pharmaceutical compositions of this invention are sterile compositions. Pharmaceutical compositions may be prepared using compounding techniques known or available to those skilled in the art.

Sterile compositions are also embraced by this invention, including compositions that comply with national and local regulations governing such compositions.

The pharmaceutical compositions and compound described herein may be formulated as solutions, emulsions, suspensions or dispersions in suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, suppositories, sachets, dragees, granules, powders, powders for reconstitution. , or capsules together with solid carriers in accordance with conventional methods known in the art, for the preparation of various dosage forms. Pharmaceutical compositions according to the invention may be administered by a suitable delivery method such as oral, parenteral, rectal, nasal, topical, or ophthalmic routes, or by inhalation. Preferably, the compositions are formulated for intravenous or oral administration.

For oral administration of a compound, the invention may be presented in solid form such as tablets or capsules, or as a solution, emulsion or suspension. For the preparation of oral compositions, the compounds of this invention may be formulated to provide a dosage of, for example, about 0.1 mg to 1 g per day, or about 1 to 50 mg per day, or about 50 to 250 mg per day, or approximately 250 mg to 1 g per day. Oral tablets may contain the active ingredient(s) in admixture with compatible pharmaceutically acceptable excipients such as diluents, disintegrants, binders, lubricants, sweeteners, agents, flavors, coloring agents and preservatives. Suitable excipients include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, cellulose, magnesium methyl stearate, mannitol, sorbitol, and the like. Illustrative liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinylpyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are exemplary disintegrants. Binders may include starch and gelatin. The lubricant, if present, may be magnesium stearate, stearic acid or talc. If desired, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or may be coated with an enteric coating.

Capsules for oral administration include hard and soft gelatin capsules. For the preparation of hard gelatin capsules, the active ingredient(s) may be mixed with a solid, semi-solid or liquid diluent. Soft gelatin capsules can be prepared by mixing the active ingredient with water, an oil such as peanut oil or olive oil, liquid paraffin, a mixture of mono- and diglycerides of short chain fatty acids, polyethylene glycol 400 or propylene glycol.

Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups, or may be lyophilized or presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically acceptable excipients such as suspending agents (eg, sorbitol, cellulose, sodium methyl alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, etc.); non-aqueous bases such as oil (eg almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol or water; preservatives (eg methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, optionally, flavoring or coloring agents.

For parenteral use, including intravenous, intramuscular, intraperitoneal, intranasal, or subcutaneous routes, the agents of this invention may

- 6 041662 be presented as sterile aqueous solutions or suspensions buffered to an appropriate pH and isotonicity or in a parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Such forms may be presented in unit dose form such as ampoules or disposable injection devices, in multiple dose forms such as vials from which the appropriate dose can be withdrawn, or in solid form or pre-concentrated which can be used to receiving the drug for injection.

Exemplary infusion doses range from about 1 to 1000 μg/kg/min of the agent in admixture with a pharmaceutical carrier over a period of several minutes to several days.

For nasal, inhalation or oral administration, the pharmaceutical compositions of the present invention may be administered using, for example, a spray formulation also containing an appropriate carrier. Compositions in accordance with this invention can be obtained for rectal administration in the form of a suppository.

For topical use, the compounds of this invention are preferably prepared as creams or ointments or a similar base suitable for topical use. For topical use, the compounds of this invention may be mixed with a pharmaceutical carrier at a concentration of from about 0.1% to about 10% drug per vehicle. Another method of using agents in accordance with this invention may use the composition for patches for transdermal delivery.

As used herein, the terms treat or treatment encompass both prophylactic and curative treatment. Prophylactic treatment is intended to indicate a delay in the development of a disease, symptom of a disease or disease, by suppressing symptoms that may occur, or by reducing the risk of development or recurrence of a disease or symptom. Therapeutic treatment includes reducing the severity or suppressing the worsening of an existing disease, symptom, or condition.

Thus, treatment includes alleviating or preventing the worsening of existing symptoms of a disease by preventing the occurrence of additional symptoms, alleviating or preventing underlying systemic causes of symptoms, inhibiting a disorder or disease, such as arresting the development of a disorder or disease, alleviating a disorder or disease, regressing a disorder or disease, alleviation of the condition caused by the disease or disorder, or cessation of the symptoms of the disease or disorder.

The term subject refers to a mammalian patient in need of such treatment, such as a human.

Exemplary diseases include cancer, pain, neurological diseases, autoimmune diseases, and inflammation. Cancer includes, for example, lung cancer, colon cancer, breast cancer, prostate cancer, hepatocellular carcinoma, renal cell carcinoma, gastric and esophageal cancer, glioblastoma, head and neck cancer, inflammatory myofibroblastic tumors, and anaplastic non-small cell lymphomas. . Pain includes, for example, pain from any source or etiology, including cancer pain, pain from chemotherapy treatment, nerve pain, pain from trauma, or other sources. Autoimmune diseases include, for example, rheumatoid arthritis, Sjögren's syndrome, type I diabetes mellitus, and lupus. Exemplary neurological diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Exemplary inflammatory diseases include atherosclerosis, allergies, and inflammation from infection or trauma.

In one aspect, the compounds and pharmaceutical compositions of this invention specifically target receptor tyrosine kinases, in particular MET, ALK, AXL, TRK and JAK. Thus, these compounds and pharmaceutical compositions can be used to prevent, reverse, slow or inhibit the activity of one or more of these kinases. In preferred embodiments of the invention, the methods of treatment are directed to cancer. In other embodiments, the methods are methods of treating lung cancer or non-small cell lung cancer.

In inhibitory methods in accordance with this invention, an effective amount means an amount sufficient to inhibit the target protein. The measurement of such target modulation can be performed using conventional analytical methods such as those described below. Such modulation is useful in a variety of situations, including in vitro assays. In such methods, the cell is preferably a cancer cell with abnormal signaling due to upregulation of Met, ALK, AXL, TRKs and/or JAK.

In the methods of treatment according to this invention, an effective amount means an amount or dose sufficient to generally obtain the desired therapeutic effect in subjects in need of such treatment. Effective amounts or doses of the compounds according to the invention can be determined using conventional methods such as modeling, escalation

- 7 041662 dose, or clinical trials taking into account conventional factors, for example, the regimen or route of administration or delivery of drugs, the pharmacokinetics of the agent, the severity and course of infection, the subject's health status, condition and weight, and the judgment of the attending physician. An exemplary dosage ranges from about 0.1 mg to 1 g per day, or about 1 to 50 mg per day, or about 50 to 250 mg per day, or about 250 mg to 1 g per day. The total dosage may be given in single or separate dosage units (eg BID, TID, QID).

Once the patient's disease has improved, the dose may be adjusted for prophylactic or maintenance treatment. For example, the dose or frequency of administration, or both, may be reduced depending on the symptoms to a level that maintains the desired therapeutic or prophylactic effect. Of course, if symptoms have been relieved to an appropriate level, treatment may be discontinued. Patients may, however, require intermittent treatment on a long-term basis for any recurrence of symptoms. Patients may also require chronic treatment on a long-term basis.

Chemical synthesis

Illustrative chemical compounds used in the methods of this invention will be described with reference to the illustrative synthetic schemes for their general preparation below and specific examples below. One of ordinary skill in the art will appreciate that, to prepare the various compounds herein, the starting materials may be appropriately selected so that the desired substituents will ultimately be carried through the reaction scheme, with or without protection as needed, to give the desired product. . Alternatively, it may be necessary or desirable to use, in place of the ultimately desired substituent, a suitable group that can be carried through the reaction scheme and replaced where appropriate by the desired substituent. In addition, one of skill in the art will appreciate that the transformations shown in the diagrams below may be performed in any order that is compatible with the functionality of particular pendant (side) groups. Each of the reactions depicted in the general schemes is preferably carried out at a temperature of from about 0° C. to the reflux temperature of the organic solvent used. Unless otherwise indicated, the variables have the meanings given above in reference to formula (I). The isotopically labeled compounds described herein are prepared according to the methods described below using appropriately labeled starting materials. Such materials are usually available from commercial suppliers of radiolabeled chemical reagents.

General method A:

It will be appreciated that the compounds of formula A or A-1 can be prepared according to the general method using appropriately functionalized starting materials and intermediates.

Step 1: To a solution of an appropriately functionalized compound A-1 (~1.00 eq.), TD, where RA and RB are groups compatible with the reaction conditions described herein, and Nu is a nucleophilic group such as an anion or group capable of forming a nucleophile, such as a halide, in a reagent capable of enhancing the coupling of A-1 and A-2, such as an acid (for example, TfOH (0.6 M)) or an alkyl halide (for example, n-BuLi ), A-2 can be added, where ^RC is a group compatible with the reaction conditions described herein, and X ² is, for example, a leaving group (~1.00 eq.) at an appropriate temperature (for example, 0°C). The mixture may be stirred at an appropriate temperature (eg 60° C.) until the reaction is complete. The reaction can then be returned to ambient temperature, the reaction mixture can be quenched, neutralized, washed, extracted, dried and/or concentrated in vacuo as needed to give A-3.

Step 2 Mixture A-3 wherein R ^A , RB and R ^C are groups compatible with the reaction conditions

- 8 041662 described in this document (in some illustrative embodiments of the invention described in this document, A-3 can be a commercially available aldehyde or ketone or A-3 can be obtained in stage 1, ~1.00 eq.) , and A-4 (A-4 may be a commercially available amine, where ^RC is a group compatible with the reaction conditions described herein (~1.50 eq.) in an appropriate solvent (for example, methanol (0. 5 M)) may be stirred at an appropriate temperature (e.g., ambient temperature) for an appropriate period of time or until the conversion is complete according to TLC or LC-MS. A reducing agent (e.g., NaBH4 (~2, 00 eq.)) in portions.The mixture may be stirred at an appropriate temperature (e.g., ambient temperature) until the reaction is indicated by TLC or LC-MS. The reaction may be quenched, washed, extracted, dried, and/or concentrate in vacuo as needed to give A-5.

Step 3: Mixture prepared or commercially available A-5, where RA, RB and ^RC are groups compatible with the reaction conditions described herein (~1 eq.), commercially available ethyl 5-chloropyrazolo[1,5 -a]pyrimidine-3-carboxylate (A-6, ~1 eq.) and the appropriate base (e.g. diisopropylethylamine (~5 eq.)) in an appropriate solvent (e.g. butanol (0.4 M)) can be stirred at appropriate temperature (eg 110° C.) for a specified period of time or until completion of the reaction is demonstrated. The reaction can be returned to ambient temperature and diluted with water as needed. The mixture can be extracted, washed, dried, concentrated under reduced pressure and/or purified by chromatography as needed to give A.

In some exemplary embodiments of the invention, general method A may be performed as follows:

NaBH4

R ^B η Methanol ίΐ ₊ 1 ---- £ J _R c ⁺ H2N-R ^d -----raA^ R ^c>< CI o

A-1 A-2 A-3 A-4

Step 1 To a solution of A-1 (1.00 eq.) in TfOH (0.6 M), A-2 (1.00 eq.) can be added at 0°C. The mixture may be stirred at 60° C. for 4 hours or until the reaction is complete. After cooling to ambient temperature, the reaction mixture can be poured into ice water (wt./wt. = 1/1), neutralized with NaHCO ₃ to pH ~9 and extracted with EtOAc three times as needed. The combined organic layers can be washed with brine, dried over anhydrous Na ₂ SO ₄ as needed and concentrated to give A-3.

Step 2 A mixture of A-3 (commercially available aldehyde or ketone or from step 1, 1.00 eq.) and commercially available amine A-4 (1.50 eq.) in methanol (0.5 M) can be stirred at ambient temperature for 2 h or until completion of imine formation is indicated by TLC or LC-MS data. NaBH4 (2.00 eq.) may be added to the reaction solution in portions. The mixture may be stirred at ambient temperature until TLC or LC-MS indicates completion of the reaction. The reaction can be quenched with water and can be extracted three times with dichloromethane as needed. The combined organic phase can be washed with brine, dried with anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to give A-5.

Step 3: Prepared or commercially available A-5 (1 eq.), ethyl 5-chloropyrazolo[1,5a]pyrimidine-3-carboxylate (A-6, 1 eq.) and diisopropylethylamine (5 eq.) in butanol (0 ,4 M) may be heated at 110°C for 30 minutes or until completion of the reaction is demonstrated. The reaction can be cooled and diluted with water. The mixture can be extracted with dichloromethane four times (as needed) and the combined extracts can be dried over anhydrous sodium sulfate. After filtration, the mixture can be concentrated under reduced pressure and the residue can be purified using flash chromatography to give A.

- 9 041662

Alternative general method A:

AA-1 AA-2 A-6

AA-3 AA-4

Coupling Step 1: A mixture of suitably functionalized AA-1 (~1.00 eq), suitably functionalized vinyl coupling agent (~1.00-1.50 eq) and palladium catalyst (~0.05 eq) in appropriate reaction conditions can be heated to ambient temperature (for example, ~90°C) for an appropriate period of time in an inert atmosphere until TLC shows complete consumption of starting material. The reaction mixture can be poured into H2O as needed.

The mixture can be extracted and the organic phase washed, dried, concentrated and purified by silica gel column chromatography as needed to give AA-2.

Coupling Step 2: A mixture of type AA-2 compound (~1.00 eq.), ethyl 5-chloropyrazolo[1,5a]pyrimidine-3-carboxylate (A-6, ~1.00 eq.) and palladium catalyst in the respective reaction conditions can be heated to ambient temperature (for example, 120°C) for an appropriate period of time in an inert atmosphere until TLC shows complete consumption of starting material. The reaction mixture can be poured into H2O as needed. The mixture can be extracted and the organic phase can be washed, dried, concentrated and purified by silica gel column chromatography as needed to give AA-3.

Step 3 To a mixture of AA-3 (~1.00 eq.) and 4-methylbenzenesulfonohydrazide (molar excess) in a suitable solvent, the appropriate base (molar excess) can be added at the appropriate temperature under an inert atmosphere. The mixture may be warmed to ambient temperature (eg 65° C.) and stirred for an appropriate period of time until TLC indicates completion of the reaction. The mixture can be cooled and concentrated under reduced pressure as needed. The concentrated reaction mixture can be diluted with water as needed and extracted. The combined organic phase can be washed, dried, filtered, concentrated in vacuo and purified to give AA-4.

General method B:

^rc nh ₂

restorative

NH ______agent______

PG Solvent

θ^-^NHPG if

B-4 A- ₆

Step 1. Aldehyde solution B-1 (~1.0 eq) where R ^A and R ^B are groups compatible with the reaction conditions described herein, B-2 (~1.0 eq) where X ¹ is a leaving group and PG is a protecting group, a suitable base (in molar excess) and a catalyst in a suitable solvent can be heated and stirred for an appropriate period of time to complete the reaction. Additional B-2 may be added and additional heat applied as needed. The mixture can be cooled to ambient temperature and diluted with water as needed. The mixture can be extracted and the combined extracts can be washed, dried and concentrated under reduced pressure as needed. The crude reaction product can be purified using flash chromatography to give B-3.

Step 2. Aldehyde B-3 (~1.0 eq.) and an appropriately functionalized amine (~2.0-4.0 eq.), where R ^A is a group compatible with the reaction conditions described in

- 10 041662 of this document, in an appropriate solvent can be heated and mixed for an appropriate period of time. Can the mixture be cooled to ambient temperature? and an appropriate reducing agent (~1.0 eq.) may be added. The mixture can be stirred for an appropriate period of time and then quenched by adding water as needed. The mixture can be extracted into an appropriate organic solvent and the combined extracts can be washed, dried and concentrated under reduced pressure as needed. The crude reaction product can be purified by flash chromatography as needed to give B-4.

Step 3 Compound B-4 (~1.0 eq.), Ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (A-6, ~1.0 eq.) and a suitable base (in molar excess) in a suitable solvent can be heated for an appropriate period of time. The reaction can be cooled and diluted with water as needed. The mixture can be extracted with a suitable organic solvent and the combined extracts dried and concentrated under reduced pressure as needed. The crude reaction product can be purified using flash chromatography to give B1.

In some illustrative embodiments of the invention, the general method B can be performed as follows:

Step 1. Aldehyde solution B-1 (~1.0 eq) where R ^A and R ^B are groups compatible with the reaction conditions described herein, B-2 (~1.0 eq) where X ¹ is a leaving group and PG is a protecting group, potassium carbonate (in molar excess) and potassium iodide (catalytic amount) in DMF, can be heated to 60°C and stirred for ~15 hours. Additional B-2 chloride may be added and additional heat of 80° C. may be applied as needed until completion of the reaction is demonstrated. The mixture can be cooled to ambient temperature and diluted by adding water (250 ml) as needed. The mixture can be extracted with ethyl acetate (3x300 ml) and the combined extracts washed with water (200 ml) and brine (100 ml), dried over sodium sulfate and concentrated under reduced pressure as needed. The crude reaction product can be purified using flash chromatography to give B-3.

Step 2 Aldehyde B-3 (~1.0 eq.) and methylamine (~2.5 eq.) in methanol can be heated to 60° C. and stirred for ~1 hour. The mixture can be cooled to ambient temperature , and sodium borohydride (~1.0 eq.) may be added. The mixture can be stirred for ~30 min and then quenched by adding water (200 ml) as needed. The mixture can be extracted with dichloromethane and the combined extracts can be washed with brine (50 ml), dried over sodium sulfate and concentrated under reduced pressure as needed. The crude reaction product can be purified by flash chromatography to give B-4.

Step 3. Amine B-4 (~1.0 eq.), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (A-6, ~1.0 eq.) and Hunig's base (in molar excess) in butanol can be heated at 110°C for ~25 min. The reaction can be cooled and diluted with water (250 ml). The mixture can be extracted with dichloromethane and the combined extracts dried over sodium sulfate as needed. The mixture can be concentrated under reduced pressure as needed. The crude reaction product can be purified by flash chromatography to give B.

- 11 041662

General method C:

Step 1 To a solution of C-1 (~1.0 eq.) where R ^A , R ^B , ^RC , RD and RE are groups compatible with the reaction conditions described herein, X1AlkNHPG (~1.5 -2.0 eq.), where X ¹ is a leaving group, Alk is an appropriately functionalized alkyl group, and PG is a protecting group in a suitable solvent, a suitable base (~3.0 eq.) can be added. The mixture may be heated to ambient temperature for an appropriate period of time under an inert atmosphere until complete conversion of the starting material into the product indicated by LC-MS. The mixture can be cooled to ambient temperature, diluted with water and extracted with a suitable organic solvent as needed. The combined organic extracts can be washed with water and brine, dried over Na ₂ SO ₄ , and concentrated as needed. The resulting residue can be purified by silica gel column chromatography as needed to give C-2.

Step 2 To a solution of C-2 (1 eq) where R ^A , R ^C , RD and RE are groups compatible with the reaction conditions described herein, Alk is an appropriately functionalized alkyl group and PG is protecting group in a suitable solvent, a suitable base (in molar excess) may be added. The solution may be heated to ambient temperature for an appropriate period of time. The reaction can be neutralized with a suitable acid to pH<5, the reaction mixture can be extracted with a suitable organic solvent. The combined organic layers can be washed and dried as needed. The crude product of the reaction mixture can be filtered, concentrated under reduced pressure and dried under high vacuum as needed to give C-3.

Step 3 To a solution of C-3 (~1.0 eq) in a suitable organic solvent, an appropriate acid (~4 eq) can be added at an appropriate temperature (eg 0°C). The reaction mixture may be stirred at an appropriate temperature for an appropriate period of time until completion of the reaction is demonstrated by LC-MS. The crude product may be filtered, washed and may be dried under high vacuum to give C-4.

Stage 4a. To a solution of C-4 (~1.0 eq.) in a suitable solvent, a suitable base (in molar excess) can be added. The solution can be cooled in an ice water bath and a suitable coupling agent (~1.5 eq) can be added to give the activated ester. The solution may be warmed to ambient temperature slowly and stirred until the starting material is shown to have been converted to the desired product by LC-MS. The mixture can be diluted with water and extracted with a suitable organic solvent as needed. The combined organic extracts can be washed, dried and concentrated under reduced pressure as needed. The resulting residue can be purified by silica gel column chromatography to give C.

In some illustrative embodiments of the invention, the general method C can be performed as follows:

- 12 041662

With

Step 1 To a solution of C-1 (~1.0 eq.) where R ^A , R ^B , ^RC , RD and RE are groups compatible with the reaction conditions described herein, X1AlkNHPG (~1.5 -2.0 eq.), where X ¹ is a leaving group, Alk is an appropriately functionalized alkyl group and PG is a protecting group in DMF (0.5 M), K ₂ CO ₃ (~3, 0 equiv.). The mixture can be heated at ~80° C. for ~2 hours or until complete conversion of starting material to product is indicated by LC-MS. The mixture can be cooled to ambient temperature, diluted with water as needed, and extracted three times with EtOAc as needed. The combined organic layers can then be washed with water and brine, dried over Na ₂ SO ₄ and concentrated as needed. The resulting residue can be purified by silica gel column chromatography eluting with EtOAc/hexane (5-100%, 10CV) to give C-2.

Step 2. To a solution of C-2 (~1 equiv.) in methanol/THF/H ₂ O (3:1:1, 0.2 M) can be added LiOH-H ₂ O (~5.0 equiv.) . The solution can be heated at ~70°C for ~2 hours. The reaction can be neutralized at -0°C aq. HCl (2 M) to pH <5 and extracted four times with CH ₂ Cl ₂ as needed. The combined organic extracts can be washed with brine and dried over Na ₂ SO ₄ as needed. The crude product mixture can be filtered, concentrated under reduced pressure and dried under high vacuum as needed to give C-3.

Step 3 To a solution of C-3 (~1.0 eq.) in CH ₂ Cl ₂ (0.25 M), HCl in dioxane (4 M, ~4 eq.) can be added at ~0°C. The reaction may be stirred and allowed to warm from 0° C. to room temperature for approximately 27 hours or until completion of the reaction is demonstrated by LC-MS. The resulting reaction mixture can be filtered, washed with CH ₂ Cl ₂ and dried under high vacuum as needed to give C-4.

Stage 4a. Cyclization of HATU.

To a solution of C-4 (~1.0 eq.) in ~10 ml DMF (~0.005 M) DIPEA (~5.0 eq.) can be added. The solution can be chilled in an ice water bath and HATU (~1.5 eq.) can be added. The solution can be allowed to warm to ambient temperature and stirred until complete conversion of the starting material to the desired product is indicated by LC-MS. The mixture can be diluted with water and extracted three times with EtOAc as needed. The combined organic extracts can be washed with water and brine, dried over Na ₂ SO ₄ and concentrated under reduced pressure as needed. The resulting residue can be purified by silica gel column chromatography (0-5% MeOH/DCM) to give C.

Stage 4b. Cyclization of FDPP.

To a solution of DIPEA (~5 eq.) in DMF/CH ₂ Cl ₂ (3:1, ~0.005 M) C-4 (~1.00 eq.) can be added. After C-4 is completely dissolved, pentafluorophenyl diphenyl phosphinate (FDPP, ~1.05 eq.) can be added. The combination can be allowed to stir for 30 minutes or until the reaction is shown to be complete by LC-MS. The reaction solution mixture can be diluted with CH ₂ Cl ₂ , washed three times with water, aqueous Na ₂ CO ₃ (2 M) and brine, and dried over Na ₂ SO ₄ as needed. After filtration and concentration under reduced pressure, the residue can be purified by silica gel column chromatography eluting with MeOH/CH ₂ Cl ₂ (0-5%) to give C.

