WO2023214325A1 - Pyrazolopyrimidine derivatives and uses thereof as tet2 inhibitors - Google Patents

Pyrazolopyrimidine derivatives and uses thereof as tet2 inhibitors Download PDF

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Publication number
WO2023214325A1
WO2023214325A1 PCT/IB2023/054596 IB2023054596W WO2023214325A1 WO 2023214325 A1 WO2023214325 A1 WO 2023214325A1 IB 2023054596 W IB2023054596 W IB 2023054596W WO 2023214325 A1 WO2023214325 A1 WO 2023214325A1
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alkyl
pyrazolo
tetrazol
pyrimidin
amine
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PCT/IB2023/054596
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French (fr)
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William D. HASTINGS
Ayako Honda
Rajesh Karki
Mitsunori Kato
Kathryn Taylor Linkens
Saravanan Parthasarathy
Scott Vaughan PLUMMER
Duncan Shaw
Ritesh Bhanudasji Tichkule
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Novartis Ag
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Publication of WO2023214325A1 publication Critical patent/WO2023214325A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • TET2 PYRAZOLOPYRIMIDINE DERIVATIVES AND USES THEREOF AS TET2 INHIBITORS FIELD OF THE INVENTION
  • the present disclosure relates to pyrazolopyrimidine compounds and compositions and their use for the treatment of proliferative diseases or disorders where the inhibition of TET2 can ameliorate a disease or disorder.
  • TET2 Ten-Eleven Translocation 2
  • TET2 is one of three members of the TET family of dioxygenases found in mammals.
  • TET2 contains a C-terminal catalytic domain that catalyzes the oxidation of methylated DNA (5-methyl cytosine, 5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidizes this to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). These reactions initiate the process of DNA demethylation, which is a known mechanism of transcriptional regulation (Schubeler, D., (2015), Nature.517:321-26).
  • TET2 is a 2002 amino acid protein that consists of an N-terminal domain, cysteine-rich region, and C-terminal catalytic domain.
  • TET2 does not contain a CXXC domain that targets the protein to CpG sequences in DNA. This region of TET2 separated from the protein due to chromosomal inversion and evolved as a separate gene (IDAX) which is thought to interact with TET2 and regulate binding to DNA sequences (Jio, C., (2020), J Biosci.45:21). TET2 utilizes alpha-keto glutarate, reduced iron, and molecular oxygen, as well as vitamin C as a cofactor, to oxidize 5mC, 5hmC, and 5fC and produce carbon dioxide and succinate as byproducts of the reaction.
  • IDAX chromosomal inversion
  • TET2 utilizes alpha-keto glutarate, reduced iron, and molecular oxygen, as well as vitamin C as a cofactor, to oxidize 5mC, 5hmC, and 5fC and produce carbon dioxide and succinate as byproducts of the reaction.
  • TET2 is expressed ubiquitously, while TET1 expression is more restricted to embryonic cells (Pastor, W.A., (2013), Nat Rev Mol Cell Biol.14:341-56).
  • TET2 genetic ablation is associated with enhanced survival of hematopoetic stem cells and biased differentiation of myeloid cells, as opposed to that of other lineages such as T, B, and erythroid cells (Ko, M., (2011), Proc Nat Acad Sci USA.108:145666-71).
  • germline loss of function of TET2 in 3 children was associated with immunodeficiency and lymphoma, with altered T cell development and loss of class-switch recombination in B cells (Spegarova, J., (2020), Blood.
  • TET2 mutations are commonly found in patients with blood malignancies and in clonal hematopoiesis of indeterminate potential (CHIP), however in mouse models deletion of TET2 alone leads to an incomplete oncogenic phenotype, with deletion of both TET2 and TET3 necessary for driving fully penetrant malignancies (An, J., (2015), Nat Commun.6: 10071). Genetic perturbation of TET2 has been shown to be associated with enhanced development of memory CD8+ cell responses in mice (Carty, S., (2016), J. of Immunol.200: 82-91).
  • TET2 Disruption of TET2 in a single CD19- targeting CART cell in a CLL patient, via integration of the CART lentivirus into intron 9, led to eradication of the tumor cells and long term remission/cure of the cancer (Fraietta, J., (2016), Nature.558: 307-12). Due to a missense mutation in the other TET2 allele it is reasonable to assume an additive loss of TET2 protein expression and function in this cell clone, which expanded to 94% of the CART cells at the peak of response.
  • TET2 Compared to CART cells from other complete responders, these cells consisted of a high proportion of central memory cells, which have been associated with more durable and effective responses of T cells in adoptive therapy models of cancer (Gattinoni, L., (2011), Nat Med.17: 1290-97). Genetic knockdown of TET2 in CART cells from normal healthy donors enhanced in vitro expansion when stimulated with CD19 expressing tumor cells, thus phenocopying the results observed in vivo with this patient’s CART cells.
  • a TET2-specific enzymatic inhibitor has the potential to enhance anti-tumor T cell therapies by enhancing memory and stemness of the T cells, leading to more durable and effective responses.
  • the invention provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, which compounds are TET2 inhibitors.