Examples

The following examples are offered to illustrate and not limit the present invention. One skilled in the art will recognize that the following synthetic reactions and schemes may be modified by selection of suitable starting materials and reagents to prepare other compounds of the invention. Bicyclic heteroaromatic groups with suitable functionalities for use in synthetic methods are commercially available.

Abbreviations.

The examples described herein use materials including, but not limited to, those described with the following abbreviations known to those skilled in the art:

Abbreviation Name those Thin layer chromatography PLC preparative liquid chromatography HPLC high performance liquid chromatography LCMS, LC-MS liquid chromatography mass spectrometry LRESIMS mass spectrometry ionization method low resolution electrospray ELISA Enzyme-associated immunoassay method DCM dichloromethane anm DMSO dimethyl sulfoxide DIPEA, DIEA diisopropylethylamine GDI 1,1'-carbonyldiimidazole THF tetrahydrofuran Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene TBSC1 tert-butyl dimethylsilyl chloride DMF N,N-dimethylformamide HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate ACN acetonitrile EtOAc ethyl acetate DTAD Di-tert-butyl azodicarboxylate FDFF pentafluorophenyl diphenyl phosphinate FBS Fetal bovine serum BSA bovine serum albumin PBS Phosphate buffered saline DMEM medium Needle, modified according to the method Dulbecco EDTA Ethylenediaminetetraacetic acid RIPA Radioimmunoprecipitation analysis HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

Reference Example A6.

A6-3 A6-4 A6-5

Step 1 To a solution of 5-fluoro-2-hydroxybenzaldehyde (500.00 mg, 3.57 mmol, 1.00 eq.) in MeOH (20.00 mL) was added 1-methylpyrrolidin-3-amine (357.43 mg , 3.57 mmol, 1.00 eq.) in one portion at 16° C. under N2. The mixture was stirred at 16°C for 10 hours under N2. Then NaBH4 (270.00 mg, 7.14 mmol, 2.00 eq.) was added and the mixture was stirred at 16° C. for 6 hours under N ₂ atmosphere. TLC (DCM:MeOH = 15:1) indicated completion of the reaction. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was diluted with water (50 ml) and extracted with DCM (3x20 ml). The combined organic layers were washed with brine (50 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give A6-5 (350.00 mg, 1.56 mmol, 43.71% yield) as a yellow solid substances.

1H NMR (300 MHz, DMSO-a ₆ ) δ 6.94 (dd, J=2.7, 9.3 Hz, 1H), 6.86 (dt, J=3.0, 8.6 Hz, 1H ), 6.67 (dd, J=4.7, 8.7 Hz, 1H), 3.71 (s, 2H), 3.24-3.09 (m, 1H), 2.58 (dd, J=7.1, 8.8 Hz, 1H), 2.48-2.32 (m, 2H), 2.30-2.17 (m, 4H), 2.05-1.82 (m, 1H), 1.60-1.43 (m, 1H).

Step 2 To a solution of A6-5 (300.00 mg, 1.34 mmol, 1.00 eq) and ethyl 5-chloropyrazolo[1,5a]pyrimidine-3-carboxylate (302.34 mg, 1.34 mmol, 1.00 eq.) in n-BuOH (40.00 ml) was added DIPEA

- 14 041662 (1.04 g, 8.04 mmol, 6.00 eq.) at 16° C. under N ₂ atmosphere. The mixture was stirred at 120°C for 2 hours.

TLC (PE:EtOAc = 1:1) indicated completion of the reaction. The mixture was poured into water (50 ml) and extracted

DCM (3x50 ml). The mixture was purified with pre-PLC to give the A6 formic acid salt (290.00 mg, 701.43 µmol, 52.35% yield) as a white solid.

Reference Example A8.

To a solution of ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (1.25 g, 5.54 mmol) and (R)-2-(1-aminoethyl)-4-fluorophenol HCl salt (purchased from NetChem, Inc.) in EtOH (15.83 ml) was added Hunig's base (3.58 g, 27.70 mmol) and heated to 70°C for 1.5 hours. The reaction was evaporated to dryness, suspended in water, extracted with DCM (5x50 ml). The combined extracts were dried over Na ₂ SO ₄ and concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel (40 g), 0-5% methanol in dichloromethane) provided A8 (1.89 g, 5.49 mmol, 99% yield).

Reference Example A9.

Step 1 To a solution of 4-fluorophenol (2.00 g, 17.84 mmol, 1.00 eq.) in TfOH (30.00 ml) was added propanoyl chloride (1.65 g, 17.84 mmol, 1.00 equiv.) at 0°С. The mixture was stirred at 60° C. for 4 h. TLC indicated completion of the reaction. The mixture was cooled to 25°C, poured into ice water (w/w = 1/1) (120 ml), neutralized with NaHCO ₃ to pH ~9, and extracted with EtOAc (3x120 ml). The combined organic layers were washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ and concentrated to give A9-3 (1.80 g, 10.70 mmol, 59.98% yield) as a colorless oil.

¹ H NMR (400 MHz, CDCl. J δ 12.09 (s, 1H), 7.45 (dd, J=3.0, 9.0 Hz, 1H), 7.26-7.20 (m, 1H), 6.97 (dd, J=4.5, 9.0 Hz, 1H), 3.02 (sq., J=7.3 Hz, 2H), 1.27 (t, J=7, 2 Hz, 3H).

Step 2 Ammonia vapor was purged through MeOH (20 ml) at -78°C for 10 minutes. A9-3 (1.00 g, 5.95 mmol, 1.00 eq.) was added to the solution and stirred at 25°C for 1 h. Ti(i-PrO) ₄ (1/63 g , 7.14 mmol, 1.20 eq.) and the mixture was stirred for another 1 h. Then NaBH ₄ (449.93 mg, 11.89 mmol, 2.00 eq.) was added. The mixture was stirred at 25° C. for 12 hours until TLC showed complete consumption of the starting material. The residue was poured into water (50 ml) and stirred for 30 minutes. The mixture was filtered and the filtrate was adjusted to pH ~1 with HCl (1 M) and extracted with EtOAc (2x50 ml). Sodium bicarbonate was added to the aqueous phase to adjust the pH ~9 and was extracted with DCM (2x50 ml). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give A9-5 (310.00 mg, 1.83 mmol, 30.79% yield) as a yellow solid.

1H NMR (400 MHz, CDCl ₃ ) δ 6.86 (dt, J=3.0, 8.4 Hz, 1H), 6.79-6.74 (m, 1H), 6.67 (dd, J =2.9, 8.9 Hz, 1H), 3.98 (t, J=7.0 Hz, 1H), 1.92-1.81 (m, 1H), 1.80-1.68 ( m, 1H), 0.95 (t, J=7.4 Hz, 3H).

Step 3 A9-5 was coupled with ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate in the presence of DIPEA in n-BuOH to give A9 as described in General Method A.

Reference Example A13-5. Preparation of 2-(1-amino-2-cyclopropylethyl)-4-fluorophenol.

Step 1. To a mixture of 2-cyclopropylacetic acid (4.47 g,

44.60 mmol, 1.00 eq) in DCM

- 15 041662 (150.00 mL) CDI (7.96 g, 49.10 mmol, 1.10 eq.) was added in one portion at 25°C under N2 atmosphere. The mixture was stirred at 25° C. for 1 hour. Then N-methoxymethanamine hydrochloride (4.79 g, 49.06 mmol, 1.10 eq.) was added. The mixture was stirred at 25° C. for another 12 hours. The reaction was quenched with 1N. aqueous hydrochloric acid (50 ml) and separated into layers. The aqueous layer was extracted with DCM (2x30 ml). The combined organic layer was washed with 50% saturated aqueous sodium carbonate (50 ml) and brine (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to give 2-cyclopropyl-N-methoxy-N-methylacetamide (6 .00 g, 41.91 mmol, 93.96% yield) as an oil.

1H NMR (400 MHz, CDCI3) δ 3.65 (s, 1H), 3.18 (s, 1H), 2.33 (d, J=6.8 Hz, 2H), 1.13-1.02 (m, 1H), 0.570.49 (m, 2H), 0.19-0.11 (m, 2H).

Step 2 n-BuLi (2.5 M, 12 .88 ml, 1.10 eq.) dropwise at -78° C. under N2. The mixture was stirred at -78°C for 10 minutes. The mixture was then treated with 2-bromo-4-fluoro-1-methoxybenzene (4.19 g, 29.27 mmol, 1.00 eq.) in THF (20 ml) over a period of 20 minutes. After stirring at -78° C. for 1 hour, the mixture was allowed to warm to 25° C. and stirred for another 1 hour. TLC indicated completion of the reaction. The mixture was poured into a 10% aqueous HCl solution (100 ml) and stirred for 10 minutes. The aqueous phase was extracted with ethyl acetate (3x300 ml). The combined organic phase was washed with brine (200 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 50/1, 10/1) to give 2-cyclopropyl-1-(5-fluoro-2-methoxyphenyl)ethan-1one (2.4 g, 39.38 % yield) as a colorless oil.

1H NMR (400 MHz, CDCl ,) δ 7.42 (dd, J=3.3, 8.8 Hz, 1H), 7.15 (dd, J=3.3, 7.5, 9.0 Hz, 1H), 6.91 (dd, J=4.0, 9.0 Hz, 1H), 3.91-3.85 (m, 3H), 2.89 (d, J=6.8 Hz , 2H), 1.18-1.05 (m, 1H), 0.61-0.50 (m, 2H), 0.20-0.09 (m, 2H).

Step 3 To a solution of 2-cyclopropyl-1-(5-fluoro-2-methoxyphenyl)ethan-1-one (500.00 mg, 2.40 mmol, 1.00 eq.) in DCM (10.00 ml) BCl ₃ (1 M, 3.00 ml, 1.25 eq.) was added dropwise at -78° C. under N2 atmosphere. The mixture was stirred at -78°C for 2 hours. TLC indicated the reaction was complete. The mixture was heated to 25° C. and poured into ice water (w/w=1/1) (10 ml) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate (3x30 ml). The combined organic phase was washed with brine (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to give 2-cyclopropyl-1-(5-fluoro-2-hydroxyphenyl)ethan-1-one (430, 00 mg, 2.21 mmol, 92.3% yield) as an oil.

1H NMR (400 MHz, CDC13) δ 12.12 (s, 1H), 7.40 (dd, J=3.0, 8.8 Hz, 1H), 7.24 (dd, J=3.0, 7.8, 9.0 Hz, 1H), 6.98 (dd, J=4.5, 9.3 Hz, 1H), 2.88 (d, J=6.8 Hz, 2H), 1, 23-1.11(m, 1H), 0.70-0.63(m, 2H), 0.25(q, J=5.0Hz, 2H).

Step 4 To a solution of 2-cyclopropyl-1-(5-fluoro-2-hydroxyphenyl)ethan-1-one (400.00 mg, 1.92 mmol, 1.00 eq.) in MeOH (20.00 ml) NH2OH-HC1 (160.18 mg, 2.31 mmol, 1.20 eq.) and AcONa (189.09 mg, 2.31 mmol, 1.20 eq.) were added at 25°C under N2 atmosphere for 12 h. TLC (petroleum ether/ethyl acetate = 3:1) did not show complete consumption of starting material. The reaction was quenched with water and then extracted with DCM (3x30 ml). The combined organic phase was washed with brine (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to give the pure product 2-cyclopropyl-1-(5-fluoro-2-hydroxyphenyl)ethan-1-one oxime ( 400.00 mg, 1.79 mmol, 93.32% yield) as a white solid. The solid was used for the next step without further purification.

Step 5 To a solution of 2-cyclopropyl-1-(5-fluoro-2-hydroxyphenyl)ethan-1-one oxime (260.00 mg, 1.16 mmol, 1.00 eq) in MeOH/HCl (10, 00 ml, 4N) was added Pd-C (10%, 100 mg) under N2 atmosphere. The suspension was degassed in vacuo and purged with H ₂ several times. The mixture was stirred in H ₂ (50 psi) at 50°C for 12 hours LC-MS did not show complete consumption of starting material. The reaction mixture was filtered and the filtrate was concentrated to give 2-(1-amino-2-cyclopropylethyl)-4-fluorophenol (200.00 mg, 955.75 µmol, 82.39% yield) as a white solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ 10.44-9.82 (m, 1H), 8.52 (br. s, 2H), 7.36 (dd, J=2.8, 9, 5 Hz, 1H), 7.076.93 (m, 2H), 4.49 (d, J=5.5 Hz, 1H), 1.82-1.72 (m, 2H), 0.67-0, 55 (m, 1H), 0.43-0.28 (m, 2H), 0.12-0.06 (m, 1H), (-0.03)-(-0.09) (m, 1H ).

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Reference Example A14-5. Preparation of 2-(amino(phenyl)methyl)-4-fluorophenol.

Step 1. To a solution of A14-3 (2.00 g, 9.25 mmol, 1.00 eq.) and AcOK (1.10 g, 11.20 mmol, 1.20 eq.) in ethanol (30.00 ml) was added NH ₂ OH-HCl (642.80 mg, 9.25 mmol, 1.00 eq.) in one portion at 25° C. under N 2 atmosphere. The mixture was stirred at 25° C. for 30 minutes, then heated to 90° C. and stirred for 5 hours. TLC indicated completion of the reaction. The mixture was concentrated and water (50 ml) was added. The mixture was extracted with ethyl acetate (3x50 ml). The combined organic phase was washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give (5-fluoro-2-hydroxyphenyl)(phenyl)methanone oxime (1.50 g, 6.49 mmol, 70 , 13% yield) as a yellow solid.

1H NMR (400 MHz, CDCl ₃ ) δ 7.50-7.37 (m, 5H), 7.19-7.07 (m, 2H), 6.71 (dd, J=2.9, 8, 9 Hz, 1H).

A17

Step 2 To a mixture of (5-fluoro-2-hydroxyphenyl)(phenyl)methanone oxime (900.00 mg, 4.18 mmol, 1.00 eq) and Zn powder (1.09 g, 16.73 mmol, 4 eq.) in THF (10.00 mL) was added NH4Cl (2.24 g, 41.82 mmol, 10.00 eq.) in one portion at 25° C. under N2 atmosphere. The mixture was stirred at 25° C. for 30 min, then heated to 60° C. and stirred for 15 h. The mixture was concentrated and water (100 ml) was added followed by extraction with ethyl acetate (3x50 ml). The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give A14-5 (630.00 mg, 2.90 mmol, 69.38% yield) as a yellow solid.

1 H NMR (400 MHz, CDCl ₃ ) δ 7.42 (d, J=7.5 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H), 7.27-7.20 ( m, 1H), 6.93-6.80 (m, 2H), 6.70 (dd, J=4.9, 8.7 Hz, 1H), 5.28 (s, 1H).

Step 1 To a solution of ethyl 5-((2-bromo-5-fluorobenzyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (prepared according to General Method A) (300.00 mg, 0.736 mmol, 1.00 eq.), 2-methylpropane-2-thiol (166.10 mg, 1.84 mmol, 2.50 eq.), Pd ₂ (dba) ₃ (84.72 mg, 0.147 mmol, 0 ,20 eq.) in dioxane (8.00 ml) were added XantPhos (127.87 mg, 0.221 mmol, 0.30 eq.) and K ₂ CO ₃ (101.81 mg, 0.736 mmol, 1.00 eq.) . The mixture was degassed and heated to 120° C. for 24 h under N ₂ . TLC (petroleum ether/ethyl acetate = 1:1) showed complete consumption of starting material. The reaction mixture was poured into H ₂ O (20 ml) and extracted with ethyl acetate (3x50 ml). The organic phase was washed with brine (30 ml), dried over anhydrous Na ₂ SO ₄ , concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2:1-1:1) to give ethyl 5-((2- (tert-Butylthio)-5-fluorobenzyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (200.00 mg, 0.48 mmol, 65.18% yield) as a yellow solid.

1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 8.29 (br. s, 1H), 7.60 (dd, J=5.9, 8.4 Hz, 1H), 7 .00 (t, J=7.7 Hz, 1H), 6.29 (br.s, 2H), 5.00 (br.s, 2H), 4.37 (d, J=6.8 Hz, 2H), 3.41 (br. s, 3H), 1.36-1.20 (m, 12H).

Step 2 To a solution of ethyl 5-((2-(tert-butylthio)-5-fluorobenzyl)(methyl)amino)pyrazolo[1,5a]pyrimidine-3-carboxylate (300.00 mg, 0.720 mmol, 1.00 equiv.) in DCM (8.00 ml) was added BBr ₃ (902.21 mg, 3.60 mmol, 5.00 equiv.) dropwise at 0° C. under N2 atmosphere. The mixture was stirred at 0° C. for 2.5 hours. TLC (petroleum ether/ethyl acetate = 1:1) indicated the reaction was complete. The mixture was poured into water (20 ml). The aqueous phase was extracted with dichloromethane (3x50 ml). The combined organic phase was washed with brine (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified pre-HPLC (column: Phenomenex Synergi C18 150x30 mmx4 µm and condition: 0.05% HCl-ACN) and lyophilized to give the A17 HCl salt (38.00 mg, 0.098 mmol, 13.61% yield) as white solid.

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Reference Example A18.

A18

Step 1: A mixture of 2-bromo-4-fluorophenol (10.00 g, 52.36 mmol, 1.00 eq.), trifluoro(vinyl)borane potassium salt (9.84 g, 66.50 mmol, 1.27 eq.), Cs ₂ CO ₃ (51.18 g, 157.08 mmol, 3.00 eq.) and Pd(PPh ₃ ) ₂ Cl ₂ (1.84 g, 2.62 mmol, 0.05 eq. ) in THF (90.00 ml) and H ₂ O (10.00 ml) was degassed and then heated to 90° C. for 12 h under N ₂ atmosphere. TLC (petroleum ether/ethyl acetate = 10/1) indicated complete consumption of the starting material. The reaction mixture was poured into H ₂ O (100 ml). The mixture was extracted with ethyl acetate (3x300 ml). The organic phase was washed with brine (200 ml), dried over anhydrous Na ₂ SO ₄ , concentrated and purified by silica gel column chromatography (eluted with EtOAc/petroleum ether = 1/30) to give 4-fluoro-2-vinylphenol (3, 50 g, 25.34 mmol, 48.39% yield) as a colorless oil.

¹ H NMR (400 MHz, CDCla) δ 7.12 (dd, J=3.0, 9.5 Hz, 1H), 6.89-6.81 (m, 1H), 6.79-6.73 (m, 1H), 5.75 (d, J=17.6 Hz, 1H), 5.64 (s, 1H), 5.39 (d, J=11.3 Hz, 1H).

Step 2 A mixture of 4-fluoro-2-vinylphenol (1.95 g, 14.12 mmol, 1.00 eq.), TBSCl (6.38 g, 42.35 mmol, 3.00 eq.) and 1H- imidazole (5.77 g, 84.70 mmol, 6.00 eq.) in DCM (20.00 ml) was stirred at 20° C. for 5 h under N ₂ atmosphere. TLC (petroleum ether/ethyl acetate = 10:1) showed complete consumption of starting material. The reaction mixture was poured into H ₂ O (30 ml). The mixture was extracted with dichloromethane (3x50 ml). The organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, concentrated and purified by silica gel column chromatography, eluting with petroleum ether to give tributyl(4-fluoro-2-vinylbenzyl)silane (2.30 g, 9 .11 mmol, 64.54% yield) as a colorless oil.

Step 3 A mixture of tri-butyl(4-fluoro-2-vinylbenzyl)silane (2.30 g, 9.11 mmol, 1.00 eq.), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3- carboxylate (2.06 g, 9.11 mmol, 1.00 eq.), Pd(PhCN) ₂ Cl ₂ (118.20 mg, 0.455.63 mmol, 0.05 eq.) and tris-o-tolylphosphamide ( 277.36 mg, 0.911 mmol, 0.10 eq.), DIPEA (7.07 g, 54.68 mmol, 6.00 eq.) in DMF (25.00 ml) was degassed and then heated to 120°C in for 24 h in an atmosphere of N ₂ . TLC (petroleum ether/ethyl acetate = 1:1) showed complete consumption of starting material. The reaction mixture was poured into H ₂ O (30 ml). The mixture was extracted with ethyl acetate (3x100 ml). The organic phase was washed with brine (30 ml), dried over anhydrous Na2SO4, concentrated and purified by silica gel column chromatography (EtOAc/petroleum ether = 1:3) to give ethyl (E)-5-(5-fluoro-2 -hydroxystyryl)pyrazolo[1,5a]pyrimidine-3-carboxylate (1.00 g, 2.26 mmol, 24.86% yield) as a white solid.

1H NMR (400 MHz, CDCl3) δ 9.29 (br. s, 1H), 8.50 (d, J=7.0 Hz, 1H), 8.28 (br. s, 1H), 7.84 (d, J=16.6 Hz, 1H), 7.20-7.04 (m, 3H), 6.69 (d, J=5.8 Hz, 2H), 4.20 (q., J = 6.9 Hz, 2H), 1.30-1.19 (m, 3H).

Step 4 To a mixture of ethyl (E)-5-(5-fluoro-2-hydroxystyryl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (378.22 mg, 1.04 mmol, 1.00 eq. ) and 4-methylbenzenesulfonohydrazide (3.29 g, 17.68 mmol, 17.00 eq.) in THF (4.00 ml) was added NaOAc (1.71 g, 20.80 mmol, 20.00 eq.) portion at 20°C in an atmosphere of N ₂ . The mixture was then heated to 65° C. and stirred for 12 hours. TLC indicated completion of the reaction. The mixture was cooled to 20°C and concentrated under reduced pressure at 45°C. Water (100 ml) was added to the residue. The aqueous phase was extracted with ethyl acetate (2x300 ml). The combined organic phase was washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered, concentrated in vacuo and purified pre-HPLC (column: Phenomenex Synergi Max-RP 250 x 50 x mm x 10 μm, 0.225% FA-ACN) to give A18 ( 120.00 mg, 0.347 mmol, 33.42% yield) as a white solid.

Reference Example A20.

A20

To a mixture of 4-fluoro-2-methylaminomethyl-phenol (305.2 mg, 1.97 mmol) and 6-chloroimidazo[1,2-b]pyridazine-3-carboxylic acid ethyl ester (230 mg, 1.02 mmol ) in DMSO (5 ml) was added

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KF (180 mg, 3.01 mmol). The reaction mixture was stirred at 120° C. for 18 hours under nitrogen.

The solution was then cooled to ambient temperature, diluted with water (20 ml) and extracted with EtOAc (3x50 ml). The combined organic layers were washed with additional water (3x50 ml) and brine (50 ml), dried over Na ₂ SO ₄ and concentrated. The residue was then purified with a silica gel column eluting with EtOAc/hexane (0-50%, 10 CV) to give the title product as a white solid (240 mg, 69%).

Reference Example A22.

A22-1 _A22

A21-1 was prepared according to general method A. To a solution of A22-1 (150 mg, 0.387 mmol) in ethanol (2 ml) was added 4 M HCl in dioxane (2 ml) and the reaction solution was heated at 75°C for 2 hours The solvents evaporated and the residue was neutralized with Et ₃ N and purified using a silica gel cartridge eluting with methanol/CH ₂ Cl ₂ (0-12.5%) to give A22 (144 mg, 100%).

Reference Example A23.

Step 1 To a mixture of (5-fluoro-2-methoxyphenyl)methanethiol (496.1 mg, 2.88 mmol) and 6-chloroimidazo[1,2-b]pyridazine-3-carboxylic acid ethyl ester (650.0 mg, 2.88 mmol) in ethanol (14.4 ml) DIPEA (1.12 g, 8.64 mmol) was added. The reaction mixture was stirred at 80°C for 1 h. The solution was cooled to ambient temperature, diluted with water (50 ml) and extracted

DCM (3x50 ml). The combined extracts were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash chromatography (ISCO system, silica gel (120 g), eluting with EtOAc/hexane (0-50%) to give A23-2 (560 mg, 54% yield). A23-2 exited the column during purification.

Step 2. To a solution of A23-2 (498.7 mg, 1.38 mmol) in methanol (100 ml) was added 4 M HCl in dioxane (10 ml) and the reaction solution was heated at 75°C for 2 h. The solvents were evaporated and the residue was neutralized with Et ₃ N and purified using a silica gel cartridge eluting with methanol/CH ₂ Cl ₂ (0-12.5%) to give A23 (470 mg, 98%).

A1-A24 were obtained in accordance with General method A and the methods described in this document.

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Example Structure Name Analytical data Al O A * y O /--Z. ethyl 5-((5-fluoro-2hydroxybenzyl) (methyl)amino)pyrazolo[1,5-а]pyrimidine3-carboxylate MS: 345.2 (M+H) ⁺ ; ² H NMR (500 MHz, Chloroform-d) δ 9.71 (br. s, 1H) , 8.32 (d, J=7.9 Hz, 1H) , 8.30 (s, 1H), 6, 98-6, 87 (m, ZN) , 6.37 (d, J=7.9 Hz, 1H) , 4.82 (s, 2H) , 4.42 (q., J=7.l Hz, 2Н) , 3.21 (s, ZN), 1.39 (t, J=7.1 Hz, ZN) , A2 O . A O f ethyl 5-(ethyl(5- fluoro-2- hydroxybenzyl)amine o) pyrazolo[1,5- a]pyrimidine-3- carboxylate MS: 359.3 (M+H) ⁺ ; ² H NMR (300 MHz, Chloroform-d) δ 9.75 (br. s, 1H) , 8.30-8.27 (m, 2H), 6.95-6.86 (m, 3H) , 6 .34 (d, J=7.9 Hz, 1H), 4.79 (s, 2H), 4.40 (sq., R=7.2 Hz, 2H), 3.56 (sq., J=7 .2 Hz, 2Н), 1.38 (t, J=7.2 Hz, ZN), 1.25 (t, J=7.2 Hz, ZN) ,

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AZ Λ __/ τ o ethyl 5-((5-fluoro-2hydroxybenzyl)amine o)pyrazolo[1,5a]pyrimidine-3carboxylate MS: 331.3 (M+H) ⁺ ; NMR (300 MHz, Chloroform-d) δ 9.61 (br. s, 1H), 8.52 (d, J=7.5 Hz, 1H), 8.28 (bt, J=5, 1 Hz, 1H), 8.13(s, 1H), 7.25-7.23(m, 1H), 6.93-6.86(m, 1H), 6.81-6.77(m, 1H) , 6.44 (d, J=7.5 Hz, 1H), 4.51 (d, J=5, 1 Hz, 2H) , 4.20 (q, J=6.9 Hz, 2H) . 1.39 (t, J=6.9 Hz, ZN), A4 o t / O ^Z 1' ethyl 5-((2- hydroxybenzyl)(methyl)amino)pyrazo lo[1,5-a]pyrimidine3-carboxylate MS: 327.5 (M+H) ⁺ ; NMR (300 MHz, Chloroform-d) δ 9.79 (s, 1H), 8.30-8.27 (m, 2H), 7.26-7.21 (m, 2H), 6.96 (d , J=7.8 Hz, 1H) , 6.84 (t, J=7.5 Hz, 1H) , 6.34 (d, J=8.l Hz, 1H), 4.85 (s, 2H ), 4.42 (sq., J=6.9 Hz, 2H) , 3.18 (s, ZN) , 1.40 (t, J=6.9 Hz, ZN) ,

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A5 O n ^t o ethyl 5-((5-fluoro-2hydroxybenzyl) (2hydroxy-2- methylpropyl)amino)p irazolo[1,5— a] pyrimidine-3-carboxylate MS: 403.2 (M+H) ⁺ ; % NMR (400 MHz, Chloroform-d) δ 8.32 (s, 1H) , 8.26 (d, J=8.0 Hz, 1H) , 7.05-6.80 (m, 3H) , 6 .59 (br. s, 1H), 5.06 (br. s, 2H) , 4.43 (q., 1=7.1 Hz, 2H) , 3.62 (br. s, 2H) , 1 .60 (s, 1H), 1.46-1.36 (m, 9H), Ab 4L about ethyl 5-((5-fluoro-2hydroxybenzyl) (1methylpyrrolidine-3y!)amino)pyrazolo[1,5-а]pyrimidine-3carboxylate MS: 414.2 (M+H) ⁺ ; S NMR (400 MHz, Chloroform-d) δ 8.67 (br. s, 2H) , 8.35 (d, J=8.0 Hz, 1H) , 8.24 (s, 1H) , 7.14 -7.07 (m, 1H) , 6.83 (dt, J=2.8, 8.4 Hz, 1H) , 6.73 (br.s, 1H) , 6.60 (br.s, 1H ), 5.13 (br.s, 1H) , 4.75-4.62 (m, 2H) , 4.34 (sq., J=6.9 Hz, 2H) , 3.88 (br.s , ZN) , 3.41 (br.s, 1H), 3.04 (br.s, ZN) , 2.54 (br.s, 2H) , 1.40 (t, J=7.2 Hz, ZN) ,

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A7 τι ,Α o O ethyl 5-((1-(5- fluoro-2- hydroxyphenyl)ethyl)amino)pyrazole[1,5a]pyrimidine-3carboxylate MS: 345.3 (M+H) ⁺ ; ² H NMR (300 MHz, Chloroform-d) δ 9.61 (br. s, 1H), 8.24 (s, 1H), 8.17 (d, J=7.2 Hz, 1H), 6, 96-6.91 (m, 2H) , 6.88-6.81 (m, 1H) , 6.09 (d, J=7.8 Hz, 1H) , 5.72-5.63 (m, 1H), 5.45 (br. d, J=8.7 Hz, 1H), 4.43 (sq., J=7.2 Hz, 2H), 1.64 (d, J=6.9 Hz , ZN) , 1.41 (t, J=7.2 Hz, ZN) , A8 A <f4 O ethyl (R)-5-((1-(5fluoro-2hydroxyphenyl)ethyl)amino)pyrazolo[1,5a]pyrimidine-3carboxylate MS: 345.2 (M+H) ⁺ .

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A9 ethyl 5-((1-(5- fluoro-2- hydroxyphenyl)propyl l)amino)pyrazolo[1,5-a]pyrimidine-3carboxylate MS: 359.2 (M+H) ⁺ ; F NMR (400 MHz, Chloroform-d) δ 8.99 (br. s, 1H), 8.27 (s, 1H), 8.20 (d, J=7.5 Hz, 1H), 6.98 (dd, J=5, 0, 8.8 Hz, 1H), 6.946.84 (m, 2H) , 6.13 (d, J=7.5 Hz, 1H) , 5.41 (br.s, 2Н), 4.57-4.40 (m, 2Н), 2.11-1.95 (m, 2Н), 1.44 (t, J=7.2 Hz, ZN), 1.02 (t , J=7.4 Hz, ZN), AYu o g ethyl 5-((1-(5- fluoro-2- hydroxyphenyl)-2methylpropyl)amino)p irazolo[1,5- a] pyrimidine-3-carboxylate MS: 373.2 (M+H) ⁺ ; F NMR (400 MHz, Chloroform-d) δ 8.25 (s, 1H) , 8.19 (d, J=7.5 Hz, 1H) , 6.99 (dd, J=5, 1, 8 .7 Hz, 1H) , 6.91 -6.81 (m, 2H) , 6.14 (d, J=7.5 Hz, 1H) , 5.11 (t, J=9.7 Hz, 1H ) , 4, 62-4.37 (m, 2H) , 2.22 (q.d., J=6.5, 17.1 Hz, 1H), 1.43 (t, J=7.2 Hz, ZN) , 1.22 (d, J=6.5 Hz, ZN) , 0.89 (d, J=6.5 Hz, ZN), All f ethyl 5- ((cyclopropyl(5- fluoro-2- hydroxyphenyl)methyl)amino)pyrazolo[1,5-a]pyrimidine-3carboxylate MS: 371.2 (M+H) ⁺ ; F NMR (400 MHz, Chloroform-d) δ 8.25 (s, 1H), 8.21 (d, R=7.5 Hz, 1H), 7.13 (dd, R=3.0, 9, 4 Hz, 1H), 7.006.94 (m, 1H) , 6.91-6.84 (m, 1H) , 6.14 (d, J=7.7 Hz, 1H) , 5.69 (d, J=8.0Hz, 1H), 4.70(t, J=8.3Hz, 1H), 4.49-4.38(m, 2H), 1.42(t, J=7,l Hz, 4H), 0.83-0.74 (m, 1H), 0.72-0.63 (m, 1H), 0.57 (q.d, J=4.8, 9.6 Hz, 1H), 0.480.40 (m, 1H),

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MS: 385.2 (M+H) ⁺ ;

cyclopropyl-1-(5fluoro-2hydroxyphenyl)ethyl)amino)pyrazolo[1,5а]pyrimidine-3carboxylate

TIL

MHz, ^g H NMR (400

Chloroform-d) δ 8 9.09 (br. s, 1H), 8.27 (s, 1H), 8.20 ethyl 5- (d, J=7.5 Hz, 1H), 6, 98 (dd, J=5, 0, 8.8 Hz, 1H), 6.91- ((cyclobutyl(5- 6.78 (m, 2H), 6.12 fluoro-2- (d, J=7.5 Hz, 1H), hydroxyphenyl)methyl 5.45 (t, J=9.4 Hz, )amino)pyrazolo[1,5 1H), 5.27 (d, -a]pyrimidine-3- J=8.4 Hz, 1H), carboxylate 4.51-4.45 (m, 2H), 2.98-2.89 (m, 1H), 2.29 (dd, J=3.8, 7.5 Hz, 1H), 2.071.90 (m, 4H), 1.75-1.66 (m, 1H), 1.45 (t, J=7.l Hz, ZN),

MS: 385.2 (M+H) ⁺ ;

NMR (400

Chloroform-d)

5-( (26, 15

1H)

2H)

2H)

2H)

Hz, (D

MHz,

8.27 (s (d, J=7.5

7.00-6.82 δ 9

0 (br

With, 1H), 1H), 8.19 Hz, 1H), (m, ZN) ,

J=7.5 Hz.

5.57 (br. s

4.52-4.40 (m,

2.01-1.77 (m,

1.44 (t,J=7.2

ZN), 0.72 (d

J=6.5 Hz,

0.56-0.41 (m,

0.24-0.07 (m,

1H)

2H)

2H)

MS: 407.2 (M+H) ⁺ ;

MHz, ^g H NMR (400

Chloroform-d) δ 9.66 (s, 1H), 8, 64-8.55 (m, 2H), ethyl 5-(((5-fluoro- 8.16 (s, 1H), 7.33 2- (d, J=4.4 Hz, 4H) , hydroxyphenyl)(feni 7.25 (q.d., J=4.3, l)methyl)amino)pyrase 8.5 Hz, 1H), 7.11 olo[1,5— (dd, J=3,l, 9.7 a]pyrimidine-3- Hz, 1H), 6.98-6.91 carboxylate (m, 1H) , 6.88-6.78 (m, 2H) , 6.58 (d, J=7.5 Hz, 1H), 4.18 (square, J=7.0 Hz, 2H), 1.30 (t, J=7.1 Hz, 4H),

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Al 5 Oh Oh ethyl 5-((1-(5- chloro-2- hydroxyphenyl)ethyl )amino)pyrazolo[1,5 -a]pyrimidine-3- carboxylate MS: 361.2 (M+H) ⁺ ; ^d H NMR (400 MHz, Chloroform-d) δ 9.42 (br. s, 1H) , 8.27 (s, 1H), 8.20 (d, J=7.5 Hz, 1H) , 7, 28 (s, 1H), 7.25 (d, J=2.5 Hz, 1H), 7.13 (dd, R=2.5, 8.8 Hz, 1H) , 6.95 (d, J \u003d 8.5 Hz, 1H) , 6.11 (d, O \u003d 7.5 Hz, 1H) , 5.75-5.64 (m, 1H) , 5.46 (d, J \u003d 8.3 Hz , 1H), 4.52-4.40 (m, 2H), 1.68 (d, J=6.8 Hz, ZN) , 1.61 (s, 2H) , 1.44 (t, J= 7.2 Hz, ZN) , Al 6 ^^/0 ⁿ \ jQCx/ o γ 4=0 ethyl 5-((1-(5- fluoro-2- hydroxyphenyl)ethyl )(methyl)amino)pyraz olo[1,5- a] pyrimidine-3-carboxylate MS: 359.2 (M+H) ⁺ ; ^g H NMR (400 MHz, Chloroform-d) δ 9.61 (s, 1H), 8.35-8.29 (m, 2H), 7.08-7.03 (m, 1H), 6.92 (dd, R=1.3, 6.1 Hz, 2H), 6.45 (q, J=6.9 Hz, 1H), 6.35 (d, J=7.9 Hz, 1H), 4.51-4.36 (m, 2H), 3.00 (s, ZN), 1.65 (d, J=7.0 Hz, ZN), 1.41 (t, J=7.2 Hz , ZN) , Al 7 XX < ^F ι y=° /N ethyl 5-((5-fluoro-2mercaptobenzyl)(methyl yl)amino)pyrazolo[1,5-а]pyrimidine-3carboxylate MS: 361.2 (M+H) ⁺ ; ^d H NMR (400 MHz, Chloroform-d) δ 9.19 (br. s, 1H), 9.09 (d, J=7.3 Hz, 1H) , 8.51 (s, 1H) , 7, 91-7.81 (m, 2H), 7.48 (dt, J=2.8, 8.5 Hz, 1H), 7.14 (d, J=7.3 Hz, 1H), 4.29 (br.s, 2H), 4.18 (q.s., J=7.0 Hz, 2H), 2.56 (br.s, ZN), 1.16 (t, J=7.2 Hz, ZN ) ,

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Al 8 ^-o I / O /- O LL ethyl 5-(5-fluoro-2hydroxyphenethyl)pyr azolo[1,5— a] pyrimidine-3-carboxylate MS: 330.2 (M+H) ⁺ ; ² H NMR (400 MHz, Chloroform-d) δ 9.42 (s, 1H), 9.14 (d, R=7.0 Hz, 1H) , 8.55 (s, 1H) , 7.18 ( d, J=7.0 Hz, 1H) , 7.05 (dd, J=3.0.9.5 Hz, 1H) , 6.866.80 (m, 1H) , 6.79-6.74 (m , 1H) , 4.30 (kb., J = 7.2 Hz, 2H), 3.21 - 3.13 (m, 2H) , 3.06-2.99 (m, 2H) , 1.33 (t, J=7.2 Hz, ZN) , Al 9 --A Ο /—z. LL ethyl 5-((5-fluoro-2hydroxybenzyl)(methyl)amino)-2methylpyrazolo[1,5а]pyrimidine-3carboxylate MS: 359.2 (M+H) ⁺ . A20 XX / ___N.J χ P ethyl 6-((5-fluoro-2hydroxybenzyl)(methyl yl)amino)imidazo[1,2-b]pyridazine-3carboxylate MS: 345.2 (M+H) ⁺ ; ² H NMR (500 MHz, Chloroform-d) δ 8.61 (s, 1H), 8.17 (s, 1H), 7.91 (d, J=10.0 Hz, 1H), 7.00- 6.86 (m, 4H) , 4.78 (s, 2H) , 4.47 (q.d., J=7.2, 0.5 Hz, 2H) , 3.17 (s, 3H) , 1 .41 (td, J=7,l, 0.5 Hz, ZN) , A21 about x O /—ζ C ethyl 5-(((5-fluoro2-hydroxypyridine- 3- yl)methyl)(methyl)amino)pyrazolo[1,5- a] pyrimidine-3-carboxylate MS: 346.2 (M+H) ⁺ . A22 O gz l LL ethyl 5-(((5-fluoro2-hydroxypyridine- 3- yl)methyl)(isopropyl)amino)pyrazolo[1,5 -a]pyrimidine-3- carboxylate MS: 374.2 (M+H) ⁺ .

- 27 041662

ethyl 5-((5-fluoro-2hydroxybenzyl)thio) pyrazolo[1,5a]pyrimidine-3carboxylate ethyl fluoro-2hydroxyphenyl)-2pyrazolo[1,5a]pyrimidine-3carboxylate

5-((1-(5hydroxyethyl)amino)MS : 361.2 (M+H) ⁺ ,

Reference Example B7.

B7-1 B7-2

B7-3

AT 7

Step 1: To a mixture of 1-(5-fluoro-2-hydroxyphenyl)ethanone (773 mg, 5.0 mmol) and (2-chloroethyl)carbamic acid tert-butyl ester (1.80 g, 10.0 mmol) KI (2.0 mg, 0.012 mmol) and CS ₂ CO ₃ (3.26 g, 10.0 mmol) were added to DMF (20 ml). The mixture was stirred at 80° C. overnight. The mixture was then cooled to ambient temperature, diluted with EtOAc and washed with 1N. NaOH (5x10 ml) until LCMS showed no 1-(5-fluoro-2-hydroxyphenyl)ethanone peak. The organic layer was dried over Na ₂ SO ₄ and concentrated. The residue was then purified with a silica gel column eluting with EtOAc/hexane (0-30%, 10 CV) to give the desired product B7-2 as a yellow solid (1.1 g, 73.8%).

LC-MS (ESI) m/z 320.3 (M+Na) ⁺ .

Step 2 To a solution of B7-2 (1.0 g, 3.36 mmol) in MeOH (10 ml) was added NaBH4 (640 mg, 16.8 mmol) in portions. The mixture was stirred at ambient temperature for 2 hours until no starting material remained as determined by LCMS. The solution was then diluted with water (50 ml) and extracted with DCM (3x20 ml). The combined DCM layers were dried over Na ₂ SO ₄ and concentrated. The residue was purified with a silica gel column eluting with EtOAc/hexane (0-50%, 10 CV) to give the desired product B7-3 as a pale yellow solid (0.75 g, 75%).

LC-MS (ESI) m/z 322.3 (M+Na) ⁺ ;

¹ H NMR (500 MHz, Chloroform-d) δ 7.11 (dd, J=9.2, 3.4 Hz, 1H), 6.89 (dd, J=9.0, 7.9, 3, 2 Hz, 1H), 6.77 (dd, J=8.9, 4.4 Hz, 1H), 5.09 (q., J=6.6 Hz, 1H), 4.92 (d, J =4.4 Hz, 1H), 4.03 (t, J=5.2 Hz, 2H), 3.62-3.50 (m, 2H), 1.49 (d, J=6.4 Hz , 3H), 1.45 (s, 9H).

Step 3 To a solution of B7-3 (600 mg, 2.0 mmol) and {2-[4-fluoro-2-(1hydroxyethyl)phenoxy]ethyl}carbamic acid tert-butyl ester (450 mg, 2.0 mmol ) in dry THF (40.0 ml) at -78°C was added NaH (60%, 80 mg, 2.0 mmol) in portions, the suspension was stirred at -78°C for 4 h and left to warm to 0°C and stirred for another 4 h. The mixture was then placed in a refrigerator at -20° C. overnight. LC-MS showed good conversion to the desired product. The mixture was then quenched with a mixture of ice and 1N. HCl and extracted with EtOAc (3x20 ml). The organic layer was dried over Na ₂ SO ₄ , concentrated and purified twice to give the desired product B7 as a yellow solid (240 mg, 25%):

B1-B7 were prepared according to general method B and the methods described in this document.

- 28 041662

Example Structure Name Analytical data Bl I^NHBoc Γϊ° > /X JX o V° XX? ethyl 5-((2-(2((tert- butoxycarbonyl )amino)ethoxy)5-fluorobenzyl)(methyl)amino)pirazolo[1,5a]pyrimidine-3carboxylate MS: 4 8 8.3, 1 (M+H) ⁺ ; ² H NMR (500 MHz, Chloroform-d) δ 8.30 (s, 1H), 8.26 (s, 1H), 6.92 (td, J=8.6, 3.3 Hz, 1H) . 6.83-6.76(m, 1H) , 6.31(s, 1H) , 4.93(s, 2H), 4.51-4.44(m, 1H), 4.36(sq. , J=7.2 Hz, 2H), 4.03 (t, J=4.9 Hz, 2H) , 3.69-3.63 (m, 1H) , 3.51 (s, 2H) , 3 .30 (s, 2H), 1.44 (s, 9H), 1.41-1.35 (t, J=7.2 Hz, ZN), AT 2 I^NHBoc XX about ethyl 5-((2-(2((tert- butoxycarbonyl) amino) ethoxy) - 5- fluorobenzyl)(ethyl)amino)pyrazolo[1,5- a] pyrimidine-3-carboxylate MS: 502.2 (M+H) ⁺ . VZ I^NHBoc rx > _/XJX o ₄ ^F i \=o XX? ethyl 5- ( (2- (2 ( (tert- butoxycarbonyl) amino) ethoxy) - 5- fluorobenzyl)(propyl)amino)pyrolo[1,5- a] pyrimidine-3-carboxylate MS: 516.3 (M+H) ⁺ . AT 4 ^XIVoc rr° > V ⁰ ethyl 5- ( (2- (2 ( (tert- butoxycarbonyl) amino) ethoxy) 5- fluorobenzyl)(cyclopropyl)amino)pyrazolo[1,5-a]pyrimidine-3carboxylate MS: 514.2 (M+H) ⁺ . AT 5 Y 2-/ O-< /4 J <kX ^{v 8} o ethyl 5- ( (2- (2 ( (tert- butoxycarbonyl) amino) ethoxy) - 5- fluorobenzyl)(2hydroxyethyl)am ino)pyrazolo[1,5-a]pyrimidine3-carboxylate MS: 518.3 (M+H) ⁺ .

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AT 6 ^^NHBoc w ethyl 5-( (6-(2( (tert- butoxycarbonyl) amino)ethoxy)2-chloro-3fluorobenzyl)(methyl)amino)pyraz olo[1,5- a] pyrimidine-3-carboxylate MS: 522.5 (M+H) ⁺ . AT 7 .NHBoc Ύ Ί ) Hi ethyl b- (1- (2 (2- ( (tert- butoxycarbonyl) amino) ethoxy) 5- fluorophenyl)ethoxy)imidazo[1,2b]pyridazine-3carboxylate LC-MS (ESI) m/z 511.6 (M+Na) ⁺ ; ^{X H} NMR (500 MHz, Chloroform-d) δ 8.16 (s, 1H), 7.90 (d, J=9.7 Hz, 1H), 7.16 (dd, J=9.0, 3 .2 Hz, 1H), 0.95 (d, J=9 _r 5 Hz, 1H), 6.90-6.88 (m, 1H), 6.81-6.78 (m, 1H), 6 .68 (sq., J=6.2 Hz, 1H), 5.84-5.68 (m, 1H), 4.38 (sq., J=7.2 Hz, 2H), 4.15- 4.09 (m, 2H), 3.60-3.52 (m, 2H), 1.65 (d, J=6.4 Hz, ZN), 1.38 (d, J=7 _r 2 Hz , 3H), 1.35 (s, 9H) .