  • the invention further provides methods of treating, preventing, or ameliorating proliferative diseases, comprising administering to a subject in need thereof an effective amount of an TET2 inhibitor.
  • Various embodiments of the invention are described herein.
  • provided herein is a compound of Formula AA:
  • FIG. 1 illustrates the Inhibition of 5hmC formation by recombinant human TET2 protein by select compounds
  • Figure 2 illustrates the Inhibition of 5hmC production in a TET2 inducible overexpressing HeLa cell line (HeLa pCR148 clone 12D) by select compounds
  • Figure 3 illustrates decreased expression of TIGIT by activated T cells in the presence of Compound Ex.
  • I-5 Figure 4 illustrates the decreased expression of FOXP3 by activated T cells in the presence of Compound Ex.
  • I-5 Figure 5 illustrates the enhanced expression of TCF7 by activated T cells in the presence of Compound Ex.
  • I-5 Figure 6 illustrates the reduced FOXP3 expression by activated T cells in the presence of Compound Ex.
  • I-83 Figure 7 illustrates the reduced FOXP3 expression by activated T cells in the presence of Compound Ex. II-10
  • Figure 8 illustrates the reduced FOXP3 expression by activated T cells in the presence of Compound Ex.
  • the invention therefore provides a compound of Formula AA: or a pharmaceutically acceptable salt thereof, wherein Ring A is selected from a 6-10 membered aryl, 6-10 membered heteroaryl, and 6-10 membered partially saturated carbocyclyl, wherein the aryl, heteroaryl and carbocyclyl are each independently unsubstituted or substituted with 0 to 5 substituents represented by R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , or R 8 ;
  • R 1 is H, NH 2 , or NH(CH 2 ) 2 N(CH 3 ) 2 ;
  • R 2 is H, halogen, -OH, C 1-6 alkyl, haloC 1-6 alkyl, CH 2 R 11 , C(R 11 ) 2 , O(CH 2 ) 1-6 R 11 , or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and
  • R 1 is H, NH 2 , or NH(CH 2 ) 2 N(CH 3 ) 2
  • R 2 is H, halogen, -OH, C 1-6 alkyl, haloC 1-6 alkyl, CH 2 R 11 , C(R 11 ) 2 , O(CH 2 ) 1-6 R 11 , or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O which is unsubstituted or substituted with one or more R 11
  • R 3 is H, halogen, C 1-6 alkyl, haloC 1-6 alkyl, OH, hydroxy(C 1-6 alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C 1-6 alkyl)(R 11 ) 2 , or OR 11
  • R 4 is H, halogen, or C 1-6 alkyl
  • R 1 is H, NH 2 , or NH(CH 2 ) 2 N(CH 3 ) 2
  • R 2 is H, halogen, -OH, C 1-6 alkyl, haloC 1-6 alkyl, CH 2 R 11 , C(R 11 ) 2 , O(CH 2 ) 1-6 R 11 , or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O which is unsubstituted or substituted with one or more R 11
  • R 3 is H, halogen, C 1-6 alkyl, haloC 1-6 alkyl, OH, hydroxy(C 1-6 alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C 1-6 alkyl)(R 11 ) 2 , or OR
  • R 1 is H, NH 2 , or NH(CH 2 ) 2 N(C H 3) 2
  • R 2 is H, -OH, halogen, C 1-6 alkyl, haloC 1-6 alkyl, CH 2 R 11 , C(R 11 ) 2 , O(C 1-6 alkyl)R 11 , or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which is unsubstituted or substituted with one or more R 11
  • R 3 is H, halogen, C 1-6 alkyl, haloC 1-6 alkyl, OH, hydroxy(C 1-6 alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C 1-6 alkyl)(R 11 ) 2 , or OR 11
  • R 4 is H, halogen, or C 1-6
  • R 1 is H, NH 2 , or NH(CH 2 ) 2 N(CH 3 ) 2
  • R 2 is H, halogen, -OH, C 1-6 alkyl, haloC 1-6 alkyl, CH 2 R 11 , C(R 11 ) 2 , O(C 1-6 alkyl)R 11 , or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R 11
  • R 3 is H, halogen, C 1-6 alkyl, haloC 1-6 alkyl, OH, hydroxy (C 1-6 alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C 1-6 alkyl)(R 11 ) 2 , or OR 11
  • R 4 is H, halogen, or C 1-6
  • R 1 is H, NH 2 , or NH(CH 2 ) 2 N(CH 3 ) 2
  • R 2 is H, halogen, -OH, C 1-6 alkyl, haloC 1-6 alkyl, CH 2 R 11 , C(R 11 ) 2 , O(C 1-6 alkyl)R 11 , or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R 11
  • R 3 is H, halogen, C 1-6 alkyl; haloC 1-6 alkyl, OH, hydroxy(C 1-6 alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C 1-6 alkyl)(R 11 ) 2 , or OR 11
  • R 4 is H, halogen,
  • R 1 is H, NH 2 , or NH(CH 2 ) 2 N(CH 3 ) 2
  • R 2 is H, halogen, -OH, C 1-6 alkyl, haloC 1-6 alkyl, CH 2 R 11 , C(R 11 ) 2 , O(C 1-6 alkyl)R 11 , or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R 11
  • R 3 is H, halogen, C 1-6 alkyl, haloC 1-6 alkyl, OH, hydroxy(C 1-6 alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C 1-6 alkyl)(R 11 ) 2 , or OR 11
  • R 4 is H, halogen, or C 1-6
  • Ring A is selected from naphthyl, benzothiophenyl, tetrahydronaphthyl, or indane.