Reference Examples 2 and 2-1.

Synthesis a.

Example 2 can be prepared as shown in the following scheme from racemic or enantiomerically enriched starting materials:

Step 1. To a mixture of compound 2A (1 equiv.) and 2B (1.2 equiv.) in anhydrous DMF (0.2 M) was added CS2CO3 (1.5 equiv.) and the reaction was heated on an oil bath at 80°C in nitrogen atmosphere all night. The mixture was cooled, poured into water and extracted with EtOAc three times. The combined organic layers were washed with water five times, washed with brine and dried over Na ₂ SO ₄ . After condensation, the residue was purified on a flash column eluting with EtOAc/hexane to give compound 2C.

Step 2 To a solution of compound 2C (1 eq.) in anhydrous THF (0.2 M) was added NaH (1.2 eq.). The reaction mixture was stirred at ambient temperature for 0.5 h. Compound 2D was added to the mixture and the reaction was heated at reflux under nitrogen atmosphere overnight. The reaction was cooled to ambient temperature and diluted with a portion of water (1/3 THF vol.) and NaOH (3 eq.). The mixture was stirred and heated at 70° C. for 2 hours or until the ester was completely hydrolyzed to the corresponding acid. After cooling, the organic layer was separated and the aqueous layer was neutralized to pH ~5. The resulting precipitate was filtered, washed with water three times and dried in vacuo to give compound 2E, which was used without further purification.

Step 3 To a solution of compound 2E (1 eq.) in CH ₂ Cl ₂ (0.2 M) was added 4 M HCl/dioxane (10 eq.) and the mixture was stirred until compound 2E was completely converted to compound 2F. The mixture was concentrated and the residue was purified by reverse phase preparative HPLC to give compound 2F.

Step 4 A solution of compound 2F (1 equiv.) and DIPEA (10 equiv.) in DMF (0.2 M) was added dropwise to a solution of HATU (1.4 equiv.) in DMF (0.1 M) at 0° WITH. After the addition was complete, the mixture was stirred at 0° C. for another 30 minutes. Water was added and the mixture was extracted with EtOAc three times. The combined organic layers were washed with saturated NaHCO ₃ twice, then with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified on a silica gel column eluting with EtOAc/hexane to give Example 2.

Synthesis V.

Examples 2 and 2-1 can also be prepared according to the following scheme using racemic or enantiomerically enriched starting materials:

Step 1 Compound 2C is reacted with compound 2G under the conditions described in Synthesis A, Step 2 to give compound 2H.

Step 2 Compound 2H is converted to compound 2I under the conditions described in Synthesis A, Step 3.

Step 3 To a solution of compound 2I (1 eq.) and DIPEA (2 eq.) in toluene (0.01 M) was added Pd(P-tBu ₃ ) ₂ (1 eq.). The reaction mixture was heated at 100° C. at 4 bar CO overnight and then concentrated. The residue was purified on a silica gel column eluting with EtOAc/hexane to give Example 2.

Reference Examples 10 and 10-1.

Examples 10 and 10-1 can be prepared as shown in the following scheme using racemic or enantiomerically enriched starting materials:

- 31 041662

prepared from compounds 10A and 10B using the method described in Step 1. Compound 10C in Example 2, Synthesis A, Step 1.

Step 2 Compound 10E was prepared from compound 10C and 10D using the method described in Example 2, Synthesis A, Step 2.

Step 3 A mixture of compound 10E (1 eq.) and NH2-NH2 (10 eq.) in methanol (0.2 M) was heated at reflux until compound 10E was completely converted to compound 10F. The mixture was concentrated and the residue was purified using reverse phase preparative HPLC to give compound 10F.

Step 4 Compound 10F was converted to Example 10 according to the method described for Example 2, Synthesis A, Step 4.

Reference Example 11-1.

Step 1 To a solution of 2-chloro-3-fluoro-6-hydroxybenzaldehyde (175 mg, 1.0 mmol) was added bis-ethylene glycol (740 mg, 2.0 mmol) in ACN (5 ml), K ₂ CO ₃ ( 276 mg, 2.0 mmol) and KI (2 mg). The reaction mixture was stirred at 120° C. for 24 hours. The solid was filtered off and the filtrate was concentrated and purified by column chromatography to give the desired product 11-1B as a white solid. The material was used directly in the next step.

Step 2 To a solution of 11-1B (373 mg, 1 mmol) in ACN (5 ml), NaN ₃ (650 mg, 10 mmol) was added and the mixture was stirred at 120° C. for 24 h. The solid was filtered off and the residue was concentrated and purified by column chromatography to give 11-1C as a white solid (200 mg, 82%).

1H NMR (500 MHz, Chloroform-d) δ 10.49 (d, J=1.1 Hz, 1H), 7.31 (dd, J=9.2, 8.2 Hz, 1H), 6.88 (dd, J=9.2, 3.7 Hz, 1H), 4.21 (dd, J=5.4, 4.5 Hz, 2H), 3.67 (dd, J=5.4, 4 .5 Hz, 2H).

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Step 3 To a solution of 11-1C (100 mg, 0.41 mmol) in anhydrous THF (5 mL) at -78°C was added methyl magnesium bromide (1N in Et2O, 0.82 mL, 0.82 mmol). The mixture was allowed to warm to room temperature and stirred for 2 hours until TLC showed no starting material. The solution was then cooled to 0°C and quenched us. aq. NH ₄ OAc and extracted with EtOAc (3x20 ml). The combined organic layer was dried over Na ₂ SO ₄ and concentrated. Residue 11-1D was used directly in the next step.

1H NMR (500 MHz, Chloroform-d) δ 6.97 (dd, J=9.2, 8.3 Hz, 1H), 6.77 (dd, J=9.1, 4.1 Hz, 1H) , 5.27 (sq., J=6.7 Hz, 1H), 4.34-4.29 (m, 1H), 4.22-4.16 (m, 1H), 4.04-3, 98 (m, 1H), 3.95-3.88 (m, 2H), 1.51 (d, J=6.7 Hz, 3H).

Step 4 To a solution of 5-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethyl ester (100 mg, 0.44 mmol) and 11-1D (110 mg, 0.41 mmol) in anhydrous THF (5.0 ml) at -78°C was added NaH (60%, 17 mg, 0.44 mmol). The mixture was allowed to warm to room temperature and stirred for 8 hours until a sufficient amount of the desired product was formed. The mixture was then diluted with water/ice and extracted with DCM (3x20 ml). The organic layer was dried over Na ₂ SO ₄ , concentrated and purified by silica gel column chromatography to give 11-1E as a yellow liquid (20 mg, 0.045 mmol, 6%), which was used directly in the next step.

Step 5 To a solution of 11-1E (20 mg, 0.045 mmol) in MeOH (1 mL), LioH (16 mg, 0.38 mmol) was added followed by 1 mL of H ₂ O. The mixture was allowed to stir at 60° C. in for 4 hours until LCMS and TLC showed the reaction was complete. The solution was cooled to room temperature, partially concentrated and acidified with 1N. HCl to pH 2-3. The aqueous mixture was extracted with DCM (3x10 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated. Residue 11-1F was used directly in the next step.

Step 6 To a solution of 11-1F (20 mg, 0.045 mmol) in DCM (5 ml) was added PPh ₃ (24 mg, 0.09 mmol). The solution was stirred for 1 hour until TLC showed complete conversion of the starting material to the desired product. The mixture was then used directly in the next step without further characterization.

11-1G MS ESI ⁺ m/z 417.7 (M+Na) ⁺ .

Step 7 DIPEA (0.20 ml, 1.15 mmol) was added to the 11-1G solution obtained in the previous DMF steps (10 ml). The solution was cooled in a dry ice/acetone bath and HATU (40.0 mg, 0.11 mmol) was added. The solution was allowed to warm to room temperature slowly and LCMS showed complete conversion of the starting material to the desired product. The mixture was then diluted with water (50 ml) and extracted with EtOAc (3x50 ml). The combined organic layer was washed with water (3x50 ml) and brine (50 ml) and dried over Na ₂ SO ₄ . The solvent was removed and the resulting residue was purified by silica gel column chromatography (0-5% MeOH/DCM) to give the desired product as a white solid (2.6 mg, 20% yield).

Reference Examples 14 and 14-1.

Examples 14 and 14-1 can be prepared according to the following scheme using racemic or enantiomerically enriched starting materials:

Step 1. To a mixture of compound 14A (1 eq.) and 14B (1.2 eq.) in anhydrous DMF (0.2 M) was added

Cs ₂ CO ₃ (1.5 eq.) and the reaction was heated in an oil bath at 80° C. under nitrogen overnight. Mixture

- 33 041662 was cooled, poured into water and extracted with EtOAc three times. The combined organic layers were washed with water five times, washed with saline and dried over Na ₂ SO ₄ . After condensation, the residue was purified with a silica gel flash column eluting with EtOAc/hexane to give 14C.

Step 2: To a cooled (-78° C.) solution of 14C (1 eq.) in anhydrous THF (0.2 M) was added MeMgBr (3 eq., 3 M in diethyl ether). The reaction was stirred for 2 h from -78 to 0°C, extinguished with saturated aqueous NH ₄ Cl and then was extracted with EtOAc (2x). The organic layers were dried over MgSO ₄ , filtered and concentrated. This residue was purified by silica gel column chromatography eluting with EtOAc/hexane to give 14D.

Step 3 To a solution of compound 14D (1 equiv.) in anhydrous THF (0.2 M) was added NaH (1.2 equiv.). The reaction mixture was stirred at ambient temperature for 0.5 h. 14E was added to the mixture and the reaction was heated to reflux under nitrogen overnight. The reaction was cooled to ambient temperature and then poured into water. The product was extracted with EtOAc three times. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ and concentrated. The residue was purified with a silica gel column eluting with EtOAc/hexane to give product 14F.

Step 4 To a solution of compound 14F (1 eq.) in CH ₂ Cl ₂ (0.2 M) was added 4 M HCl/dioxane (10 eq.) and the mixture was stirred until all 14F was converted to 14G. After concentration, the residue was purified by reverse phase preparative HPLC to give 14G.

Step 5 To a solution of 14G (1 equiv.) and DIPEA (2 equiv.) in toluene (0.01 M) was added Pd(Pt-Bu ₃ ) ₂ (1 equiv.). The reaction mixture was heated to 100° C. at 4 bar CO overnight and then concentrated. The residue was purified on a silica gel column eluting with EtOAc/hexane to give 14.

Reference Examples 15 and 15-1.

Examples 15 and 15-1 can be prepared according to the following scheme using racemic or enantiomerically enriched starting materials:

Step 1. To a suspension of 15A (1.0 eq.) in THF (0.15 M) was added a solution of 2.0 M aqueous NaOH (3 eq.). The homogeneous reaction mixture was stirred overnight and then the organic layers were removed under reduced pressure. The aqueous residue was adjusted to pH ~4 with 1.0 M aqueous HCl. The resulting precipitate was collected by filtration and washed with H ₂ O to give a solid 15B. The filtrate was extracted with EtOAc (2x) and the organic layers were concentrated under reduced pressure to give an additional portion of 15B.

Step 2 A stock solution of Jones reagent (2.67 M) was prepared by carefully adding concentrated H2SO4 (2.3 ml) in CrO ₃ (2.67 g) and then diluting to 10 ml with H ₂ O. To suspension 15B (1, 0 eq.) in acetone (0.067 M) was slowly added Jones reagent (1.2 eq.). The reaction mixture was stirred for 15 min and then quenched with i-PrOH and filtered through a pad of diatomaceous earth, rinsing with acetone. The filtrate was concentrated to give 15C which was used without further purification.

Step 4 To a solution of 15C (1.0 eq.) in DMF (0.40 M) was added NaH (60% in mineral oil, 1.5 eq.) at 0°C. The reaction mixture was stirred at room temperature for 30 min and then

- 34 041662 was again cooled to 0°C and slowly added 2-(trimethylsilylethoxymethyl)chloride (4.3 ml, 1.2 eq.).

The reaction mixture was warmed to room temperature, stirred for 1 h and then quenched.

H ₂ O and was extracted with EtOAc (3x). The combined organic layers were washed with H ₂ O (3x) and brine and then dried over MgSO ₄ and concentrated. The residue was purified by silica gel flash chromatography eluting with 20-30% EtOAc/hexane to give 15D.

Step 5. To the reaction mixture of 14D (1.0 eq.), copper(I) iodide (0.05 eq.), 8-hydroxyquinoline (0.1 eq.) and tribasic potassium phosphate (2.0 eq.) in DMF (0.2 M) under nitrogen atmosphere was added 15D (1.2 eq.), the reaction mixture was heated at 120°C for 24 hours, the Reaction mixture was cooled to room temperature and then diluted with EtOAc. The mixture was filtered through a pad of diatomaceous earth and filtered by evaporating in vacuo. The crude residue was purified on a silica gel column eluting with EtOAC/hexane to give 15E.

Step 6 A suspension of 15E (1.0 eq.) in 1,4-dioxane (0.062 M) and water (1/3 THF) was treated with sulfamic acid (6.0 eq.). A solution of sodium chlorite (1.3 eq.) and potassium dihydrogen phosphate (12 eq.) in water (1.2 M) were added via addition funnel over 20 minutes. After the addition was complete, the ice bath was removed, the reaction mixture was stirred at room temperature for 3 hours. THF was added, the reaction mixture was stirred at room temperature for another 3 hours. The reaction mixture was diluted with water and extracted with EtOAc (2x). The combined organic layers were washed with water and brine and then dried over Na ₂ SO ₄ , filtered and concentrated. The residue was triturated with ethyl acetate/hexane to give 15F.

Step 7 To a solution of compound 15F (1 eq.) in CH ₂ Cl ₂ (0.2 M) was added 4 M HCl/dioxane (10 eq.) and the mixture was stirred until all 15F was converted to 15G. After concentration, the residue was purified by reverse phase preparative HPLC to give 15G.

Step 8 A solution of compound 15G (1 equiv.) and DIPEA (10 equiv.) in DMF (0.2 M) was added dropwise to a solution of HATU (1.4 equiv.) in DMF (0.1 M) at 0° WITH. After the addition was complete, the mixture was stirred at 0° C. for another 30 minutes. Water was added and the mixture was extracted three times with EtOAc. The combined organic layers were washed with saturated NaHCO ₃ twice, brine, dried over Na ₂ SO ₄ and evaporated. The residue was purified with a silica gel column eluting with EtOAc/hexane to give 15.

Reference Examples 18 and 18-1.

Examples 18 and 18-1 can be prepared according to the following scheme using racemic or enantiomerically enriched starting materials:

13V ^1SC

Step 1. NaH ( 3.0 equiv.). The reaction mixture was heated at 120°C for 24 h, then cooled to room temperature and diluted with EtOAc. The mixture was filtered through a pad of diatomaceous earth and filtered by evaporating in vacuo. The crude residue was purified on a silica gel column eluting with EtOAc/hexane to give 18B.

Step 2 To the reaction mixture of 18B (1.0 eq.) in DMF (0.2 M) were added KOH (2 eq.) and I ₂ (1.1 eq.). The reaction mixture was stirred at room temperature for 1 h, then quenched with NaHSO ₃ and extracted with EtOAc. The combined organic layers were washed with saturated NaHCO ₃ twice, brine, dried over Na ₂ SO ₄ and evaporated. The residue was purified with a silica gel column eluting with EtOAc/hexane to give 18C.

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Step 3 To a solution of compound 18C (1 eq.) in CH2C12 (0.2 M) was added 4 M HCl/dioxane (10 eq.) and the mixture was stirred until all 18C was converted to 18D. After concentration, the residue was purified by reverse phase preparative HPLC to give 18D.

Step 4 To a solution of 18D (1 eq.) and DIPEA (2 eq.) in toluene (0.01 M) was added Pd(Pt-Bu ₃ ) ₂ (1 eq.). The reaction mixture was heated at 100° C. at 4 bar CO overnight and then concentrated. The residue was purified on a silica gel column eluting with EtOAc/hexane to give 18.

Reference example 20.

Example 20 was obtained in accordance with the following scheme:

Step 1: tert-Butyl (2-(4-fluoro-2-formylphenoxy)ethyl)carbamate (20C).

Solution of aldehyde 20A (1.5 g, 11 mmol), chloride 20B (2.1 g, 12 mmol), potassium carbonate (7.4 g, 54 mmol) and potassium iodide (36 mg, 0.2 mmol) in DMF (11 ml) was heated to 60° C. and stirred for 15 h. Addition of additional chloride 20B (1.0 g, 6 mmol) and additional heating at 80° C. for 5 h completed the reaction. The mixture was cooled to room temperature and diluted by adding water (250 ml). The mixture was extracted with ethyl acetate (3x300 ml) and the combined extracts were washed with water (200 ml) and brine (100 ml), dried over sodium sulfate and concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel, 0-20% ethyl acetate in hexanes) gave 20C (3.0 g, 99%) as a viscous oil.

LRESIMS m/z 306.1 [M+Na]+, calculated on C^^NiN^ 306.1.

Step 2: tert-Butyl (2-(4-fluoro-2-((methylamino)methyl)phenoxy)ethyl)carbamate (20D).

Aldehyde 20C (2.5 g, 8.8 mmol) and methylamine (0.69 g, 22 mmol) in methanol (88 ml) were heated to 60° C. and stirred for 1 h. The mixture was cooled to room temperature and borohydride was added sodium (.33 g, 8.8 mmol). The mixture was stirred for 30 minutes and then quenched by adding water (200 ml). The mixture was extracted with dichloromethane (4x100 ml) and the combined extracts were dried with brine (50 ml), sodium sulfate and concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel, 0-100% (10% methanol in ethyl acetate) in hexane) gave the compound (2.1 g, 80%) as a gel.

LRESIMS m/z 299.2 [M+H] ⁺ , calculated as C ₁₅ H ₂₄ F1N ₂ O ₃ 299.2.

Step 3 Ethyl 5-((2-(2-((tert-butoxycarbonyl)amino)ethoxy)-5-fluorobenzyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylate (20F).

Amine 20D (2.1 g, 7.0 mmol), ester 20E (1.59 g, 7.0 mmol) and Hunig's base (7.0 ml, 5.2 g, 40 mmol) in butanol (17 ml ) was heated at 110°C for 25 min. The reaction was cooled and diluted with water (250 ml). The mixture was extracted with dichloromethane (4x100 ml) and the combined extracts were dried over sodium sulfate. The mixture was concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel, 20-100% ethyl acetate in hexanes) gave the title compound (2.1 g, 75%) as a solid.

LRESIMS m/z 488.3 [M+H] ⁺ , calculated as C24H31F1N5O5 488.2.

Step 4 5-((2-(2-((tert-Butoxycarbonyl)amino)ethoxy)-5-fluorobenzyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (20G).

Sodium hydroxide (40 ml, 2 M in water) was added to a stirring solution of ester 20F (2.1 g, 4.3 mmol) in tetrahydrofuran:methanol (3.2, 100 ml) at room temperature. The reaction was heated to 60° C. and stirred for 6.5 h. The mixture was cooled to 0° C. and acidified with hydrochloric acid (45 ml, 2 M in water), then diluted with water (100 ml). The mixture was extracted with ethyl acetate (4x150 ml) and the combined extracts were dried with brine (50 ml) and sodium sulfate. The mixture was concentrated under reduced pressure to give the title compound (1.92 g, 97%) as a solid.

LRESIMS m/z 460.2 [M+H] ⁺ , calculated as C ₂₂ H ₂₇ F1N ₅ O ₅ 460.2.

Step 5 5-((2-(2-Aminoethoxy)-5-fluorobenzyl)(methyl)amino)pyrazolo[1,5-a]pyrimidine-3carboxylic acid (20H).

Hydrochloric acid (5 ml, 4 M in dioxane) was added to a stirring solution of carboxylic acid 20G (1.92 g, 4.2 mmol) in dichloromethane (25 ml) at room temperature. The reaction was stirred for 2 hours and then concentrated under reduced pressure to give the title compound as a solid.

LRESIMS m/z 360.2 [M+H] ⁺ , calculated as C ₁₇ H ₁₀ F1N ₅ O ₃ 360.2.

Step 6 Under argon, HATU (1.67 g, 4.4 mmol) was added to a stirring solution of carboxylic acid 20N (1.50 g, 4.2 mmol) and Hunig's base (7.28 ml, 5.40 g, 41.8 mmol) in DMF:dichloromethane (5:1, 60 ml) at -78°C. The reaction was slowly warmed to room temperature and stirred for 3 h, and then extinguished with water (300 ml). The mixture was extracted with ethyl acetate (3x100 ml) then dichloromethane (2x100 ml) and the combined extracts were dried with brine (50 ml) and sodium sulfate. The mixture was concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel, 1-4% methanol in dichloromethane) followed by recrystallization from ethyl acetate/methanol provided Example 20 (0.98 g, 68%, 2 steps) as a solid.

LRESIMS m/z 342.2 [M+H] ⁺ , calculated as C17H17F1N5O2 342.1;

1H NMR (500 MHz, DMSO-06) δ 9.43 (dd, J=6.9, 2.7 Hz, 1H), 8.76 (d, J=7.9 Hz, 1H), 8.10 (s, 1H), 7.197.25 (m, 1H), 7.03-7.07 (m, 2H), 6.72 (d, J=7.9 Hz, 1H), 5.64 (dd, J=14.9, 1.5 Hz, 1H), 4.48 (dt, J=10.2, 4.3 Hz, 1H), 4.04-4.10 (m, 2H), 3.81 -3.87(m, 1H), 3.58(s, 3H), 3.38-3.46(m, 1H).

Alternative synthesis of example 20.

Example 20 was also obtained from the following alternative route:

^B1 20

Step 1. Ethyl 5-oxo-4H-pyrazolo[1,5-a]pyrimidine-3-carboxylate (20J).

Cs ₂ CO ₃ (656.77 g, 2.02 mol) in one portion at 20°C under N2 atmosphere. The mixture was stirred at 110° C. for 6 hours. The mixture was cooled to 20° C. and filtered through a diatomaceous earth pad. The filter cake was washed with ethyl acetate (3x30 ml). The filtrate was added to H ₂ O (2 L) and acidified with HOAc to pH 4. The resulting precipitate was filtered to give 20J (173.00 g, 834.98 mmol, 86.36% yield) as a white solid.

- 37 041662 ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.54 (d, J=7.91 Hz, 1H), 8.12 (s, 1H), 6.13 (d, J=7 .91 Hz, 1H), 4.27 (sq.

J=7.11 Hz, 2H), 1.28 (t, J=7.09 Hz, 3H).

Stage 2. 5-Chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (20K).

To a mixture of 20J (158.00 g, 762.59 mmol) in MeCN (1.6 L) was added POCl ₃ (584.64 g, 3.81 mol) at 20°C under N2 atmosphere. The mixture was stirred at 100°C for 2 hours. The mixture was cooled to 20°C and poured into ice water (5000 ml) in portions at 0°C and stirred for 20 minutes. The precipitate was filtered and dried to give 20K (110.00 g, 487.52 mmol, 63.93% yield) as a white solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ 9.33 (d, J=7.28 Hz, 1H), 8.66 (s, 1H), 7.41 (d, J=7.15 Hz, 1H), 4.31 (sq., J=7.15 Hz, 2H), 1.32 (t, J=7.09 Hz, 3H).

Stage 3. 4-Fluoro-2-methylaminomethyl-phenol (20M).

To a solution of 20 L (5.00 g, 35.69 mmol, 1.00 eq.) in MeOH (50.00 ml) was added aqueous methanamine (8.8 ml, 71.38 mmol, 25%, 2.00 eq. .) in one batch at 25°C under N2. The mixture was stirred at 25°C for 3 h, then NaBH ₄ (2.70 g, 71.38 mmol, 2.00 eq.) was added in portions. The mixture was then stirred at 25° C. for an additional 9 hours. TLC indicated completion of the reaction. The mixture was concentrated under reduced pressure at 45°C. The residue was poured into water (50 ml). The aqueous phase was extracted with dichloromethane (3x200 ml) and the combined organic phase was washed with brine (200 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo to give 20M (5.10 g, 32.87 mmol, 92.09 % yield) as a colorless solid.

1H NMR (400 MHz, CDCl ,) δ 6.86 (dt, J=3.0, 8.7 Hz, 1H), 6.78-6.69 (m, 2H), 3.93 (s, 2H), 2.48 (s, 3H).

Step 4 5-[(5-Fluoro-2-hydroxybenzyl)methylamino]pyrazolo[1,5a]pyrimidine-3-carboxylic acid ethyl ester (A1).

To a suspension of 20M (33.70 g, 217.17 mmol, 1.00 eq.) and 20K (49.00 g, 217.17 mmol, 1.00 eq.) in n-BuOH (740.00 ml), DIPEA (159.98 g, 1.24 mol, 5.70 eq.) was added. The reaction mixture was stirred at 120° C. for 2 hours under nitrogen. TLC showed completion of the reaction. The solution was cooled to 25°C and then the solvent was removed. The residue was diluted with water (500 ml) and extracted with dichloromethane (3x500 ml). The combined organic extracts were washed with brine (300 ml), dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. The residue was triturated with EtOAc (100 mL) to give A1 (60.00 g, 174.25 mmol, 80.24% yield) as a white solid.

1H NMR (500 MHz, Chloroform-d) δ 9.71 (s, 1H), 8.32 (d, J=7.9 Hz, 1H), 8.30 (s, 1H), 6.98-6 .87 (m, 3H), 6.37 (d, J=7.9 Hz, 1H), 4.82 (s, 2H), 4.42 (sq., J=7.1 Hz, 2H), 3.21 (s, 3H), 1.39 (t, J=7.1 Hz, 3H).

Step 5 5-{[2-(2-tert-Butoxycarbonylaminoethoxy)-5-fluorobenzyl]methylamino}pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethyl ester (B1).

To a solution of A1 (102.85 g, 298.6 mmol, 1 eq.), (2-chloroethyl)carbamic acid tert-butyl ester (56.33 g, 313.5 mmol, 1.05 eq.) in DMF ( 854 ml) K2CO3 (206.41 g, 1493 mmol, 5.0 eq.) was added. The mixture was heated at 80° C. for 20 hours with -85% conversion of starting material to product by LC-MS. Additional portions of (2-chloroethyl)carbamic acid tert-butyl ester (5.633 g, 31.35 mmol, 0.1 eq.) and K2CO3 (41.282 g, 298.6 mmol, 1 eq.) were added to the reaction flask. The reaction was continued at 80° C. for another 21 h. The mixture was then cooled to room temperature, quenched with water (1000 ml) and extracted with EtOAc (3x900 ml). The combined organic extracts were then washed with water (3x700 ml) and brine (500 ml), dried over Na ₂ SO ₄ and concentrated. The resulting residue was purified with a silica gel column eluting with EtOAc/hexane (0-70% to give B1 as a white solid (128.74 g, 96.7% yield).

LC-MS (ESI) m/z 510.1 (M+Na)+;

¹ H NMR (500 MHz, Chloroform-d) δ 8.30 (s, 1H), 8.26 (s, 1H), 6.92 (td, J=8.6, 3.3 Hz, 1H), 6.83-6.76(m, 1H), 6.31(s, 1H), 4.93(s, 2H), 4.51-4.44(m, 1H), 4.36(sq. , J=7, 2 Hz, 2H), 4.03 (t, J=4.9 Hz, 2H), 3.69-3.63 (m, 1H), 3.51 (s, 2H), 3 .30 (s, 2H), 1.44 (s, 9H), 1.41-1.35 (t, J=7.2 Hz, 3H).

Step 6. 11-Fluoro-14-methyl-6,7,13,14-tetrahydro-1,15-ethenopyrazolo[4,3-1][1,4,8,10]benzoxatriazacyclotridecin-4(5H)-one (20).

To a solution of B1 (128.74 g, 264.07 mmol, 1 eq.) in methanol (750 ml) and THF (250 ml) was added LiOH-H ₂ O (55.40 g, 1320 mmol, 5.0 eq. ) in H ₂ O (250 ml). The clear solution was heated at 70° C. for 2 hours. The reaction was neutralized at 0° C. aq. HCl (2 M, 250 ml) until pH <5 and then extracted with CH ₂ Cl ₂ (1x1000 ml, 3x500 ml). The combined organic layers were washed with brine (300 ml) and dried over Na2SO4. Filtration, evaporation and drying under high vacuum gave a white solid (126.47 g, 275.25 mmol, 104% yield). To a solution of the acid (121.30 g, 264 mmol) in CH ₂ Cl ₂ (996 ml) was added HCl in dioxane (4 M, 204 ml) at 0°C. Stirring was continued from 0° C. to room temperature for 27 h until de-Boc was complete by LC-MS. The white solid was filtered, washed with DCM (400 ml), dried under high vacuum to give a white solid of the amine HCl salt (123.55 g) which was used directly without further purification. _{Ki _}

- 38 041662 amino HCl salt (22.92 g, 49.0 mmol, 1.00 eq.). After complete dissolution of the solid salt, pentafluorophenyl diphenyl phosphinate (FDPP) in CH ₂ Cl ₂ (1.1 M, 19.76 g, 51.44 mmol, 1.05 eq.) was added. The coupling was completed in 30 minutes by LC-MS and then the second portions of the salt and FDPP were added following the same procedure as the first portion. Salt addition followed by FDPP was repeated every 30 minutes and monitored by LC-MS for each addition cycle. All of the salt (123.55 g, 264 mmol, 1.00 eq.) and FDPP (106.44 g, 277 mmol, 1.05 eq.) were added to the reaction flask in portions. The reaction solution was concentrated to a volume of ~500 mL, and a large amount of precipitate formed. Solid 20 was filtered and washed with DMF (3x50 ml). The filtrate was poured into water (2 L) and the additional product precipitated. The solid was filtered and washed with water (3x100 ml). The combined solid was dried, redissolved in 10% methanol in dichloromethane (1.5 L) and then ethyl acetate (1 L) was added. The solution was condensed to ~500 ml and a large amount of white solid formed. Filtration and drying in high vacuum gave a white solid 20 (74.58 g, 83% yield).

Powder X-Ray Diffraction (PXRD) Example 20.

A sample of Example 20, crystalline polymorph Form 1, was transferred to a background calibration plate for PXRD analysis. PXRD data were obtained using a Bruker D8 X-ray diffractometer according to the procedures recommended by the manufacturer. Scan parameters: 2-theta range: 4.5-39.1°; stage size: 0.02°; stage time: 1 s; analysis time: 180 s.

Diffraction peaks were typically measured with an error of ±0.1° (2θ).

The results are illustrated in FIG. 1. The data are summarized in Table. 1.

Table 1

2-Θ(degrees) d-value Peak intensity (units) Intensity peak (%) 10, 68 9, 611 31, 15 5, 2 11.96 8.586 19, 11 2, 9 15, 26 6, 737 20, 92 4.4 19, 64 5, 244 27.57 6, 4 21.94 4,701 452.41 100 23, 96 4.309 91, 85 18.2 26, 82 3,857 10.92 2.2

Differential Scanning Calorimetry (DSC) Example 20.

DSC measurements illustrated in FIG. 2 were carried out using a Seiko Model SSC/5200 differential scanning calorimeter. 7.92 mg of a sample of Example 20, crystalline polymorph Form 1, was equilibrated at 36° C. and then the temperature was increased linearly to 380° C. at a rate of 10° C./min. A sample of Example 20, crystalline polymorph Form 1, showed a melting point of 298.9°C.

Reference example 26.

- 39 041662

Example 26 can be obtained in accordance with the following scheme:

Step 1. Titanium(IV) isopropoxide (1.3 eq.) was added to a commercially available solution of methylamine in methanol (2 M, 3 eq.) followed by the addition of the starting aldehyde 14C (1.0 eq.). The reaction mixture was stirred at ambient temperature for 5 hours, after which sodium borohydride (1.0 eq.) was added and the resulting mixture was further stirred for an additional 2 hour period. The reaction was then quenched by adding water, the resulting inorganic precipitate was filtered and washed with EtOAc. The organic layer was separated and the aqueous layer was further extracted with EtOAc (x2). The combined extracts were dried (K ₂ CO ₃ ) and concentrated in vacuo to give 26A.

Step 2 A mixture of compound 26A (1 eq.) and DIPEA (2 eq.) in n-BuOH (0.2 M) was heated at 120° C. overnight, cooled to ambient temperature and then concentrated. The residue was purified with a silica gel column eluting with EtOAc/hexane to give product 26B.

Step 3 To a solution of compound 26B (1 eq.) in CH ₂ Cl ₂ (0.2 M) was added 4 M HCl/dioxane (10 eq.) and the mixture was stirred until all 26B was converted to 26C. After concentration, the residue was purified by reverse phase preparative HPLC to give 26C.

Step 4 To a solution of 26C (1 equiv.) and DIPEA (2 equiv.) in toluene (0.01 M) was added Pd(Pt-Bu ₃ ) ₂ (1 equiv.). The reaction was heated at 100° C. at 4 bar CO overnight and then concentrated. The residue was purified on a silica gel column eluting with EtOAc/hexane to give 26.

Reference Examples 37 and 37-1.

Examples 37 and 37-1 can be prepared according to the following scheme from racemic or enantiomerically enriched starting materials:

O

37C ³⁷

Step 1 Compound 37B was prepared from compound 2C and compound 37A using the method described in Example 2 Synthesis A Step 2.

Step 2 Compound 37C was prepared from compound 37B using the method described in Example 2, Synthesis A, Step 3.

Step 3 Example 37 was prepared from compound 37C using the method described in Example 2, Synthesis A, Step 4.

Reference Examples 38 and 38-1.

Examples 38 and 38-1 can be prepared according to the following scheme from racemic or enantiomerically enriched starting materials:

Step 1 Compound 38B was prepared from compounds 2C and 38A as described in Example 2, Synthesis A, Step 2.

Step 2 Compound 38C was prepared from compound 38B using the method described in Example 2, Synthesis A, Step 3.

Step 3 Example 38 was prepared from compound 38C using the method described in Example 2, Synthesis B, Step 4.

Reference example 39.

- 41 041662

Example 39 was prepared according to the following scheme:

Step 1 2-(3-Chloro-4-fluoro-2-formylphenoxy)ethyl]carbamic acid tert-butyl ester (39B).

To a solution of 2-chloro-3-fluoro-6-hydroxybenzaldehyde (39A, 53 mg, 0.3 mmol) and (2-chloroethyl)carbamic acid tert-butyl ester (135 mg, 0.75 mmol) in DMF (5 ml) was added KI (2.0 mg, 0.012 mmol) and K ₂ CO ₃ (105 mg, 0.75 mmol). The mixture was subjected to microwaves at 100° C. for 2 hours. The mixture was then diluted with water (20 ml) and extracted with EtOAc (3x20 ml). The combined organic layers were washed with water (3x20 ml) and brine (20 ml), dried over Na ₂ SO ₄ and concentrated to give 39B. The crude residue was used directly in the next step.

LC-MS: (ESI) m/z 340.3 (M+Na)+.

Step 2: {[2-(3-Chloro-4-fluoro-2-methylaminomethylphenoxy)ethyl]carbamic acid tert-butyl ester (39C).

To a solution of 39B (95.4 mg, 0.3 mmol) in MeOH (3 ml) was added methylamine hydrochloride (50.7 mg, 0.75 mmol). The mixture was stirred at 60° C. for 30 minutes. The solution was then cooled to ambient temperature and NaBH ₄ (11.1 mg, 0.3 mmol) was added. The mixture was stirred at ambient temperature for 2 hours. The solution was then diluted with water (50 ml) and extracted with DCM (3x20 ml). The combined organic layers were dried over Na2SO4 and concentrated to give 39C. The crude residue was used directly in the next step.

LC-MS: (ESI) m/z 333.3 (M+H) ⁺ .

Step 3 5-{[6-(2-tert-Butoxycarbonylaminoethoxy)-2-chloro-3fluorobenzyl]methylamino}pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethyl ester (3 9D).

DIEA (1.0 ml) was added to a solution of 20K (67.5 mg, 0.3 mmol) and 39C (99.9 mg, 0.3 mmol) in n-BuOH (2.0 ml). The mixture was heated under microwave at 150° C. for 2 hours. The mixture was then diluted with water and extracted with DCM (3x20 ml). The organic layer was dried over Na ₂ SO ₄ , concentrated and purified by silica gel column chromatography to give 17 as a yellow liquid.

LC-MS: (ESI) m/z 522.5 (M+H) ⁺ .

Step 4. 5-{[6-(2-tert-Butoxycarbonylaminoethoxy)-2-chloro-3-fluorobenzyl]methylamino}pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (39E).

To a solution of 39D (40 mg, 0.0776 mmol) in MeOH (1 ml) was added LioH (16 mg, 0.38 mmol) and H ₂ O (1 ml). The mixture was stirred at 60° C. for 4 hours. The solution was cooled to ambient temperature, partially concentrated and acidified with aqueous HCl (1N) to pH 2-3. The aqueous mixture was extracted with DCM (3x10 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated to give 39E. The crude residue was used directly in the next step.

LC-MS: (ESI) m/z 494.3 (M+H)+.

Step 5 5-{[6-(2-Aminoethoxy)-2-chloro-3-fluorobenzyl]methylamino}pyrazolo[1,5-a]pyrimidine-3 carboxylic acid (39F).

- 42 041662

To a solution of 39E (40 mg, 0.0776 mmol) in DCM (2 ml) was added TFA (0.4 ml). The solution was stirred for 1 hour. The solvent was removed using a rotary evaporator. The residue was redissolved

DCM and re-concentrated (3x) to give 39F as a foamy liquid.

LC-MS: (ESI) m/z 393.5 (M+H)+.

Step 6 To a solution of 39F (36 mg, 0.078 mmol) in 10 ml DCM was added DIEA (0.20 ml, 1.15 mmol). The solution was cooled in a dry ice/acetone bath and HATU (40.0 mg, 0.11 mmol) was added. The solution was allowed to slowly warm to ambient temperature. The mixture was diluted with water (50 ml) and extracted with EtOAc (3x50 ml). The combined organic layer was washed with water (3x50 ml) and brine (50 ml), dried over Na ₂ SO ₄ and concentrated. The resulting residue was purified using a silica gel column (0-5% MeOH/DCM) to give Example 39 as a white solid (6.2 mg, 23.4%).

LC-MS (ESI) m/z 376.5 (M+H) ⁺ .

¹ H NMR (500 MHz, chloroform-d) δ 9.51 (s, 1H), 8.40-8.33 (m, 2H), 7.03 (ddd, J=8.9, 8.0, 0.7 Hz, 1H), 6.78 (dd, J=9.3, 4.2 Hz, 1H), 6.40 (d, J=7.9 Hz, 1H), 5.97 (dd, J=15.0, 2.1Hz, 1H), 4.49-4.43(m, 1H), 4.31(ddd, J=10.9, 6.4, 4.5Hz, 1H) , 4.12-4.03 (m, 1H), 3.91 (d, J=14.9 Hz, 1H), 3.72-3.63 (m, 1H), 3.56 (s, 3H ).

Reference example 40.

Example 40 was prepared as shown in the following scheme:

A19 40A

40V ₄₀

Step 1. 5-[(5-Fluoro-2-hydroxybenzyl)methylamino]-2-methylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (40B).

To a solution of 19A (75 mg, 0.14 mmol) in MeOH (2 ml) was added LiOH (60 mg, 1.4 mmol) and H ₂ O (2 ml). The mixture was stirred at 60° C. for 4 hours. The solution was cooled to ambient temperature, partially concentrated and acidified with aqueous HCl (1N) to pH 2-3. The resulting suspension was extracted with EtOAc (3x20 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated to give 40A.

LC-MS (ESI) m/z 331.6 (M+H) ⁺ .

Step 2. 5-[(5-Fluoro-2-hydroxybenzyl)methylamino]-2-methylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (2-hydroxyethyl)amide (40B).

DIEA (0.20 ml, 1.15 mmol) and HATU (380 .0 mg, 1.0 mmol). The solution was slowly allowed to warm to ambient temperature. The mixture was then diluted with water (25 ml) and extracted with EtOAc (3x25 ml). The combined organic layers were washed with HCl (1N, 3x20 ml) and brine (50 ml), dried over Na ₂ SO ₄ and concentrated. The resulting residue was purified with a silica gel column eluting with 0-5% MeOH/DCM (10 CV) to give 40B as a white solid (74 mg, 47%).

LC-MS (ESI) m/z 374.3 (M+H) ⁺ .

Stage 3. To a solution of 40B (74 mg, 0.2 mmol) in THF (3 ml) and DCM (3 ml) at 0°C was added PPh ₃ (131 mg, 0.5 mmol) and di-tert-butyl azodicarboxylate (DTAD) (115 mg, 0.5 mmol). The mixture was allowed to warm to ambient temperature and stirred for another 4 h. The solvent was then removed and the residue was purified by column on silica gel eluting with 0-10%, MeOH/DCM (10 CV), followed by preparative TLC to give example 40 in as a white solid (15 mg).

LC-MS (ESI) m/z 356.5 (M+H) ⁺ ;

1H NMR (500 MHz, chloroform-d) δ 8.12 (d, J=7.7 Hz, 1H), 6.93 (ddd, J=9.0, 3.1.0.9 Hz, 1H) , 6.78 (ddd, J=9.0, 7.3, 3.0 Hz, 1H), 6.71 (dd, J=9.1, 4.5 Hz, 1H), 6.28 (d , J=7.7 Hz, 1H), 5.77 (dd, J=15.2, 1.7 Hz, 1H), 4.38-43 041662

4.33 (m, 1H), 3.98 (s, 1H), 3.91 (d, J=1.4 Hz, 1H), 3.78 (dd, J=15.1, 0.9 Hz , 1H), 3.45 (s, 3H), 3.43-3.36 (m, 1H),

2.45 (s, 3H).

Reference example 41.

Example 41 was prepared using the method shown in the following scheme:

[2-(2-Acetyl-4-fluorophenoxy)ethyl]carbamic tert-Butyl ester

Step 1. acids (41B).

To a mixture of 1-(5-fluoro-2-hydroxyphenyl)ethanone (41A, 773 mg, 5.0 mmol) and (2-chloroethyl)carbamic acid tert-butyl ester (1.80 g, 10.0 mmol) in DMF (20 ml) was added KI (2.0 mg,

0.012 mmol) and Cs ₂ CO ₃ (3.26 g, 10.0 mmol). The mixture was stirred at 80° C. overnight. The mixture was then cooled to ambient temperature, diluted with EtOAc and washed with 1N. NaOH (5x10 ml) until LCMS showed no 1-(5-fluoro-2-hydroxyphenyl)ethanone peak. The organic layer was dried over Na ₂ SO ₄ and concentrated. The residue was then purified with a silica gel column eluting with EtOAc/hexane (0-30%, 10 CV) to give the title product 41B as a yellow solid (1.1 g, 73.8%).

LC-MS (ESI) m/z 320.3 (M+Na)+.

Step 2: tert-Butyl (2-(4-fluoro-2-(1-hydroxyethyl)phenoxy)ethyl)carbamate (41C).

To a solution of 41B (1.0 g, 3.36 mmol) in MeOH (10 ml) was added NaBH ₄ (640 mg, 16.8 mmol) in portions. The mixture was stirred at ambient temperature for 2 hours. The solution was then diluted with water (50 ml) and extracted with DCM (3x20 ml). The combined DCM layers were dried over Na ₂ SO ₄ and concentrated. The residue was purified with a silica gel column eluting with EtOAc/hexane (0-50%, 10 CV) to give the title product as a pale yellow solid (0.75 g, 75%).

LC-MS (ESI) m/z 322.3 (M+Na) ⁺ ;

¹ H NMR (500 MHz, chloroform-d) δ 7.11 (dd, J=9.2, 3.4 Hz, 1H), 6.89 (dd, J=9.0, 7.9, 3, 2 Hz, 1H), 6.77 (dd, J=8.9, 4.4 Hz, 1H), 5.09 (q., J=6.6 Hz, 1H), 4.92 (d, J =4.4 Hz, 1H), 4.03 (t, J=5.2 Hz, 2H), 3.62-3.50 (m, 2H), 1.49 (d, J=6.4 Hz , 3H), 1.45 (s, 9H).

Step 3. 6-{1-[2-(2-tert-Butoxycarbonylaminoethoxy)-5fluorophenyl]ethoxy}imidazo[1,2-b]pyridazine-3-carboxylic acid ethyl ester (41D).

To a solution of 41C (600 mg, 2.0 mmol) and {2-[4-fluoro-2-(1hydroxyethyl)phenoxy]ethyl}carbamic acid tert-butyl ester (450 mg, 2.0 mmol) in dry THF ( 40.0 ml) at -78 °C NaH (60%, 80 mg, 2.0 mmol) was added in portions. The suspension was stirred at -78°C for 4 h and left to warm at 0°C and stirred for another 4 h. The mixture was then placed in a freezer at -20°C overnight. The mixture was then quenched with a mixture of ice and 1N. HCl and extracted with EtOAc (3x20 ml). The organic layer was dried over Na ₂ SO ₄ , concentrated and purified twice to give the desired product as a yellow solid (240 mg, 25%).

LC-MS (ESI) m/z 511.6 (M+Na) ⁺ ;

1H NMR (500 MHz, chloroform-d) δ 8.16 (s, 1H), 7.90 (d, J=9.7 Hz, 1H), 7.16 (dd, J=9.0, 3, 2 Hz, 1H), 0.95 (d, J=9.5 Hz, 1H), 6.90-6.88 (m, 1H), 6.81-6.78 (m, 1H), 6, 68 (q, J=6.2 Hz, 1H), 5.84-5.68 (m, 1H), 4.38 (q, J=7.2 Hz, 2H), 4.15-4 .09 (m, 2H), 3.60-3.52 (m, 2H), 1.65 (d, J=6.4 Hz, 3H), 1.38 (d, J=12 Hz, 3H) , 1.35 (s, 9H).

Step 4 Compound 41D was converted to Example 41 using methods similar to those described herein.

MS: 34 3.2 (M+H) ⁺ ;

1H NMR (500 MHz, chloroform-d) δ 9.82 (d, J=7.0 Hz, 1H), 8.27 (s, 1H), 8.09 (d, J=9.5 Hz, 1H ), 7.18 (dd,

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J=8.9, 3.2 Hz, 1H), 7.01-6.94 (m, 2H), 6.83 (dd, J=9.0, 4.3 Hz, 1H), 6.60 -6.53(m, 1H), 4.63-4.52(m, 1H), 4.274.16(m, 1H), 4.16-4.04(m, 1H), 3.70-3 .56 (m, 1H), 1.70 (d, J=6.4 Hz, 3H).

Reference example 42.