  • R 1 is H or NH 2 .
  • R 2 is H or CH 3 .
  • R 9 is H or NH 2 .
  • R 10 is COOH,
  • A is CR 8 .
  • A is S, and , G is N, NR 5 , or CR 5 .
  • G is CR 5 .
  • G is S
  • A is N, NR 8 , or CR 8 .
  • Specifica compounds include:
  • One embodiment is a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition further comprising at least one additional pharmaceutical agent.
  • the pharmaceutical composition is for use in the treatment of a disease or disorder that is affected by the inhibition of TET2.
  • Another emobidment is a method of inhibiting TET2 comprising administering to the patient in need thereof a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof.
  • Another emobidment is a method of reducing the proliferation of a cell, the method comprising contacting the cell with a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof, and inhibition TET2.
  • Another emobidment is a method of treating cancer comprising administering to the patient in need thereof a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof.
  • Another emobidment is a method wherein the cancer is selected from non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS).
  • NSCLC non-small cell lung cancer
  • HCC Hepatocellular Carcinoma
  • HCC Hepatocellular Carcinoma
  • esophageal cancer uterine cancer
  • breast cancer bladder cancer
  • cervical cancer colorectal cancer
  • kidney cancer melanoma
  • NSCLC non-small cell lung cancer
  • adenocarcinoma adenocarcinoma, squamous cell carcinoma, large cell carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, and sarcomatoid carcinoma.
  • NSCLC non-small cell lung cancer
  • Another embodiment is the compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or disorder that is affected by the inhibition of TET2.
  • TET2 non-small cell lung cancer
  • Another embodiment is the use of a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or disorder that is affected by the inhibition of TET2.
  • the disease or disorder is selected from non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS).
  • NSCLC non-small cell lung cancer
  • HCC Hepatocellular Carcinoma
  • HCC Hepatocellular Carcinoma
  • esophageal cancer uterine cancer
  • breast cancer bladder cancer
  • cervical cancer colorectal cancer
  • kidney cancer melanoma
  • stomach castration-resistant prostate cancer
  • Another embodiment is the compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for treating a disease or disorder that is affected by the inhibition of TET2.
  • NSCLC
  • the term “compounds of the present disclosure” or “compound of the present disclosure” refers to compounds of formula (I) subformulae thereof, and exemplified compounds, and salts thereof, as well as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties.
  • the terms "Halogen”, “halide”, or, alternatively, “halo” refer to bromo, chloro, fluoro or iodo.
  • C 1-6 alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molecule by a single bond.
  • the term “C 1-4 alkyl” is to be construed accordingly. Examples of C 1-6 alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl and 1,1-dimethylethyl (t- butyl).
  • C 3-8 cycloalkyl refers to a monocyclic or polycyclic radical that contains only carbons and hydrogen, having from three to eight ring atoms, and can be saturated or partially unsaturated.
  • Examples of C 3-8 cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentyenyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • hydroxyC 1-6 alkyl refers to a C 1-6 alkyl radical as defined above, wherein one of the hydrogen atoms of the C 1-6 alkyl radical is replaced by OH.
  • hydroxyC 1-6 alkyl examples include, but are not limited to, hydroxy-methyl, 2-hydroxy-ethyl, 2-hydroxy- propyl, 3-hydroxy-propyl and 5-hydroxy-pentyl.
  • haloC 1-6 alkyl refers to C 1-6 alkyl radical, as defined above, substituted by one or more halo radicals, as defined above.
  • halo C 1-6 alkyl examples include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,3- dibromopropan-2-yl 3-bromo-2-fluoropropyl and 1,4,4-trifluorobutan-2-yl.
  • Aryl refers to an aromatic hydrocarbon ring system. Aryl groups are monocyclic ring systems or bicyclic ring systems. Monocyclic aryl ring refers to phenyl. Bicyclic aryl rings refer to naphthyl. Aryl groups may be optionally substituted with one or more substituents as defined in formula (I).
  • heterocyclic refers to a 3 to 8 membered saturated or partially unsaturated monocyclic or bicyclic ring containing from 1 to 5 heteroatoms. Heterocyclic ring systems are not aromatic. Heterocyclic groups containing more than one heteroatom may contain different heteroatoms.
  • Heterocyclic includes ring systems wherein a carbon atom is oxidized forming a cyclic ketone or lactam group. Heterocyclic also includes ring systems wherein a sulfur atom is oxidized to form SO or SO2. Heterocyclic groups may be optionally substituted with one or more substituents as defined in formula (I). Heterocyclic groups are monocyclic, spiro, or fused or bridged bicyclic ring systems. Monocyclic heterocyclic have 3 to 7 ring atoms, unless otherwise defined.
  • Examples of monocyclic heterocyclic groups include tetrahydrofuranyl, dihydrofuranyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, piperazinyl, piperidinyl, 1,3-dioxolanyl, imidazolidinyl, imidazolinyl, pyrrolinyl, pyrrolidinyl, tetrahydropyranyl, dihydropyranyl, oxathiolanyl, dithiolanyl, 1,3-dioxanyl, 1,3-dithianyl, oxathianyl, thiomorpholinyl and the like.
  • Fused heterocyclic ring systems have from 8 to 11 ring atoms and include groups wherein a heterocyclic ring is fused to a phenyl or monocyclic heteroaryl ring. Examples of fused heterocyclic rings include 3,4-dihydroquinolin-2(1H)-onyl and the like.
  • Heteroaryl refers to an aromatic ring system containing from 1 to 5 heteroatoms. Heteroaryl groups containing more than one heteroatom may contain different heteroatoms. Heteroaryl groups may be optionally substituted with one or more substituents as defined in formula (I). Heteroaryl groups are monocyclic ring systems or are fused bicyclic ring systems.
  • Monocyclic heteroaryl rings have from 5 to 6 ring atoms.
  • Bicyclic heteroaryl rings have from 8 to 10 member atoms.
  • Heteroaryl includes, but is not limited to, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, furanyl, furanzanyl, thienyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, trazinyl, tetrazinyl, tetrzolyl, indonyl, isoindolyl, indolizinyl, indazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, benzimidazolyl, benzopyranyl, benzo
  • salts refers to an acid addition or base addition salt of a compound of the present invention.
  • Salts include in particular “pharmaceutical acceptable salts”.
  • pharmaceutically acceptable salts refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable.
  • the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.
  • the compounds of the present invention may also form internal salts, e.g., zwitterionic molecules.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
  • Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table.
  • the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.
  • the present invention provides compounds of the present invention in acetate, ascorbate, adipate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, caprate, chloride/hydrochloride, chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate
  • any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds.
  • lsotopically labeled compounds have structures depicted by the formulae given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number.
  • Isotopes that can be incorporated into compounds of the disclosure include, for example, isotopes of hydrogen.
  • Formula (II) is deuterated as shown in the compound of formula (IIc): (IIc) or a pharmaceutically acceptable salt thereof, wherein R 1 , through R 10 are defined as in Formula (II), RD 1 through RD 10 are independently H or D.
  • isotopes particularly deuterium (i.e., 2 H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index or tolerability.
  • deuterium in this context is regarded as a substituent of a compound of the present disclosure.
  • concentration of deuterium may be defined by the isotopic enrichment factor.
  • isotopic enrichment factor as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope.
  • a substituent in a compound of this disclosure is denoted as being deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).
  • isotopic enrichment factor can be applied to any isotope in the same manner as described for deuterium.
  • isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 3 H, 11 C, 13 C, 14 C, 15 N, 18 F , 35 S, 36 Cl, 123 I, 124 I, 125 I respectively.
  • the disclosure includes compounds that incorporate one or more of any of the aforementioned isotopes, including for example, radioactive isotopes, such as 3 H and 14 C, or those into which non- radioactive isotopes, such as 2 H and 13 C are present.
  • Such isotopically labelled compounds are useful in metabolic studies (with 14 C), reaction kinetic studies (with, for example 2 H or 3 H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • an 18 F or labeled compound may be particularly desirable for PET or SPECT studies.
  • Isotopically-labeled compounds of the present disclosure can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.
  • the term “pharmaceutical composition” refers to a compound of the disclosure, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier, in a form suitable for oral or parenteral administration.
  • pharmaceutically acceptable carrier refers to a substance useful in the preparation or use of a pharmaceutical composition and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffering agents, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegration agents, lubricants, wetting agents, sweetening agents, flavoring agents, dyes, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22 nd Ed.
  • a therapeutically effective amount of a compound of the present disclosure refers to an amount of the compound of the present disclosure that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme, receptor, ion channel, or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc.
  • a therapeutically effective amount refers to the amount of the compound of the present disclosure that, when administered to a subject, is effective to (1) at least partially alleviate, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by TET2, or (ii) associated with TET2 activity, or (iii) characterized by activity (normal or abnormal) of TET2; or (2) reduce or inhibit the activity of TET2; or (3) reduce or inhibit the expression of TET2.
  • a therapeutically effective amount refers to the amount of the compound of the present disclosure that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of TET2; or at least partially reducing or inhibiting the expression of TET2.
  • the meaning of the term “a therapeutically effective amount” as illustrated in the above embodiment for TET2 also applies by the same means to any other relevant proteins/peptides/enzymes/receptors/ion channels.
  • the term “subject” refers to primates (e.g., humans, male or female), dogs, rabbits, guinea pigs, pigs, rats and mice.
  • the subject is a primate. In yet other embodiments, the subject is a human.
  • the term “inhibit”, “inhibition” or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
  • the term “treat”, “treating” or “treatment” of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient.
  • the term “prevent”, “preventing” or “prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder
  • a subject is “in need of” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.