Example 42 was prepared using the methods shown in the following scheme:

Step 1. 6-[(5-Fluoro-2-hydroxybenzyl)methylamino]imidazo[1,2b]pyridazine-3-carboxylic acid ethyl ester (42B).

To a mixture of 4-fluoro-2-methylaminomethylphenol (20L, 305.2 mg, 1.97 mmol) and 6-chloroimidazo[1,2-b]pyridazine-3-carboxylic acid ethyl ester (42A, 230 mg, 1.97 mmol) 02 mmol) in DMSO (5 ml) was added KF (180 mg, 3.01 mmol). The reaction mixture was stirred at 120° C. for 18 hours under nitrogen. The solution was then cooled to ambient temperature, diluted with water (20 ml) and extracted with EtOAc (3x50 ml). The combined organic layers were washed with additional water (3x50 ml) and brine (50 ml), dried over Na ₂ SO ₄ and concentrated. The residue was then purified with a silica gel column eluting with EtOAc/hexane (0-50%, 10 CV) to give the title product as a white solid (240 mg, 69%).

LC-MS (ESI) m/z 345.2 (M+H)+;

1H NMR (500 MHz, chloroform-d) δ 8.61 (s, 1H), 8.17 (s, 1H), 7.91 (d, J=10.0 Hz, 1H), 7.00-6 .86 (m, 4H), 4.78 (s, 2H), 4.47 (q.d., J=7.2, 0.5 Hz, 2H), 3.17 (s, 3H), 1, 41 (td, J=7.1, 0.5 Hz, 3H).

Step 2. 6-{[2-(2-tert-Butoxycarbonylaminoethoxy)-5fluorobenzyl]methylamino}imidazo[1,2-b]pyridazine-3-carboxylic acid ethyl ester (42C).

To a solution of 6-[(5-fluoro-2-hydroxybenzyl)methylamino]imidazo[1,2b]pyridazine-3-carboxylic acid ethyl ester (2B, 200 mg, 0.58 mmol) and tert-butyl ester (2 -chloroethyl)carbamic acid (209 mg, 1.16 mmol) in DMF (5 ml) was added K ₂ CO ₃ (200 mg, 1.45 mmol) and KI (2.0 mg, 0.012 mmol). The mixture was heated at 90° C. for 4 hours under nitrogen. The mixture was then diluted with water (20 ml) and extracted with EtOAc (3x10 ml). The combined organic layers were then washed with water (3x5 ml) and brine (2x5 ml). The organic layer was dried over Na ₂ SO ₄ and concentrated. The resulting residue was purified with a silica gel column eluting with EtOAc/hexane (0-100%, 10 CV) to give 42C as a white solid (203 mg, 76%).

LC-MS (ESI) m/z 510.1 (M+Na)+;

1H NMR (500 MHz, chloroform-d) δ (ppm) 8.16 (s, 1H), 7.85 (d, J=9.9 Hz, 1H), 7.00 (dd, J= 8.9, 3.2 Hz, 1H), 6.95-6.87 (m, 2H), 6.80 (dd, J=8.9, 4.3 Hz, 1H), 4.95 (s , 1H), 4.74 (s, 2H), 4.41 (q., J=7.2 Hz, 2H), 4.04 (t, J=5.2 Hz, 2H), 3.56- 3.50 (m, 2H), 3.26 (s, 3H), 1.43 (s, 9H), 1.40 (t, J=7.2 Hz, 3H).

Step 3 Compound 42C was converted to Example 42 using methods similar to those described herein.

MS: 342.5 (M+H)+;

¹ H NMR (500 MHz, chloroform-d) δ 10.01 (d, J=6.9 Hz, 1H), 8.17 (s, 1H), 8.04 (d, J=10.0 Hz, 1H), 7.077.04 (m, 1H), 7.00 (d, J=10.0 Hz, 1H), 6.96-6.92 (m, 1H), 6.84 (dd, J=9 .1, 4.5 Hz, 1H), 5.69 (dd, J=15.8, 1.6 Hz, 1H), 4.55 (dt, J=9.9, 3.7 Hz, 1H) , 4.20-4.09 (m, 2H), 3.98 (dd, J=15.9, 1.0 Hz, 1H), 3.66-3.62 (m, 1H), 3.61 (s, 3H).

- 45 041662

Reference Example 51-1.

Step 1: K ₂ CO ₃ (801.6 mg, 5.80 mmol) and heated at 80°C with stirring for 6 hours. The reaction was cooled to ambient temperature and diluted with DCM (3 ml), filtered through a syringe filter and concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel (12 g), 0-70% ethyl acetate in hexanes) gave 51-1A (407.4 mg, 0.836 mmol, 72% yield).

Step 2 To a solution of 51-1A (407.4 mg, 0.836 mmol) in MeOH (6 ml) and THF (4 ml) was added LiOH aqueous solution (2 M, 4.0 ml) at ambient temperature. The reaction solution was heated at 70° C. for 2 hours. The reaction flask was cooled to ambient temperature, diluted with water and methanol, and then quenched with aqueous HCl (2 M, 4 ml) to pH <5. The mixture was extracted with DCM (3x5 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure and dried under high vacuum overnight. To a solution of the acid product in DCM (6 ml) was added 4 M HCl in 1,4-dioxane (2.97 ml). The mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure and dried under high vacuum. To a solution of the de-Boc product and FDPP (352.9 mg, 0.918 mmol) in DMF (21 ml) was added Hunig's base (539.5 mg, 0.327 mmol) at room temperature. The mixture was stirred for 2.5 h and then extinguished the reaction with 2 M Na ₂ CO ₃ solution (21 ml). The mixture was stirred for 15 min and then was extracted with DCM (4x10 ml). The combined extracts were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash chromatography (ISCO system, silica gel (12 g), 0-11.25% methanol in dichloromethane) to give 51-1 (164.0 mg, 0.480 mmol, 57.55% yield in three steps).

Reference example 53.

Example 53 was prepared using the methods shown in the following scheme:

Step 1. 5-[1-(5-Fluoro-2-hydroxyphenyl)ethylamino]pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (53A).

To a solution of 5-[(5-fluoro-2-hydroxybenzyl)methylamino]pyrazolo[1,5a]pyrimidine-3-carboxylic acid ethyl ester (20 M, 300 mg, 0.87 mmol) in MeOH (5 ml) was added LiOH (420 mg, 10 mmol), followed by the addition of 5 ml H ₂ O. The mixture was left to stir at 60°C for 4 hours. The solution was cooled to ambient temperature, partially concentrated and acidified

1n. HCl to pH 2-3. The resulting suspension was extracted with EtOAc (3x20 ml). The combined organic layers were dried over Na ₂ SO ₄ and concentrated. The remainder was used directly in the next step.

LCMS (ESI+) m/z 317.4 (M+H)+.

Step 2 3-({5-[(5-Fluoro-2-hydroxybenzyl)methylamino-pyrazolo[1,5a]pyrimidine-3-carbonyl-amino)-2-hydroxy-propionic acid methyl ester (53B).

To a solution of 53A (80 mg, 0.25 mmol) and 3-amino-2 hydrochloride methyl ester

- 46 041662 hydroxypropionic acid (70 mg, 0.5 mmol) in DCM (5 ml) at 0°C was added DIPEA (1.0 ml, 5.7 mmol) followed by the addition of HATU (140.0 mg, 0.5 mmol). The solution was allowed to slowly warm to ambient temperature. The mixture was diluted with water (25 ml) and extracted with EtOAc (3x25 ml). The combined organic layers were washed with 1N. HCl (3x20 ml) and brine (50 ml) and dried over Na ₂ SO ₄ . The solvent was removed and the resulting white solid was used directly in the next step.

LC-MS (ESI+) m/z 418.4 (M+H) ⁺ .

Step 3. Methyl 11-fluoro-14-methyl-4-oxo-4,5,6,7,13,14-hexahydro-1,15-ethenopyrazolo[4,3f][1,4,8,10]benzoxatriazacyclotridecine -7-carboxylate (53C).

To a solution of 53B (83 mg, 0.2 mmol) in DCM (5 mL) was added PPh ₃ (263 mg, 1.0 mmol) followed by CBr ₄ (332 mg, 1.0 mmol). The mixture was stirred at ambient temperature overnight. The solvent was removed and the residue was redissolved in DMF (5 ml) followed by the addition of K ₂ CO ₃ (116.8 mg, 0.84 mmol). The mixture was then stirred at 80° C. until the desired product was formed. The mixture was then diluted with EtOAc and washed with water. The organic layer was dried over Na ₂ SO ₄ and concentrated. The residue was purified using a silica gel column (0-10%, MeOH/DCM) to give 53C as a white solid (40 mg).

LC-MS (ESI+) m/z 400.2 (M+H) ⁺ .

Step 4 To 53C (20 mg, 0.05 mmol) was added NH ₃ in MeOH solution (7N, 2 ml). The mixture was stirred at 60° C. overnight. The solvent was removed and the residue was purified using a silica gel column (0-10%, MeOH/DCM) to give Example 53 as an off-white solid (8 mg).

LC-MS (ESI ⁺ ) m/z 385.5 (M+H) ⁺ ;

¹ H NMR (300 MHz, chloroform-d) δ 8.41 (s, 1H), 8.34 (d, J=7.9 Hz, 1H), 8.17 (s, 1H), 6.99- 6.92 (m, 2H), 6.77 (dd, J=6.2, 3.5 Hz, 1H), 6.38 (d, J=7.9 Hz, 1H), 5.63-5 .44 (m, 2H), 5.09 (dd, J=11.0, 8.4 Hz, 1H), 4.38 (dd, J=14.7, 11.0 Hz, 1H), 4, 28-4.17 (m, 1H), 4.17-4.07 (m, 2H), 3.22 (s, 3H).

Reference example 54.

Example 54 was obtained using the method shown in the following diagram:

To a solution of compound 53C (20 mg, 0.05 mmol) in MeOH (2 ml) was added NaBH ₄ (19 mg, 0.5 mmol) in portions. The mixture was stirred for 4 hours. The solvent was removed and the residue was purified using a silica gel column (0-10%, MeOH/DCM) to give the desired product as a white solid (8 mg).

LC-MS (ESI ⁺ ) m/z 372.5 (M+H) ⁺ ;

¹ H NMR (300 MHz, Chloroform-d) δ 8.39 (s, 1H), 8.32 (d, J=7.9 Hz, 1H), 7.01-6.85 (m, 3H), 6.35 (d, J=8, 0 Hz, 1H), 5.55-5.43 (m, 1H), 4.92-4.82 (m, 1H), 4.09-3.98 ( m, 2H), 3.80-3.70 (m, 3H), 3.23 (s, 3H).

Example 93.

Step 1 To a solution of tert-butyl (R)-2-hydroxypropyl carbamate (1.00 g, 5.71 mmol) and tosyl chloride (1.14 g, 6.00 mmol) in DCM (29 ml) was added triethylamine (1. 44 g, 14.28 mmol) and the mixture was stirred at room temperature for 48 hours. The reaction solution was concentrated under reduced

- 47 041662 pressure and the residue was purified using flash chromatography (ISCO system, silica gel (40 g), 0-20% ethyl acetate in hexane) to obtain (R)-1-((tert-butoxycarbonyl)amino)propan-2- yl 4-methylbenzenesulfonate (1.12 g, 3.40 mmol, 59.54% yield).

Step 2 To a solution of A8 (100.00 mg, 0.290 mmol) and (R)-1-((tert-butoxycarbonyl)amino)propan-2yl-4-methylbenzenesulfonate (143.50 mg, 0.436 mmol) in DMF (1 45 ml) was added K ₂ CO ₃ (200.7 mg, 1.45 mmol) and heated at 80°C with stirring for 16 h. The reaction was cooled to ambient temperature and diluted with DCM (3 ml), filtered through a syringe filter and concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel (12 g), 0-60% ethyl acetate in hexanes) gave 93A (32.90 mg, 0.0656 mmol, 22.59% yield).

Step 3 To a solution of 93A (32.90 mg, 0.0656 mmol) in MeOH (3 ml) and THF (2 ml) was added LiOH aqueous solution (2 M, 2 ml) at ambient temperature. The reaction solution was heated at 70° C. for 2 hours. The reaction flask was cooled to ambient temperature, diluted with water and methanol, and then quenched with HCl aqueous solution (2 M, 2 ml) to pH <5. The mixture was extracted with DCM (3x5 ml), dried over Na ₂ SO ₄ , concentrated under reduced pressure and dried under high vacuum overnight. To a solution of the acidic product DCM (4 ml) was added 4 M HCl in 1,4-dioxane (2.0 ml). The mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure and dried under high vacuum. To a solution of de-Boc product and FDPP (27.62 mg, 0.0719 mmol) in DMF (1.6 mL) was added Hunig's base (42.23 mg, 0.327 mmol) at room temperature. The mixture was stirred for 2.5 h and then extinguished the reaction with 2 M Na ₂ CO ₃ solution (2 ml). The mixture was stirred for 15 min and then was extracted with DCM (4x10 ml). The combined extracts were dried over Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by flash chromatography (ISCO system, silica gel (12 g), 0-10% methanol in dichloromethane) to give 93 (10.1 mg, 0.0284 mmol, 43.49% yield over three steps).

Reference Examples 104, 106 and 107.

106 107

Step 1 To a solution of A17 HCl (38 mg, 0.096 mmol) and tert-butyl (2-chloroethyl) carbamate (12.9 mg, 0.072 mmol) in DMF (0.5 ml) was added K ₂ CO ₃ (33.1 mg, 0.24 mmol) and heated at 80° C. with stirring for 1.5 hours. The reaction was cooled to ambient temperature and diluted with DCM (3 ml), filtered through a syringe filter and concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel (12 g), 0-60% ethyl acetate in hexanes) gave 104A (20.8 mg, 0.0413 mmol, 86.3% yield).

Step 2 Solution 104 was prepared according to General Method C from 104A as a white solid.

Step 3 To a solution of 104 (4.6 mg, 0.0129 mmol) in DCM (0.3 mL) was added methyl 3-chlorobenzoperoxoate (2.2 mg, 0.0129 mmol) and the reaction was stirred for 20 min, followed by adding saturated NaHCO ₃ solution (3 ml) and extracting with DCM (4x4 ml). The combined extracts were dried over Na ₂ SO ₄ and concentrated under reduced pressure. Flash chromatography (ISCO system, silica gel (12 g), 0-12.5% methanol in dichloromethane) gave 106 (0.5 mg, 10.4% yield) and 107 (1.7 mg, 33.9 % exit).

The following examples were obtained using methods similar to those described in this document, in particular the General methods A, B and C, as described in this document.

Etc. Analytical data 11-1 MS: 377.7 (M+H) ⁺ ; NMR (500 MHz, chloroform-d) δ 8.49 (d, J=7.9 Hz, 1H) , 8.29 (s, 1H) , 7.13 (dd, J=9.2, 7.8 Hz, 1H), 7.02 (d, J=7.3 Hz, 1H), 6.92 (dd, J=9.4, 3.9 Hz, 1H), 6.82 (d, J=7 .7 Hz, 1H), 4.63-4.55 (m, 1H), 4.45 (dd, J=10.8.5.4 Hz, 1H) , 4.31-4.23 (m, 1H), 4.00 (dd, J=16.2, 8.7 Hz, 1H), 1.70 (d, J=6.9 Hz, ZN).

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20 MS: 342.2 [M+H] ⁺ , ³ H NMR (500 MHz, flMCO-d ₆ ) 9.43 (dd, J=6.9, 2.7 Hz, 1H) , 8.76 (d, J=7.9 Hz, 1H), 8.10 (s, 1H), 7.19-7.25 (m, 1H), 7.03-7.07 (m, 2H), 6.72 (d , J=7.9 Hz, 1H) , 5.64 (dd, J=14.9, 1.5 Hz, 1H) , 4.48 (dt, J=10.2, 4.3 Hz, 1H) , 4.04-4.10 (m, 2H), 3.81-3.87 (m, 1H), 3.58 (s, 3H), 3.38-3.46 (m, 1H). 39 LC-MS (ESI) m/z 376.5 (M+H) ⁺ , ³ H NMR (500 MHz, chloroformci) δ 9.51 (s, 1H), 8.40-8.33 (m, 2H) , 7.03 (ddd, J=8.9, 8.0, 0.7 Hz, 1H), 6.78 (dd, J=9.3, 4.2 Hz, 1H), 6.40 (d , J=7.9 Hz, 1H) , 5.97 (dd, J=15.0, 2.1 Hz, 1H) , 4.49-4.43(m, 1H) , 4.31 (dd, J=10.9, 6.4, 4.5 Hz, 1H) , 4.124.03 (m, 1H) , 3.91 (d, J=14.9 Hz, 1H) , 3.72-3.63 (m, 1H), 3.56 (s, 3H). 40 MS: 356.5 (M+H) ⁺ ; ³ H NMR (500 MHz, chloroform-d) δ 8.12 (d, J=7.71 Hz, 1H), 6.93 (ddd, J=9.0, 3.1, 0.9 Hz, 1H ) , 6.78 (ddd, J=9.0, 7.3, 3.0 Hz, 1H) , 6.71 (dd, J=9.l, 4.5 Hz, 1H) , 6.28 ( d, J=7.7 Hz, 1H), 5.77 (dd, J=15.2, 1.7 Hz, 1H), 4.38-4.33 (m, 1H), 3.98 (s , 1Н) , 3.91 (d, J=l.4 Hz, 1Н) , 3.78 (dd, J=15.l, 0.9 Hz, 1Н) , 3.45 (s, ZN) , 3 .43-3.36 (m, 1H), 2.45 (s, 3H). 41 MS: 343.2 (M+H) ⁺ ; ³ H NMR (500 MHz, Chloroform-d) δ 9.82 (d, J=7.0 Hz, 1H) , 8.27 (s, 1H) , 8.09 (d, J=9.5 Hz, 1H) , 7.18 (dd, J=8.9, 3.2 Hz, 1H) , 7.01-6.94 (m, 2H) , 6.83 (dd, J=9.0, 4, 3 Hz, 1H), 6.60-6.53 (m, 1H), 4.63-4.52 (m, 1H), 4.27-4.16 (m, 1H), 4.16-4 .04 (m, 1H), 3.70-3.56 (m, 1H), 1.70 (d, J=6.4 Hz, ZN). 42 MS: 342.5 (M+H) ⁺ ; ³ H NMR (500 MHz, chloroform-d) δ 10.01 (d, J=6.9 Hz, 1H) , 8.17 (s, 1H) , 8.04 (d, J=10.0 Hz, 1H), 7.07-7.04 (m, 1H) , 7.00 (d, J=10.0 Hz, 1H) , 6.966.92 (m, 1H) , 6.84 (dd, J=9 ,l, 4.5 Hz, 1H) , 5.69 (dd, J=15.8, 1.6 Hz, 1H) , 4.55 (dt, J=9.9, 3.7 Hz, 1H) , 4.204.09 (m, 2H) , 3.98 (dd, J=15.9, 1.0 Hz, 1H) , 3.66-3.62 (m, 1H) , 3.61 (s, ZN ) .

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43 MS: 356.6 (M+H) ⁺ ; ² H NMR (500 MHz, Chloroform-d) δ 8.27 (d, J=7.9 Hz, 1H) , 8.17 (s, 1H) , 6.96 (ddd, J=9.0, 3 .1, 0.9 Hz, 1H) , 6.88-6.81 (m, 1H) , 6.77 (dd, J=9.0, 4.7 Hz, 1H) , 6.41 (d, J=7.9 Hz, 1H) , 5.71-5.63 (m, 1H) , 4.43 (dt, J=10.0, 4.4 Hz, 1H) , 4.09 (ddd, J =10.3, 8.4, 4.0 Hz, 1H) , 3.96-3.92 (m, 1H) , 3.87 (dd, J=15.0, 0.8 Hz, 1H) . 3.77 (dd, J=15.0, 7.2 Hz, 1H), 3.55-3.51 (m, 2H), 1.33 (t, J=7.2 Hz, ZN), 44 MS: 370.1 (M+H) ⁺ ; ² H NMR (500 MHz, flMCO-d ₆ ) 9.28 (dd, J=5.8, 4.0 Hz, 1H) , 8.71 (d, J=7.9 Hz, 1H) , 8, 08 (s, 1H) , 7.16 (dd, J=9.5, 3.0 Hz, 1H) , 6.98-7.09 (m, 2H) , 6.82 (d, J=8, 0 Hz, 1H), 5.48 (d, J=15, 0 Hz, 1H), 4.424.51 (m, 1H), 4.16-4.23 (m, 1H), 4.04-4, 14 (m, 2H), 3.74-3.82 (m, 2H), 3.39-3.46 (m, 1H), 1.58-1.81 (m, 2H), 0.97 ( t, J=7.3 Hz, ZN) . 45 MS: 370.1 (M+H) ⁺ ; ² H NMR (500 MHz, flMCO-d ₆ ) 8.87-8.98 (m, 1H), 8.69-8.79 (m, 1H), 8.04-8.12 (m, 1H) , 7.10-7.18 (m, 1H) , 6.92-7.04 (m, ZN) , 5.09-5.18 (m, 1H) , 4.61-4.69 (m, 1H), 4.50-4.56 (m, 1H), 4.41-4.49 (m, 1H), 4.16 (d, J=15.30 Hz, 1H), 3.57-3 . 68 (m, 2H) . 1.23-1.27 (m, 6H) . 46 MS: 368.1 (M+H) ⁺ ; ² H NMR (500 MHz, flMCO-d ₆ ) 9.35 (dd, J=7.0, 2.7 Hz, 1H) , 8.81 (d, R=7.8 Hz, 1H) , 8, 07-8.15 (m, 1H) , 7.19 (dd, J=9.2, 2.3 Hz, 1H) , 7.01-7.08 (m, 2H), 6.98 (d, J=7.8 Hz, 1H), 5.53 (dd, H=15.1, 1.5 Hz, 1H) , 4.47 (dt, J=10.22, 4.25 Hz, 1H) , 4.34 (t, J=5.08 Hz, 1H) , 4.14 (d, J=15.30 Hz, 1H) , 4.02-4.10 (m, 2H) , 3.793.92 (m , 1H), 1.12-1.16 (m, 1H), 1.03-1.08 (m, 2H), 0.81-0.86 (m, 1H).