  • the term “a,” “an,” “the” and similar terms used in the context of the present disclosure are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
  • Any asymmetric atom (e.g., carbon or the like) of the compound(s) of the present disclosure can be present in racemic or enantiomerically enriched, for example the (R)-, (S)- or (R,S)- configuration.
  • each asymmetric atom has at least 50 % enantiomeric excess, at least 60 % enantiomeric excess, at least 70 % enantiomeric excess, at least 80 % enantiomeric excess, at least 90 % enantiomeric excess, at least 95 % enantiomeric excess, or at least 99 % enantiomeric excess in the (R)- or (S)- configuration.
  • Substituents at atoms with unsaturated double bonds may, if possible, be present in cis- (Z)- or trans- (E)- form.
  • a compound of the present disclosure can be in the form of one of the possible stereoisomers, rotamers, atropisomers, tautomers or mixtures thereof, for example, as substantially pure geometric (cis or trans) stereoisomers, diastereomers, optical isomers (antipodes), racemates or mixtures thereof. Any resulting mixtures of stereoisomers can be separated on the basis of the physicochemical differences of the constituents, into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and/or fractional crystallization.
  • Any resulting racemates of compounds of the present disclosure or of intermediates can be resolved into the optical antipodes by known methods, e.g., by separation of the diastereomeric salts thereof, obtained with an optically active acid or base, and liberating the optically active acidic or basic compound.
  • a basic moiety may thus be employed to resolve the compounds of the present disclosure into their optical antipodes, e.g., by fractional crystallization of a salt formed with an optically active acid, e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di-O,O'-p-toluoyl tartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid.
  • Racemic compounds of the present disclosure or racemic intermediates can also be resolved by chiral chromatography, e.g., high pressure liquid chromatography (HPLC) using a chiral adsorbent.
  • HPLC high pressure liquid chromatography
  • the disclosure further includes any variant of the present processes, in which an intermediate obtainable at any stage thereof is used as starting material and the remaining steps are carried out, or in which the starting materials are formed in situ under the reaction conditions, or in which the reaction components are used in the form of their salts or optically pure material.
  • Compounds of the present disclosure and intermediates can also be converted into each other according to methods generally known to those skilled in the art.
  • Pharmaceutical Compositions in another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
  • the pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration (e.g., by injection, infusion, transdermal or topical administration), and rectal administration. Topical administration may also pertain to inhalation or intranasal application.
  • the pharmaceutical compositions of the present disclosure can be made up in a solid form (including, without limitation, capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including, without limitation, solutions, suspensions or emulsions). Tablets may be either film coated or enteric coated according to methods known in the art.
  • the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more of: a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and e) absorbents, colorants, flavors and sweeteners.
  • diluents e.g., lactose, dextrose
  • the compounds of the present invention in free form or in pharmaceutically acceptable salt form exhibit valuable pharmacological properties, e.g., TET2 inhibition, e.g., as indicated in vitro and in vivo tests as provided in the next sections, and are therefore indicated for therapy or for use as research chemicals, e.g., as tool compounds.
  • pharmacological properties e.g., TET2 inhibition, e.g., as indicated in vitro and in vivo tests as provided in the next sections, and are therefore indicated for therapy or for use as research chemicals, e.g., as tool compounds.
  • NSCLC non-small cell lung cancer
  • HCC Hepatocellular Carcinoma
  • HCC Hepatocellular Carcinoma
  • esophageal cancer uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS).
  • NSCLC non-small cell lung cancer
  • HCC Hepatocellular Carcinoma
  • HCC Hepatocellular Carcinoma
  • esophageal cancer uterine cancer
  • breast cancer bladder cancer
  • cervical cancer colorectal cancer
  • kidney cancer melanoma
  • stomach castration-resistant prostate cancer
  • GIST gastrointestinal
  • the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt thereof in therapy.
  • the therapy is selected from a disease which may be treated by inhibition of TET2.
  • the disease is selected from the afore-mentioned list.
  • the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament.
  • the medicament is for treatment of a disease which may be treated by inhibition of TET2.
  • the disease is selected from the afore-mentioned list.
  • the compound of Formula (I) for use in the treatment of non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS).
  • NSCLC non-small cell lung cancer
  • HCC Hepatocellular Carcinoma
  • HCC Hepatocellular Carcinoma
  • esophageal cancer uterine cancer
  • breast cancer bladder cancer
  • cervical cancer colorectal cancer
  • kidney cancer melanoma
  • stomach castration
  • the pharmaceutical composition or combination of the present disclosure can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg, or about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or about 1-50 mg of active ingredients.
  • the therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
  • the above-cited dosage properties are demonstrable in vitro and in vivo tests using advantageously mammals, e.g., mice, rats, dogs, monkeys or isolated organs, tissues and preparations thereof.
  • the compounds of the present disclosure can be applied in vitro in the form of solutions, e.g., aqueous solutions, and in vivo either internally, parenterally, advantageously intravenously, e.g., as a suspension or in aqueous solution.
  • the dosage in vitro may range between about 10 -3 molar and 10 -9 molar concentrations.
  • a therapeutically effective amount in vivo may range depending on the route of administration, between about 0.1-500 mg/kg, or between about 1-100 mg/kg.
  • Combinations “Combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present disclosure and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a coope-rative, e.g., synergistic effect.
  • the single components may be packaged in a kit or separately.
  • One or both of the components e.g., powders or liquids
  • co-administration or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
  • pharmaceutical combination as used herein means a product that results from the mixing or combining of more than one therapeutic agent and includes both fixed and non- fixed combinations of the therapeutic agents.
  • fixed combination means that the therapeutic agents, e.g., a compound of the present disclosure and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage.
  • non-fixed combination means that the therapeutic agents, e.g., a compound of the present disclosure and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient.
  • cocktail therapy e.g., the administration of three or more therapeutic agents.
  • pharmaceutical combination refers to either a fixed combination in one dosage unit form, or non-fixed combination or a kit of parts for the combined administration where two or more therapeutic agents may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect.
  • composition therapy refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure.
  • administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients.
  • administration encompasses co-administration in multiple, or in separate containers (e.g., tablets, capsules, powders, and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to a desired dose prior to administration.
  • administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.
  • the compound of the present disclosure may be administered either simultaneously with, or before or after, one or more other therapeutic agent.
  • the compound of the present disclosure may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents.
  • a therapeutic agent is, for example, a chemical compound, peptide, antibody, antibody fragment or nucleic acid, which is therapeutically active or enhances the therapeutic activity when administered to a patient in combination with a compound of the present disclosure.
  • the disclosure provides a product comprising a compound of the present disclosure and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy.
  • the therapy is the treatment of a disease or condition mediated by inhibition of TET2.
  • Products provided as a combined preparation include a composition comprising the compound of the present disclosure and the other therapeutic agent(s) together in the same pharmaceutical composition, or the compound of the present disclosure and the other therapeutic agent(s) in separate form, e.g. in the form of a kit.
  • the disclosure provides a pharmaceutical composition comprising a compound of the present disclosure and another therapeutic agent(s).
  • the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, as described above.
  • the disclosure provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of the present disclosure.
  • the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet.
  • kits are blister pack, as typically used for the packaging of tablets, capsules and the like.
  • the kit of the disclosure may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another.
  • the kit of the disclosure typically comprises directions for administration.
  • the compound of the present disclosure and the other therapeutic agent may be manufactured and/or formulated by the same or different manufacturers.
  • the compound of the present disclosure and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g.
  • the disclosure provides the use of a compound of the present disclosure for treating a disease or condition mediated by inhibition of TET2, wherein the medicament is prepared for administration with another therapeutic agent.
  • the disclosure also provides the use of another therapeutic agent for treating a disease or condition mediated by inhibition of TET2, wherein the medicament is administered with a compound of the present disclosure.
  • the disclosure also provides a compound of the present disclosure for use in a method of treating a disease or condition mediated by inhibition of TET2, wherein the compound of the present disclosure is prepared for administration with another therapeutic agent.
  • the disclosure also provides another therapeutic agent for use in a method of treating a disease or condition mediated by inhibition of TET2, wherein the other therapeutic agent is prepared for administration with a compound of the present disclosure.
  • the disclosure also provides a compound of the present disclosure for use in a method of treating a disease or condition mediated by inhibition of TET2, wherein the compound of the present disclosure is administered with another therapeutic agent.
  • the disclosure also provides another therapeutic agent for use in a method of treating a disease or condition mediated inhibition of TET2, wherein the other therapeutic agent is administered with a compound of the present disclosure.
  • the disclosure also provides the use of a compound of the present disclosure for treating a disease or condition mediated by inhibition of TET2, wherein the patient has previously (e.g., within 24 hours) been treated with another therapeutic agent.
  • the disclosure also provides the use of another therapeutic agent for treating a disease or condition mediated by inhibition of TET2, wherein the patient has previously (e.g., within 24 hours) been treated with compound of the present disclosure.
  • the other therapeutic agent is selected from: anti-cancer agents, anti-nausea agents (or anti-emetics), a chemotherapy, pain relievers, cytoprotective agents, and combinations thereof.
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof of the present disclosure are administered in combination with one or more second agent(s) selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a cytokine, an A2A antagonist, a GITR agonist, a TIM-3 inhibitor, a STING agonist, and a TLR7 agonist, to treat a disease, e.g., cancer.
  • a second agent(s) selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a cytokine, an A2A antagonist, a GITR agonist, a TIM-3 inhibitor, a STING agonist, and a TLR7 agonist
  • one or more chemotherapeutic agents are used in combination with the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, for treating a disease, e.g., cancer
  • said chemotherapeutic agents include, but are not limited to, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5- fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arab
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more other anti-HER2 antibodies, e.g., trastuzumab, pertuzumab, margetuximab, or HT-19 described above, or with other anti-HER2 conjugates, e.g., ado-trastuzumab emtansine (also known as Kadcyla®, or T- DM1).
  • anti-HER2 antibodies e.g., trastuzumab, pertuzumab, margetuximab, or HT-19 described above
  • other anti-HER2 conjugates e.g., ado-trastuzumab emtansine (also known as Kadcyla®, or T- DM1).