- 50 041662

47 MS: 372.1 (M+H) ⁺ ; ² H NMR (500 MHz, flMCO-d ₆ ) 9.25 (t, J=4.9 Hz, 1H) , 8.71 (d, R=7.9 Hz, 1H) , 8.07 (s, 1H), 7.22 (dd, J=9.5, 3.0 Hz, 1H) , 7.05 -7.11 (m, 1H) , 6.96-7.04 (m, 1H) , 6 .83 (d, J=8.0 Hz, 1H) , 5.51 (d, J=14.6 Hz, 1H) , 4.96 (t, J=5.4 Hz, 1H) , 4.42 -4.51 (m, 1H) , 4.24 (ddd, J=10.9, 6.8, 4.2 Hz, 1H) , 4.09-4.20 (m, 2H) , 3.91 (dt, J=15.2, 5.5 Hz, 1H) , 3.67-3.82 (m, ZN) , 3.39-3.51 (m, 1H) . 48 MS: 356.1 (M+H) ⁺ ; ² H NMR (500 MHz, flMCO-d ₆ ) 9.70 (d, J=8.6 Hz, 1H) , 8.76 (d, J=8.0 Hz, 1H) , 8.09 (s, 1H) , 7.25 (dd, J=9.5, 3.0 Hz, 1H) , 7.01-7.11 (m, 1H) , 6.947.00 (m, 1H) , 6.71 (d , J=8.0 Hz, 1H) , 5.64-5.73 (m, 1H) , 4.34 (d, J=9.6 Hz, 1H) , 4.28 (t, J=8, 9 Hz, 1H) , 4.10 (d, J=15.0 Hz, 1H) , 3.94 (dd, J=9.6, 3.6 Hz, 1H) , 3.58 (s, ZN) , 1.36 (d, J=6.8 Hz, ZN). 49 MS: 324.1 (M+H) ⁺ ; ² H NMR (500 MHz, flMCO-d ₆ ) 9.52 (d, J=4.5 Hz, 1H) , 8.74 (d, J=7.9 Hz, 1H) , 8.09 (s, 1H) , 7.44 (d, J=7.6 Hz, 1H) , 7.18-7.25 (m, 1H) , 7.02 (d, J=7.9 Hz, 1H) , 6, 93 (t, J=7.4 Hz, 1H) , 6.71 (d, J=7.9 Hz, 1H) , 5.69 (d, J=14.8 Hz, 1H) , 4.47 ( dt, J=10.l, 4.1 Hz, 1H), 4.01-4.13 (m, 2H), 3.83-3.90 (m, 1H), 3.54-3.61 ( m, ZN) , 3.38-3.46 (m, 1H) . 50 MS: 328.1 (M+H) ⁺ ; ² H NMR (500 MHz, flMCO-d ₆ ) 9.80 (d, J=7.82 Hz, 1H) , 8.89 (t, J=6.00 Hz, 1H) , 8.58 (d, J=7.62 Hz, 1H) , 8.03-8.08 (m, 1H) , 7.12-7.18 (m, 1H) , 6.99-7.05 (m, 2H), 6 .39 (d, J=7.62 Hz, 1H) , 5.13-5.21 (m, 1H) , 4.46-4.53 (m, 1H), 3.87-4.00 (m , 4H). 51 MS: 342.3 (M+H) ⁺ ; ² H NMR (500 MHz, chloroform-d with CD ₃ OD) δ 8.14 (s, 1H) , 7.81 - 7.72 (m, 1H) , 7.10 (dd, J=9.0, 3.0 Hz, 1H) , 6.88 (ddd, J=9.0, 7.6, 3.0 Hz, 1H) , 6.80 (dd, J=9.2, 4.4 Hz, 1H) , 6.20 (d, J=7.4 Hz, 1H), 5.75 (td, J=7.2, 1.9 Hz, 1H), 4.52-4.46 (m, 1H), 4 .09 (tdd, J=9.6, 6.4, 3.9 Hz, 2H) , 3.60-3.52 (m, 1H) , 1.52 (d, J=7.0 Hz, ZN ) .

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51-1 MS: 342.2 ( _M +H ^{) +} . br.d, J=6.86 Hz, 1H) , 8.57 (d, J=7.41 Hz, 1H) , 8.04 (s, 1H) , 7.11-7.22 (m, 1H ) , 6.96-7.04 (m, 2H) , 6.36 (d, J=7.68 Hz, 1H) , 5.63 (br.dd, J=6.86, 5.49 Hz, 1H) , 4.50 (dt, J=10.15, 3.98 Hz, 1H) , 4.01 (td, J=9.61, 3.84 Hz, 1H) , 3.87 (dt, J =10.09, 3.74 Hz, 1H), 3.35-3.46 (m, 1H), 1.45 (d, J=7.14 Hz, ZN). 52 MS: 376.5 (M+H) ⁺ ; ² H NMR (500 MHz, Chloroform-d) δ 9.92 (s, 1H) , 8.29-8.18 (m, 2H) , 7.01 (dd, J=9.2, 8.2 Hz , 1H) , 6.77 (dd, J=9.2, 4.2 Hz, 1H) , 6.37-6.26 (m, 1H) , 6.19 (d, J=7.6 Hz, 1H) , 6.12 (s, 1H) , 4.53-4.45 (m, 1H) , 4.14 (d, J=6.3 Hz, 1H) , 4.04-3.98 (m , 1H) , 3.57 (s, 1H), 1.74 (d, J=7.3 Hz, ZN). 55 MS: 385.6 (M+H) ⁺ ; ² H NMR (300 MHz, Methanol-d ₄ ) 8.35 (d, J=7.6 Hz, 1H) , 8.24 (s, 1H) , 7.24-6.96 (m, 1H) . 6.82 (m, 2H) , 6.41 (dd, J=7.7, 4.8 Hz, 1H) , 5.59 (m, 1H) , 5.315.05 (m, 1H), 4.39 -4.21 (m, 1H), 3.17-3.02 (m, 1H), 1.58 (d, J=6.9 Hz, ZN). 56 MS: 372.3 (M+H) ⁺ ; ² H NMR (300 MHz, Methanol-d ₄ ) δ 8.35 (d, J=7.6 Hz, 1H) , 8.18 (s, 1H) , 7.05 (d, J=9.4 Hz , 1H) , 6.82 (dd, J=6.5, 1.8 Hz, 1H) , 6.39 (d, J=7.6 Hz, 1H) , 5.60 (m, 1H) , 4 .92 (m, 2H) , 4.08 (dd, J=13,l, 9.9 Hz, 1H) , 3.91-3.81 (m, 2H) , 3.73 (dd, J=12 .6, 5.1 Hz, 1H) , 1.58 (d, J=6.9 Hz, ZN). 57 MS: ^371.4 (M+H ₎ ⁺ . 7.00 (dd, J=9.l, 2.9 Hz, 1H) 6.88-6.78 (m, 2H) 6.58 (d, J=7.7 Hz, 1H) .5 .20 (s, 1H), 4.65 (s, 2H), 3.49 (sq., J=7.3 Hz, 2H), 58 MS: 358.5 (M+H) ^{+ ,} ) ⁺ . ) , 6.90 (d, J=7.5 Hz, ZN) , 6.10 (d, J=7.6 Hz, 1H) , 5.88 (s, 1H) , 5.11-4.85 (m, ZN) , 4.20 (dd, J=15,l, 5.7 Hz, 1H), 4.05 (dd, J=14.0, 9.9 Hz, 1H) , 3, 83- 3.68 (m, 3H) , 3.44 (d, J=7.3 Hz, 1H) .

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59 MS: 386.1 (M+H) ⁺ ; and NMR (500 MHz, flMCO-d ₆ ) 9.97 (s, 1H), 8.57 (d, J=7.6 Hz, 1H), 8.40 (d, J=5.9 Hz, 1H ), 8.10 (s, 1H) , 6.85 (dd, J=8.9, 4.8 Hz, 1H) , 6.60 (d, J=7.6 Hz, 1H) , 7.23 (dd, J=9.3, 3.2 Hz, 1H) , 7.00 (td, J=8.6, 3.2 Hz, 1H) , 5.90 (d, J=6.4 Hz, 1H) , 4.27-4.34 (m, 2H) , 3.90 (t, J=9.33 Hz, 2H) , 3.66 (s, 3H) . 60 MS: 371.1 (M+H) ⁺ ; and NMR (500 MHz, flMCO-d ₆ ) 9.98 (br. s, 1H) , 8.54 (d, J=7.6 Hz, 1H) , 8.33 (d, J=6.24 Hz , 1H) , 8.07 (s, 1H) , 7.44 (br.s, 1H) , 7.28 (br.s, 1H) , 7.18 (dd, J=9.6, 3.2 Hz, 1H) , 6.94 (td, J=8.5, 3.2 Hz, 1H) , 6.83 (dd, J=8.9, 4.9 Hz, 1H) , 6.66 (d , J=7.5 Hz, 1H) , 5.86 (d, J=6.4 Hz, 1H) , 4.22-4.36 (m, 2H) , 3.84-3.97 (m, 2H) . 61 MS: 343.2 (M+H) ⁺ , H NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.22 (dd, J=6.87, 2.86 Hz, 1H), 8.78 (d, J 7.45 Hz, 1H), 8.10 (s, 1H), 8.06 (d, J=3.44 Hz, 1H), 7.80 (dd, J=8.59, 2 .86 Hz, 1H) , 6.74 (d, J=8.02 Hz, 1H) , 5.44 (dd, J=14.89, 1.72 Hz, 1H) , 4.69 (ddd, J =10, 88, 8.59, 4.58 Hz, 1H) , 4.32-4.39 (m, 1H) , 4.21 (d, J=15.47 Hz, 1H) , 3.80- 3.88 (m, 1H), 3.58 (s, 3H), 3.41-3.49 (m, 1H). 62 MS: 371.2 (M+H) ⁺ , SH NMR (500 MHz, flMCO-d ₆ ) δ ppm 8.728.80 (m, 2H) , 8.08 (s, 1H) , 8.01 (d , J=2.74 Hz, 1H), 7.49 (dd, J=8.78, 2.74 Hz, 1H), 7.00 (d, J=8.23 Hz, 1H), 4.94 -5.06(m, 2H) 4.57-4.68(m, 1H) 4.26-4.39(m, 2H) 3.66-3.77(m, 1H) 3 .49-3.55 (m, 1H), 1.56 (d, J=6.59 Hz, ZN), 1.22 (d, J=6.60 Hz, ZN). 66 MS: 368.2 (M+H) ⁺ . and NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.56 (dd, J=6.87, 2.86 Hz, 1H), 9.02 (d, J=6.87 Hz, 1H) , 8.58 (d, J=8.02 Hz, 1H) , 8.03 (s, 1H) , 7.18 (dd, J=9.74, 2.86 Hz, 1H) , 6, 97- 7.08(m, 2H) , 6.41(d, J=7.45Hz, 1H) , 4.68-4.80(m, 1H), 4.48(dt, J=10.60, 4.15 Hz, 1H) , 4.05 (ddd, J=10.45, 8.45, 4.01 Hz, 1H) , 3.75-3.84 (m, 1H) , 3.36-3 .43 (m, 1H) , 1.26-1.38 (m, 1H) , 0.63 (tt, J=8.74, 4.44 Hz, 1H), 0.37-0.49 (m , 2H), 0.28 (dkv., J=9.31.4.53 Hz, 1H).

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67 MS: 370.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.75 (br.d, J=6.30 Hz, 1H), 8.78 (d, J=7.45 Hz, 1H) , 8.57 (d, J=8.02 Hz, 1H) 8.04 (s, 1H) , 7.06 (dt, J=9.16, 1.43 Hz , 1H) , 6.98-7.02 (m, 2H) , 6.39 (d, J=7.45 Hz, 1H) , 5.13 (ddd, J=10.02, 7.73, 1 .72 Hz, 1H), 4.51 (dt, J=9.88, 3.65 Hz, 1H) 3.94 (td, J=9.88, 3.72 Hz, 1H), 3.82- 3.90 (m, 1H) , 3.39-3.43 (m, 1H) , 1.96-2.09 (m, 1H) , 1.12 (d, J=6.30 Hz, ZN) , 0.68 (d, J=6.30 Hz, ZN) , 75 MS: 356.2 (M+H) ⁺ , NMR (500 MHz, flMCO- ^J6 ) δ ppm 8.73 (d, J=8.02 Hz, 1H) , 8.25 (t, J=4 .30 Hz, 1H), 8.10 (s, 1H), 7.14-7.21 (m, 1H), 7.00-7.04 (m, 2H), 6.68 (d, J= 8.02 Hz, 1H), 5.75 (br.d, J=14.32 Hz, 1H), 4.33-4.43 (m, 1H), 4.22 (br.d, J=6 .87 Hz, 1H), 4.05 (br. d, J=14.89 Hz, 1H), 3.59-3.68 (m, 1H), 3.59-3.68 (m, 1H) , 3.37-3.45 (m, 1H), 1.98-2.17 (m, 2H), 76-1 MS: 356.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 8.68 (d, J=7.45 Hz, 1H), 8.53 (d, J= 7.45 Hz, 1H), 8.40 (s, 1H), 8.03 (s, 1H), 7.11-7.18 (m, 1H), 6.96-7.00 (m, 2H ), 6.32 (d, J=7.45 Hz, 1H), 5.65-5.74 (m, 1H), 4.29-4.36 (m, 1H), 4.20-4, 26 (m, 1H), 3.54-3.62 (m, 1H), 3.39-3.47 (m, 1H), 1.98-2.17 (m, 2H), 1.41 ( d, J=7.45 Hz, ZN), 84 MS: 358.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 8.78 (d, J=6.79 Hz, 1H), 8.58 (d, J= 7.62 Hz, 1H), 8.05 (s, 1H), 7.36 (d, J=2.61 Hz, 1H), 7.21 (dd, J=8.85, 2.68 Hz, 1H) , 7.03 (d, J=8.85 Hz, 1H) , 6.36 (d, J=7.68 Hz, 1H) , 5.62 (sq., J=6.90 Hz, 1H ) , 4.52 (dt, J=10.15, 3.98 Hz, 1H), 3.98-4.11 (m, 1H), 3.80-3.92 (m, 1H), 3, 35-3.47 (m, 1H), 1.45 (d, J=7.07 Hz, ZN),

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85 MS: 356.2 (M+H) ⁺ , ^g H NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.73 (br.d, J=5.49 Hz, 1H) , 8.74 ( d, J=7.14 Hz, 1H) , 8.57 (d, J=7.68 Hz, 1H) , 8.04 (s, 1H) , 7.06-7.14 (m, 1H) , 6.977.03(m, 2H) , 6.37(d, J=7.68 Hz, 1H) , 5.33-5.45(m, 1H) , 4.51(dt, J=10, 15, 3.43 Hz, 1H), 3.98 (td, J=9.88, 3.84 Hz, 1H), 3.82-3.93 (m, 1H), 3.39 (td, J \u003d 9.61, 2.74 Hz, 1H) , 1.85-1.99 (m, 1H) , 1.62-1.76 (m, 1H) , 0.87 (t, J \u003d 7.14 Hz, ZN), 86 MS: 382.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.74 (dd, J=7.16, 2.00 Hz, 1H) , 8.64 ( e, J=6.87 Hz, 1H) , 8.57 (d, J=7.45 Hz, 1H) 8.05 (s, 1H) , 6, 95-7.06 (m, ZN) , 6 .38 (d, J=8.02 Hz, 1H) , 5.47 (ddd, J=10.60, 7.16, 1.15 Hz, 1H) , 4.54 (dt, J=10.17 , 3.79 Hz, 1H), 4.01 (td, J=9.59, 3.72 Hz, 1H), 3.80-3.90 (m, 1H), 3.39-3.48 ( m, 1H), 2.66-2.77 (m, 1H) , 2.12-2.23 (m, 1H) , 1.83 (br. d, J=2.29 Hz, ZN), 1 .55-1.73 (m, 2H), 87 MS: 346.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.40 (s, 1H) , 8.77 (d, J=8.23 Hz, 1H) , 8.10 (s, 1H) , 7, ΙΟΙ.26 (m, 1H) , 7.01-7.08 (m, 2H) , 6.72 (d, J=8.23 Hz, 1H) , 5.64 (dd, J=15.09, 1.37 Hz, 1H), 4.08 (d, J=14.82 Hz, 1H), 3.58 (s, 3H), 88 MS: 404.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.70 (dd, J=6.87, 2.86 Hz, 1H), 9.26 ( d, J=7.45 Hz, 1H), 8.66 (d, J=7.45 Hz, 1H) , 8.09 (s, 1H) , 7.35-7.45 (m, 4H) , 7.28-7.34 (m, 1H), 7.15 (dd, J=9.16, 3.44 Hz, 1H), 7.097.13 (m, 1H), 7.04-7.09 ( m, 1H) , 6.92 (d, J=6, 87 Hz, 1H) , 6.52 (d, J=7.45 Hz, 1H) , 4.56 (dt, J=10.31, 4 .01 Hz, 1H), 4.08-4.14 (m, 1H), 3.87 (ddt, J=13.75, 7.59, 3.94, 3.94 Hz, 1H), 3, 44-3.49 (m, 1H),

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89 MS: 382.2 (M+H) ⁺ . ² H NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.76 (dd, J=7.45, 2.29 Hz, 1H), 8.77 (d, J=7.45 Hz, 1H ) , 8.58 (d, J=8.02 Hz, 1H), 8.05 (s, 1H), 7.09 (dt, J=9.74, 1.72 Hz, 1H) , 7.00 (dd, J=6.30, 1.72 Hz, 2H) , 6.38 (d, J=7.45 Hz, 1H) , 5.56-5.63 (m, 1H) , 4.51 ( dt, J=10.17, 3.79 Hz, 1H) , 3.99 (td, J=9.59, 3.72 Hz, 1H) , 3.86 (ddt, J=13.75, 7, 45, 3.72, 3.72 Hz, 1H) , 3.38-3.43 (m, 1H) , 1.94 (ddd, J=13.89, 7.88, 6.30 Hz, 1H) 1.44 (dt, J=14.03, 7.30 Hz, 1H) , 0.63-0.73 (m, 1H) , 0.37-0.45 (m, 1H) , 0.27- 0.34 (m, 1H), 0.18 (dq, J=9.24, 4.75 Hz, 1H), -0.12-0.04 (m, 1H), 90 _MS : 372.2 (M+H ^{) +} . J=7.45 Hz, 1H) , 8.36 (t, J=4.01 Hz, 1H) , 8.04 (s, 1H) , 7.16 (dd, J=9.45, 3.15 Hz, 1H) , 7.06 (dd, J=9.17, 4.58 Hz, 1H) , 6.95-7.02 (m, 1H) , 6.30 (d, J=8.02 Hz , 1H) , 5.66-5.75 (m, 1H) , 5.45 (d, J=4.58 Hz, 1H) , 4.12-4.25 (m, 2H) , 4.05 ( d, J=9.16 Hz, 1H) , 3.60-3.67 (m, 1H) , 3.28-3.31 (m, 1H) , 1.42 (d, J=6.87 Hz , ZN), 91 MS: 372.2 (M+H) ⁺ , ² H NMR (500 MHz, flMCO-d ₆ ) δ ppm 8.70 (d, J=6, 87 Hz, 1H) , 8.53 (d, J=7.45 Hz, 1H) , 8.47 (dd, J=8.31, 2.00 Hz, 1H) , 8.03 (s, 1H) , 7.07-7.13 (m, 1H ) , 6, 97-7.03 (m, 2H) , 6.34 (d, J=7.45 Hz, 1H), 5.60 (sq., J=7.02, 7.02, 7, 02, 7.02, 1.72 Hz, 1H) , 5.36 (d, J=4.01 Hz, 1H) , 4.42 (br. d, J=10.88 Hz, 1H) , 4, 01-4.14 (m, 2H) , 3.88-3.97 (m, 1H) , 3.10-3.17 (m, 1H) , 1.41 (d, J=7.45 Hz, ZN),

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92 MS: 356.2 (M+H) ^{+ .2} H NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.41 (dd, J=6.01, 3.72 Hz, 1H), 8, 71 (d, J=7.45 Hz, 1H), 8.58 (d, J=7.45 Hz, 1H), 8.06 (s, 1H), 7.14 (dd, J=9.74 , 3.44 Hz, 1H) , 7.07 (dd, J=9.17, 4.58 Hz, 1H) , 6.96 (dd, J=9, 17, 8.02, 3.44 Hz, 1H), 6.35 (d, J=7.45 Hz, 1H), 5.63-5.74 (m, 1H), 4.77-4.89 (m, 1H), 3.73-3 .85 (m, 1H), 3.52-3.58 (m, 1H) , 1.43 (d, J=6, 87 Hz, ZN) , 1.19 (br.l, J=6.30 Hz, ZN), 93 MS: 356.2 (M+H) ^{+ .2} H NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.82 (dd, J=8.02, 2.29 Hz, 1H), 8, 81 (d, J=6.87 Hz, 1H), 8.58 (d, J=7.45 Hz, 1H) , 8.04 (s, 1H) , 7.12 (dd, J=9.45 , 3.15 Hz, 1H) , 6.99-7.05 (m, 1H) , 6.94-6.99 (m, 1H) , 6.36 (d, J=7.45 Hz, 1H) , 5.53 (sq., J=6, 87, 6, 87, 6, 87, 6, 87, 1.15 Hz, 1H) , 4.45-4.52 (m, 1H) , 3.90 (ddd, J=13.46, 8.31, 4.01 Hz, 1H) , 3.10-3.17 (m, 1H) , 1.46 (d, J=6.30 Hz, ZN), 1.44 (d, J=7.45 Hz, ZN), 94 MS: 356.2 (M+H) ^{+ .2} H NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.49 (dd, J=7.45, 2.86 Hz, 1H), 8, 77 (d, J=7.45 Hz, 1H), 8.09 (s, 1H), 7.15 (dd, J=9.45, 3.15 Hz, 1H) , 7, 04-7.09 (m, 1H) , 6.97-7.03 (m, 1H) , 6.73 (d, J=8.02 Hz, 1H) , 5.54 (dd, J=14.89, 1.72 Hz, 1H), 4.55 (ddd, J=7.59, 5, 87, 4.30 Hz, 1H), 4.08 (d, J=14.89 Hz, 1H), 3, 85-3 , 92 (m, 1H) , 3.59 (s, ZN) , 3.16 (ddd, J=13, 60, 7, 88, 3, 15 Hz, 1H), 1.45 (d, J=6 .30 Hz, ZN), 95 MS: 356.2 (M+H) ^{+ .2} H NMR (500 MHz, DMSO-dg) δ ppm 9.71 (d, J=8.59 Hz, 1H) 8.76 (d, J= 8.02 Hz, 1H) 8.09 (s, 1H) 7.25 (dd, J=9.45, 3.15 Hz, 1H) 7.02-7.09 (m, 1H) 6.957.00 ( m, 1H) 6.71 (d, J=8.02 Hz, 1H) 5.68 (dd, J=14.89, 1.15 Hz, 1H) 4.34 (dd, J=9, 45, 1.43 Hz, 1H) 4.24-4.30 (m, 1H) 4.10 (d, J=14.89 Hz, 1H) 3.94 (dd, J=9.74, 4.01 Hz , 1Н) 3.58 (s, ZN) 1.36 (d, J=6.87 Hz, ZN),