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more tyrosine kinase inhibitors, including but not limited to, EGFR inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors, for treating a disease, e.g., cancer.
  • one or more tyrosine kinase inhibitors including but not limited to, EGFR inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors, for treating a disease, e.g., cancer.
  • tyrosine kinase inhibitors include but are not limited to, Erlotinib hydrochloride (Tarceva®); Linifanib (N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N'-(2-fluoro-5-methylphenyl)urea, also known as ABT 869, available from Genentech); Sunitinib malate (Sutent®); Bosutinib (4- [(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1- yl)propoxy]quinoline-3-carbonitrile, also known as SKI-606, and described in US Patent No.
  • Tarceva® Erlotinib hydrochloride
  • Linifanib N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N'-(2-fluoro-5-methylphenyl
  • Epidermal growth factor receptor (EGFR) inhibitors include but are not limited to, Erlotinib hydrochloride (Tarceva®), Gefitinib (Iressa®); N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3''S'')- tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®); (3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1- f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); Canertinib dihydrochloride (CI-1033); 6- [4-[(4-Ethyl-1-pipe
  • EGFR antibodies include but are not limited to, Cetuximab (Erbitux®); Panitumumab (Vectibix®); Matuzumab (EMD-72000); Nimotuzumab (hR3); Zalutumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1).
  • HER2 inhibitors include but are not limited to, Neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4- [(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2- enamide, and described PCT Publication No.
  • HER3 inhibitors include but are not limited to, LJM716, MM-121, AMG-888, RG7116, REGN- 1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A.
  • MET inhibitors include but are not limited to, Cabozantinib (XL184, CAS 849217-68-1); Foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); Tivantinib (ARQ197, CAS 1000873-98-2); 1- (2-Hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2- phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458); Cryzotinib (Xalkori®, PF-02341066); (3Z
  • IGFR inhibitors include but are not limited to, BMS-754807, XL-228, OSI-906, GSK0904529A, A- 928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. See e.g., Yee, JNCI, 104; 975 (2012) for review.
  • the compounds of Formula (I) of the present disclosure are used in combination with one or more proliferation signalling pathway inhibitors, including but not limited to, MEK inhibitors, BRAF inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTOR inhibitors, and CDK inhibitors, for treating a disease, e.g., cancer.
  • one or more proliferation signalling pathway inhibitors including but not limited to, MEK inhibitors, BRAF inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTOR inhibitors, and CDK inhibitors, for treating a disease, e.g., cancer.
  • mitogen-activated protein kinase (MEK) inhibitors include but are not limited to, XL- 518 (also known as GDC-0973, CAS No.1029872-29-4, available from ACC Corp.); 2-[(2-Chloro- 4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (also known as CI-1040 or PD184352 and described in PCT Publication No.
  • N-[3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)-2,3- dihydroxypropyl]- cyclopropanesulfonamide also known as RDEA119 or BAY869766 and described in PCT Publication No.
  • BRAF inhibitors include, but are not limited to, Vemurafenib (or Zelboraf®), GDC-0879, PLX-4720 (available from Symansis), Dabrafenib (or GSK2118436), LGX 818, CEP-32496, UI-152, RAF 265, Regorafenib (BAY 73-4506), CCT239065, or Sorafenib (or Sorafenib Tosylate, or Nexavar®), or Ipilimumab (or MDX-010, MDX-101, or Yervoy).
  • Phosphoinositide 3-kinase (PI3K) inhibitors include, but are not limited to, 4-[2-(1H-Indazol-4-yl)- 6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC0941, RG7321, GNE0941, Pictrelisib, or Pictilisib; and described in PCT Publication Nos.
  • mTOR inhibitors include but are not limited to, Temsirolimus (Torisel®); Ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30- dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4- azatricyclo[30.3.1.04,9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No.
  • CDK inhibitors include but are not limited to, Palbociclib (also known as PD-0332991, Ibrance®, 6-Acetyl-8-cyclopentyl-5-methyl-2- ⁇ [5-(1-piperazinyl)-2-pyridinyl]amino ⁇ pyrido[2,3-d]pyrimidin- 7(8H)-one).
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, of the present disclosure are used in combination with one or more pro-apoptotics, including but not limited to, IAP inhibitors, BCL2 inhibitors, MCL1 inhibitors, TRAIL agents, CHK inhibitors, for treating a disease, e.g., cancer.
  • IAP inhibitors include but are not limited to, LCL161, GDC-0917, AEG-35156, AT406, and TL32711.
  • IAP inhibitors include but are not limited to those disclosed in WO04/005284, WO 04/007529, WO05/097791, WO 05/069894, WO 05/069888, WO 05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO 06/010118, WO 06/017295, and WO08/134679, all of which are incorporated herein by reference.
  • BCL-2 inhibitors include but are not limited to, 4-[4-[[2-(4-Chlorophenyl)-5,5-dimethyl-1- cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(4-morpholinyl)-1- [(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide (also known as ABT-263 and described in PCT Publication No.