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96 MS: 372.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 8.64 (d, J=8.23 Hz, 1H) 8.27 (br.s, 1H ) 8.08 (s, 1H) 7.15 (br.l, J=6.59 Hz, 1H) 7.04-7.10 (m, 1H) 6.96-7.02 (m, 1H) 6.66 (d, J=8.23 Hz, 1H) 5.11 (br.s, 1H) 4.28 (br.s, 2H) 4.15 (br.s, 1H) 4.06 (br.s, 1H) .s, 1H) 3.90 (br.s, 2H) 3.57 (s, 3N) 3.29 (br.s, 1H), 97 MS: 356.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.49 (dd, J=7.45, 2.86 Hz, 1H), 8.77 ( d, J=8.02 Hz, 1H), 8.09 (s, 1H) , 7.15 (dd, J=9.74, 2.86 Hz, 1H) , 7.04-7.10 (m , 1H) , 6.97-7.03 (m, 1H) , 6.73 (d, J=8.02 Hz, 1H) , 5.54 (dd, J=14.89, 1.72 Hz, 1H), 4.50-4.60 (m, 1H) , 4.08 (d, J=15.47 Hz, 1H) , 3.84-3.92 (m, 1H) , 3.59 (s , ZN) , 3.16 (ddd, J=13.46, 7.73, 2.86 Hz, 1H) , 1.45 (d, J=6.30 Hz, ZN) , 98 MS: 358.2 (M+H) ⁺ . NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.76 (dd, J=7.45, 2.29 Hz, 1H) , 8.82 (d, J=6.87 Hz, 1H) . 8.58 (d, J=7.45 Hz, 1H) , 8.05 (s, 1H) , 7.06-7.15 (m, 1H) , 6.99-7.04 (m, 2H) , 6.45 (d, J=8.02 Hz, 1H) , 5.57-5.66 (m, 1H) , 5.16-5.25 (m, 1H) , 4.52 (dt, J =10.17, 3.79 Hz, 1H), 3.99 (td, J=9.74, 4.01 Hz, 1H), 3.87 (ddt, J=13, 82, 7.52, 3, 94, 3, 94 Hz, 1H), 3.71 (ddd, J=ll,17, 8, 31, 6.30 Hz, 1H), 3.59 (dt, J=ll.17, 5.01 Hz, 1H), 3.36-3.45 (m, 1H), 99 MS: 372.2 (M+H) ⁺ , NMR (500 MHz, flMCO-d ₆ ) δ ppm 8.72 (d, J=8.02 Hz, 1H), 8.53 (d, J= 7.45 Hz, 1H), 8.40 (t, J=4.01 Hz, 1H), 8.04 (s, 1H), 7.09 (dd, J=9.16, 2.86 Hz, 1H) , 6.95-7.05 (m, 2H) , 6.42 (d, J=7.45 Hz, 1H) , 5.635.72 (m, 1H) , 5.16 (t, J=5 .44 Hz, 1H) , 4.29-4.37 (m, 1H) , 4.19-4.27 (m, 1H) , 3.65 (ddd, J=11, 17 , 8.31.6 .30 Hz, 1H), 3.53-3.61 (m, 2H), 3.41-3.48 (m, 1H), 2.00-2.18 (m, 2H),

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100 MS: 356.2 (M+H) ⁺ , F NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.46 (dd, J=7.16, 2.58 Hz, 1H), 8.77 (d, J=8.02 Hz, 1H) , 8.11 (s, 1H) , 7.22 (dd, J=9.74, 2.29 Hz, 1H) , 7.01-7.06 ( m, 2H) , 6.74 (d, J=8.02 Hz, 1H) , 6.20-6.30 (m, 1H) , 4.50 (dt, J=10.31, 4.01 Hz , 1H), 4.05 (ddd, J=10.31, 9.16, 4.01 Hz, 1H), 3.85 (ddt, J=13, 68, 7.52, 3.72, 3, 72 Hz, 1H), 3.38-3.49 (m, 4H), 1.53 (d, J=7.45 Hz, ZN), 101 MS: 400.2 (M+H) ⁺ , F NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.58 (dd, J=7.45, 2.86 Hz, 1H), 8.50 (s, 1H) , 8.01 (s, 1H) , 7.36 (dd, J=9.16, 2.86 Hz, 1H) , 7.00-7.14 (m, 2H) , 5, 61 (dd, J=14.61, 1.43 Hz, 1H) , 4.44-4.52 (m, 1H) , 4.14 (d, J=12.60 Hz, 1H) , 4.00 -4.09 (m, 2H) , 3.81-3.92 (m, 2H) , 3.39-3.47 (m, 1H), 1.40 (s, 3N), 1.38 (s , ZN), 102 MS: 327.2 (M+H) ⁺ , F NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.589.70 (m, 1H) , 9.09 (d, J=6, 87 Hz, 1H ) , 8.42 (s, 1H) , 7.22 (dd, J=9.74, 2.86 Hz, 1H), 7.11 (d, J=7.45 Hz, 1H), 6.84 -6.97 (m, 2H) , 4.37-4.50 (m, 1H) , 3.90-4.06 (m, WN) , 3.42-3.64 (m, WN), 2 .54-2.62 (m, 1H), 103 MS: 341.2 (M+H) ⁺ , F NMR (500 MHz, flMCO-d ₆ ) δ ppm 9.04 (d, J=6.87 Hz, 1H) , 8.56 (t, J =4.01 Hz, 1H) , 8.40 (s, 1H) , 7.19 (dd, J=9, 74, 2.86 Hz, 1H) , 7.06 (d, J=6, 87 Hz , 1H) , 6.81-6.96 (m, 2H) , 4.19-4.29 (m, 2H) , 3.53-3.63 (m, 4H), 3.24-3.31 (m, 2H), 2.09-2.21 (m, 2H), 104 MS: 358.2 (M+H) ⁺ , F NMR (500 MHz, flMCO-d ₆ ) δ ppm 8.79 (d, J=8.02 Hz, 1H), 8.70 (dd, J =7.45, 2.86 Hz, 1H), 8.07 (s, 1H), 7.59 (dd, J=8.59, 5.73 Hz, 1H), 7.10 (td, J= 8.59, 2.86 Hz, 1H) , 7.04 (dd, J=10.02, 2.58 Hz, 1H) , 6.78 (d, J=8.02 Hz, 1H) , 5, 79 (dd, J=15.75, 1.43 Hz, 1H) , 4.17 (d, J=16.04 Hz, 1H) , 3.73-3.82 (m, 1H) , 3.59 (s, ZN), 3.52-3.58 (m, 1H), 3.26-3.30 (m, 1H), 3.18-3.23 (m, 1H), 105 MS: 411.2 (M+H) ⁺ .

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106 MS: 374.2 (M+H) ⁺ , NMR (500 MHz, DMSO-D ₆ ) δ ppm 8.84 (d, J=8.02 Hz, 1H) , 8.09-8.19 ( m, 2H) , 8.07 (s, 1H) , 7.35 (td, J=8.45, 2.58 Hz, 1H) , 7.22 (dd, J=10.31, 2.29 Hz , 1H) , 6.86 (d, J=8.02 Hz, 1H) , 5.75 (d, J=16.61 Hz, 1H) , 4.57 (d, J=16.61 Hz, 1H ) , 4.11-4.15 (m, 1H) , 3.793.87 (m, 2H) , 3.59 (s, 3H), 3.48-3.57 (m, 1H), 107 MS: 390.2 (M+H) ⁺ , SH NMR (500 MHz, DMSO-D ₆ ) δ ppm 8.83 (d, J=8.02 Hz, 1H), 8.12 (dd, J \u003d 9.16, 5.73 Hz, 1H), 8.07 (s, 1H), 7.82 (br. t, J=5.16 Hz, 1H) , 7.39 (td, J=8, 59, 2.86 Hz, 1H) , 7.14-7.21 (m, 1H) , 6.84 (d, J=7.45 Hz, 1H), 5.37-5.54 (m, 1H ), 4.61-4.76 (m, 1H), 3.83-3.93 (m, 1H), 3.57-3.63 (m, 5H) , 3.46-3.54 (m , 1Н) , 108 MS: 371.2 (M+H) ⁺ , SH NMR (500 MHz, DMSO-D ₆ ) δ ppm 9.73 (br.d, J=6, 87 Hz, 1H) 9.09 (d, J=8.00 Hz, 1H) 8.41 (s, 1H) 7.20 (dd, J=9, 74, 2.86 Hz, 1H) 7.11 (d, J=6, 87 Hz, 1H ) 6.94 (dd, J=9.16, 4.58 Hz, 1H) 6.78-6.88 (m, 1H) 4.44 (dd, J=8.88, 5.44, 4, 01 Hz, 1H) 3.99-4.06 (m, 1H) 3.88-3.97 (m, 1H) 3.67-3.73 (m, 1H) 3.47-3.53 (m , 1H) 3.12-3.21 (m, 1H) 2.54-2.62 (m, 1H) 1.43 (d, R = 6.30 Hz, ZN), 109 MS: 371.2 (M+H) ⁺ , SH NMR (500 MHz, DMSO-D ₆ ) δ ppm 9.43 (d, J=2.29 Hz, 1H) 8.73 (d, J= 6, 87 Hz, 1H) 8.57 (d, J=7.50 Hz, 1H) 8.01 (s, 1H) 7.16 (dd, J=9.17, 2.86 Hz, 1H) 7 .00-7.11 (m, 2H) 6.34 (d, J=7.45 Hz, 1H) 5.61-5.73 (m, 1H) 4.37 (dd, J=10.31, 4.01 Hz, 1H) 4.00 (ddt, J=8.45, 4.30, 2.22, 2.22 Hz, 1H) 3.88-3.96 (m, 1H) 1.48 ( e, J=6.87 Hz, ZN) 1.42 (d, J=7.45 Hz, ZN), 110 MS: 371.2 (M+H) ⁺ , ^g H NMR (500 MHz, DMSO-D ₆ ) δ ppm 9.95 (d, J=8.59 Hz, 1H) 8.79 (d, J =6, 87 Hz, 1H) 8.58 (d, J=7.45 Hz, 1H) 8.04 (s, 1H) 7.16 (dd, J=9.45, 3.15 Hz, 1H) 6.98-7.05 (m, 1H) 6.92-6.97 (m, 1H) 6.34 (d, J=8.02 Hz, 1H) 5.67 (sq., J=7, 02, 7.02, 7.02, 7.02, 1.72 Hz, 1H) 4.35 (dd, J=9, 45, 1.43 Hz, 1H) 4.20-4.30 (m, 1Н) 3.93 (dd, J=9.74, 4.01 Hz, 1Н) 1.47 (d, J=7.45 Hz, ZN) 1.37 (d, J=6.87 Hz, ZN ) 111 MS: 371.2 (M+H) ⁺ . 112 MS: 345.2 (M+H) ⁺ .

Biological example 1. Biochemical analyzes of kinases.

MET/ALK/AXL/TRK enzyme inhibition can be measured using Omnia (Invitrogen Inc.) continuous fluorometric assay. Reactions are carried out in 50 μl volumes in 96-well plates at 30°C. Mixtures contain 1 nM human recombinant target kinase domain, 2 μM phosphoacceptor peptide, test compound (11-dose, 3-fold serial dilutions, 2% DMSO final) or DMSO alone, 0.2 mM DTT and 10 mM MgCl ₂ in 20 mM Hepes , pH 7.5, and reactions initiated by the addition of ATP (100 μM final concentration) followed by a 20 min pre-incubation. Initial phosphopeptide production rates are measured over 20 minutes using a Tecan Safire microplate reader with wavelength settings of 360 nm for excitation and 485 nm for emission. Ki values are calculated by fitting the data to a competitive inhibition equation using a non-linear regression method (GraphPad Prism, GraphPad Software, San Diego, CA).

Biological Example 2 Cellular Kinase Phosphorylation ELISA Assays.

Experiments are performed based on the procedures described in (Christensen, J. et al., Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma, Mol. Cancer Ther 2007, 6(12):3314-3322.) All experiments are performed under standard conditions (37° C. and 5% CO ₂ ). IC ₅₀ values are calculated from the fitting concentration/response curve using Microsoft Excel based on the four parameter method. Cells were seeded in 96-well plates in medium supplemented with 10% fetal bovine serum (FBS) and transferred to serum-free medium [with 0.04% bovine serum albumin (BSA)] 24 hours later. In experiments investigating ligand-dependent RTK phosphorylation, relevant facts

- 60 041662 growth tori added up to 20 min. After incubating the cells with the inhibitor for 1 h and/or the appropriate ligands at the designated time, the cells were washed once with HBSS supplemented with 1 mmol/l Na ₃ VO4 and protein lysates were obtained from the cells. Subsequently, phosphorylation of selected protein kinases is assessed by sandwich ELISA using specific capture antibodies for coating 96-well plates and a detection antibody specific for phosphorylated tyrosine residues. Plates coated with antibodies are (a) incubated in the presence of protein lysates at 4°C overnight, (b) washed seven times in 1% Tween 20 in PBS, (c) incubated in horseradish peroxidase-conjugated anti-total antiphosphotyrosine (PY-20 ) antibody (1:500) for 30 min, (d) washed again seven times, (e) incubated in 3,3,5,5 tetramethylbenzidine peroxidase substrate (Bio-rad) to initiate a colorimetric reaction, which was stopped by adding 0, 09n. H ₂ SO ₄ , and (f) measure the absorbance at 450 nm using a spectrophotometer. Cell lines that are used for individual kinases include A549 for MET, Karpas 299 for ALK, 293-AXL for AXL, PAET RKA for TRKA, and PAE-TRKB for TRKB.

Biological Example 3 Kinase Binding Assays.

Kinase binding assays were performed on DiscoveRx using the general KINOMEscan K _d Protocol (Fabian, MA et al., A small molecule-kinase interaction map for clinical kinase inhibitors, Nat. Biotechnol. 2005, 23(3):329-36). For most assays, kinase-tagged T7 phage strains were prepared in E. coli host derived from strain BL21.

E. coli were grown in log phase and infected with T7 phage and incubated with shaking at 32° C. until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were generated in HEK-293 cells and then labeled with DNA for qPCR detection. Streptavidin-coated magnetic beads are treated with biotinylated small ligand molecules for 30 min at room temperature to generate affinity resins for kinase assays. These liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% bovine serum albumin, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinases, liganded beads and test compounds in 1x binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). All reactions were carried out in polystyrene 96-well plates in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1 x PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1x PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR. The results for the compounds tested in this analysis are presented in table. 2. In this method, example 20 also had a binding affinity for PLK4 kinase (K _d 2.9 nM).

table 2

Etc. TRKA K _d (nM) TRKB K _d (nM) TRKC K _d (nM) JAK1 A _a (nM) JAK2 K _d (nM) JAK3 K _d (nM) ALK K _d (nM) ROS1 K _d (nM) 11-1 1900 >30000 1900 20 0.031 0.18 0.30 >1000 4.8 120 80 21 39 0.23 27 180 4.7 40 600 410 41 6.00 280 2.6 33 200 42 0.088 43 0.086 3.7 45 0.082 7.8 49 0.14 24 50 0.20 0.57 51 0.065 65 0.15 4.3 51-1 0.051 37 0.048 1.8 6.8 0.73 52 6.5 270 62 75 0.015 6.5 92 0.12 8.2 93 0.082 5.7 98 0.74 14 103 1.9 28

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Biological example 4 Ba/F3 cell proliferation assay.

TRKA Ba/F3 cell proliferation assays were performed using ACD (Advanced Cellular Dynamics). Ba/F3 cell lines were maintained in RPMI-1640 culture medium containing 10% fetal bovine serum and antibiotics. Cells in logarithmic growth phase were collected and 5000 cells were dispensed into each well of a 384-well plate in 50 μl of growth medium. 50 nl of the diluted compound was added to the respective wells in duplicate and the cells were cultured for 48 hours at 37° C. in a humidified 5% CO2 incubator. Viability was determined by adding 15 μl of CellTiter-Glo and measured luminescence, which was reported as relative light units (RLU), measured in units per second. Data (RLU) for each compound were normalized to the mean maximum response obtained in the presence of base (DMSO) alone. This data was used to obtain percent inhibition (100% of maximum response) and the mean of two data points/concentration was used to calculate _IC50 values (concentration causing half maximum inhibition of cell survival) using non-linear regression analysis using GraphPad Prism software. (GraphPad, Inc., San Diego, CA). Using this method, Example 20 inhibited TRKA Ba/F3 cell proliferation at an IC ₅₀ of 3.0 nM. Data for the compounds tested in this analysis are presented in table. 3.

Biological example 5 Establishment of a stable EML4-ALK Ba/F3 cell line and analysis of cell proliferation.

The wild type EML4-ALK gene (variant 1) was synthesized in GenScript and cloned into the PCDH-CMV-MCS-EFL-Puro plasmid (System Biosciences, Inc). A wild-type Ba/F3-EML4-ALK cell line was established by infecting Ba/F3 cells with a lentivirus containing wild-type EML4-ALK. Stable cell lines were selected by puromycin treatment followed by IL-3 recovery. 5000 cells were seeded in a 384-well white plate overnight prior to compound treatment. Cell proliferation was measured using the CellTiter-Glo luciferase-based ATP detection assay (Promega) according to the manufacturer's protocol after 48 h of varying compound incubation concentrations. IC ₅₀ determinations were made using GraphPad Prism software (GraphPad, Inc., San Diego, CA). Data for the compounds tested in this analysis are presented in table. 3.

Biological example 6 Cell proliferation assays.

Colorectal cell lines KM 12 (containing the endogenous TPM3-TrkA fusion gene) cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. 5000 cells were seeded in a 384-well white plate for 24 hours prior to compound treatment. Cell proliferation was measured using CellTiter-Glo luciferase-based ATP detection assay (Promega) according to manufacturers protocol after 72 h of incubation. IC50 determinations were made using GraphPad Prism software (GraphPad, Inc., San Diego, CA).

Alternatively, the KM12 colorectal cell line (containing the endogenous TPM3-TRKA fusion gene) was cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. Essential cells of the SET-2 thrombocythemia cell line (containing the endogenous JAK2 V618F fusion gene) or the Karpas-299 T-cell lymphoma cell line (containing the endogenous NPM-ALK fusion gene) were cultured in RPMI medium supplemented with 10% fetal bovine serum and 100 u/ ml of penicillin/streptomycin. 5000 cells were seeded in a 384-well white plate for 24 hours prior to compound treatment. Cell proliferation was measured using CellTiter-Glo luciferase-based ATP detection assay (Promega) according to manufacturer's protocol after 72 h of incubation. IC50 determinations were made using GraphPad Prism software (GraphPad, Inc., San Diego, CA).

Data for the compounds tested in these assays are presented in table. 3.

Table 3

Etc. KM 12 cells. profile IC ₅₀ (nM) SET2 cell. profile 1С ₅₀ (nM) Karpas 299 cage. profile 1С ₅₀ (nM) EML4-APK Va/EZ cell. profile 1С ₅₀ (nM) 11-1 >10000 >10000 >10000 20 0.86 2000 1000 39 3, 8 8800 3800 40 204 >10000 >10000 41 118 1500 3900

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42 4.0 2000 3400 43 2, 6 1700 2800 44 9, 9 2030 4100 45 0.35 8000 >10000 46 1.5 7000 7100 47 31 >10000 >10000 48 62 6000 6000 49 6, 7 7000 3900 50 74 6000 4100 51 3.2 425 832 51-1 1.3 234 289 248 52 52 3600 7800 59 >1000 60 >1000 61 0.6 3747 3900 62 0.9 4000 66 17.5 1543 1900 67 2.8 1231 1200 75 0.6 4436 3900 76-1 5, 8 1003 3800 84 0.8 3146 4200 85 0.9 928 1080 86 1998 1000 87 0.3 2734 1591 88 50.4 1900 3129 89 0.2 859 1398 90 18 5911 1653 91 1.8 1536 961 92 0.3 142 88.7 78.6 93 0.5 242 23.7 21.1 94 0.2 >10000 >10000 95 0.4 2673 4107 96 0.6 6000 5000 97 0.3 6500 1419 98 7.4 808 281 99 6, 3 6848 506 100 0.6 5834 5364 101 >1000 6000 >10000 102 1.2 2450 2304 103 15 >10000 1956 104 0.3 2353 5747 105 500 >10000 >5000 106 176 >10000 >10000 107 75.6 3000 >10000 108 3, 6 870 619 109 0.86 398 225 110 0.7 219 163 111 76 1996 329

Biological Example 7 Cell Mechanism of Action-TRKA Assays and Lower Signaling Target Phosphorylation Assays.

KM 12 colorectal cell lines (containing the endogenous TPM3-TrkA fusion gene) were cultured in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. 1 million cells were seeded in 6-well plates for 24 hours before treatment.

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Claims (10)

connection. Cells were washed with 1x PBS and harvested after 5 h treatment and lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS), supplemented with 10 mM EDTA, Halt protease and phosphatase inhibitors (Thermo Scientific). Protein lysates (20 µm) were resolved on 4-12% Bolt Bis-Tris pre-cured gels in MES-flow buffer (Life Technologies), transferred to nitrocellulose membranes using the Trans-Blot Turbo transfer system (Bio-rad) and detected by antibodies , phosphorylated TRK A (Cell Signaling Technology, Y496, Y680, Y681, clone C50F3; dilution 1:1000), total TRK A (Santa Cruz Biotechnology, SC-11, clone C-14, 1:2000 dilution), phosphorylated AKT ( Cell signaling, S473, D9E, #9271; 1:5000 dilution), total AKT (Cell Signaling Technology, 40D4; 1:2000 dilution), phosphorylated ERK (Cell Signaling Technology, Thr 202/204, D13.14.4E, #4370 ; 1:2000 dilution), total ERK (Cell Signaling Technology; dilution 1:1000) and tubulin (Sigma, T4026, dilution 1:5000). The antibodies are typically incubated overnight at 4° C. with gentle shaking, followed by washing and incubation with the appropriate HRP-conjugated secondary antibodies. The membranes were exposed to a chemiluminescent substrate for 5 min at room temperature (SuperSignal West Femto, Thermo Scientific). Images were obtained using the CDigit Imaging System (LI-COR Biosciences). Relative banding density was obtained directly using Licor's Image Studio Digits. Half inhibition concentration (IC ₅₀ ) values were calculated using non-linear regression analysis using GraphPad Prism software (GraphPad, Inc., San Diego, CA). Using this method, Example 20 inhibited autophosphorylation of TPM3-TRKA with an IC ₅₀ of 1.07 nM and phosphorylation of its downstream signaling targets AKT and ERK with IC ₅₀ of 2.80 and 2.00 nM, respectively, in KM12 cells. Biological example 8 Caspase activity assays. KM12 cells were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics. 500,000 cells were seeded in a 12-well plate and various concentrations of the compound were injected over 72 hours. For staurosporine treatment, 500 nM STS was added during 60 hours of incubation and 12 hours as a positive control. All cells were harvested and washed with 1x PBS twice and then lysed in lysis buffer (20 mM HEPES, 150 mM NaCl, 10 mM KCl, 5 mM EDTA, 1% NP40) supplemented with Halt protease and phosphatase inhibitors (Thermo Scientific). For caspase assays, approximately 20 μl (20 μg) of cell lysate was incubated with 20 μl of caspase3 Glo reagent (Promega), the enzymatic activity was measured by luminescence release after 20 min incubation at 37°C. For Western blotting, cell lysates were boiled and analyzed by SDS-PAGE/immunoblotting with PARP or actin antibodies. Using this method, Example 20 induced apoptosis of KM12 cells. CLAIM 1. A method of inhibiting in a cell a protein or tyrosine kinase selected from MET, ALK, ROS1, AXL, TRK and/or JAK, comprising contacting the cell with an effective amount of a compound of the formula or a pharmaceutically acceptable salt thereof, wherein the contact is in vitro, ex vivo or in vivo. 2. The method of claim 1 wherein the protein or tyrosine kinase is a MET kinase. 3. The method of claim 1 wherein the protein or tyrosine kinase is an ALK kinase. 4. The method of claim 1 wherein the protein or tyrosine kinase is a ROS1 kinase. 5. The method of claim 1 wherein the protein or tyrosine kinase is an AXL kinase. 6. The method of claim 1 wherein the protein or tyrosine kinase is a TRK kinase. 7. The method of claim 1 wherein the protein or tyrosine kinase is a JAK kinase. 8. The method of claim 1, wherein the cell is a cancer cell. 9. The method of claim 8 wherein the cancer is selected from the group consisting of lung cancer, colon cancer, breast cancer, prostate cancer, hepatocellular carcinoma, renal cell carcinoma, gastric and esophageal cancer, glioblastoma, cancer head and neck and anaplastic non-small cell lymphoma. 10. The method of claim 9 wherein the cancer is non-small cell lung cancer. -