  • Proapoptotic receptor agonists including DR4 (TRAILR1) and DR5 (TRAILR2), including but are not limited to, Dulanermin (AMG-951, RhApo2L/TRAIL); Mapatumumab (HRS-ETR1, CAS 658052-09-6); Lexatumumab (HGS-ETR2, CAS 845816-02-6); Apomab (Apomab®); Conatumumab (AMG655, CAS 896731-82-1); and Tigatuzumab(CS1008, CAS 946415-34-5, available from Daiichi Sankyo).
  • PARAs Proapoptotic receptor agonists
  • DR4 DR4
  • TRAILR2 DR5
  • Dulanermin AMG-951, RhApo2L/TRAIL
  • Mapatumumab HRS-ETR1, CAS 658052-09-6
  • Lexatumumab HS-ETR2, CAS 8458
  • Checkpoint Kinase (CHK) inhibitors include but are not limited to, 7-Hydroxystaurosporine (UCN- 01); 6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(3R)-3-piperidinylpyrazolo[1,5-a]pyrimidin-7-amine (SCH900776, CAS 891494-63-6); 5-(3-Fluorophenyl)-3-ureidothiophene-2-carboxylic acid N- [(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4-[((3S)-1-Azabicyclo[2.2.2]oct-3- yl)amino]-3-(1H-benzimidazol-2-yl)-6-chloroquinolin-2(1H)-one (CHIR 124, CAS 405168-58-3); 7-Aminodactinomycin (7-AAD), I
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more immunomodulators (e.g., one or more of an activator of a costimulatory molecule or an inhibitor of an immune checkpoint molecule), for treating a disease, e.g., cancer.
  • the immunomodulator is an activator of a costimulatory molecule.
  • the agonist of the costimulatory molecule is selected from an agonist (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion) of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand.
  • the immunomodulator is an inhibitor of an immune checkpoint molecule.
  • the immunomodulator is an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFRbeta.
  • the inhibitor of an immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3 or CTLA4, or any combination thereof.
  • the term “inhibition” or “inhibitor” includes a reduction in a certain parameter, e.g., an activity, of a given molecule, e.g., an immune checkpoint inhibitor.
  • inhibition of an activity e.g., a PD-1 or PD-L1 activity
  • inhibition need not be 100%.
  • Inhibition of an inhibitory molecule can be performed at the DNA, RNA or protein level.
  • an inhibitory nucleic acid e.g., a dsRNA, siRNA or shRNA
  • the inhibitor of an inhibitory signal is a polypeptide e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof, that binds to the inhibitory molecule; e.g., an antibody or fragment thereof (also referred to herein as “an antibody molecule”) that binds to PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR beta, or a combination thereof.
  • a polypeptide e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof, that binds to the inhibitory molecule; e.g., an antibody or fragment thereof (also referred to herein as “an antibody molecule”) that binds to PD-1, PD
  • the antibody molecule is a full antibody or fragment thereof (e.g., a Fab, F(ab')2, Fv, or a single chain Fv fragment (scFv)).
  • the antibody molecule has a heavy chain constant region (Fc) selected from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, selected from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, more particularly, the heavy chain constant region of IgG1 or IgG4 (e.g., human IgG1 or IgG4).
  • Fc heavy chain constant region
  • the heavy chain constant region is human IgG1 or human IgG4.
  • the constant region is altered, e.g., mutated, to modify the properties of the antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).
  • the antibody molecule is in the form of a bispecific or multispecific antibody molecule.
  • the bispecific antibody molecule has a first binding specificity to PD-1 or PD-L1 and a second binding specificity, e.g., a second binding specificity to TIM-3, LAG-3, or PD-L2.
  • the bispecific antibody molecule binds to PD-1 or PD-L1 and TIM-3. In another embodiment, the bispecific antibody molecule binds to PD-1 or PD- L1 and LAG-3. In another embodiment, the bispecific antibody molecule binds to PD-1 and PD- L1. In yet another embodiment, the bispecific antibody molecule binds to PD-1 and PD-L2. In another embodiment, the bispecific antibody molecule binds to TIM-3 and LAG-3.
  • any combination of the aforesaid molecules can be made in a multispecific antibody molecule, e.g., a trispecific antibody that includes a first binding specificity to PD-1 or PD-1, and a second and third binding specificities to two or more of TIM-3, LAG-3, or PD-L2.
  • the immunomodulator is an inhibitor of PD-1, e.g., human PD-1.
  • the immunomodulator is an inhibitor of PD-L1, e.g., human PD-L1.
  • the inhibitor of PD-1 or PD-L1 is an antibody molecule to PD-1 or PD-L1.
  • the PD-1 or PD-L1 inhibitor can be administered alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of LAG-3, TIM-3 or CTLA4.
  • the inhibitor of PD-1 or PD-L1, e.g., the anti-PD-1 or PD-L1 antibody molecule is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule.
  • the inhibitor of PD-1 or PD-L1, e.g., the anti-PD-1 or PD-L1 antibody molecule is administered in combination with a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule.
  • the inhibitor of PD-1 or PD-L1, e.g., the anti-PD-1 antibody molecule is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule, and a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule.
  • a LAG-3 inhibitor e.g., an anti-LAG-3 antibody molecule
  • a TIM-3 inhibitor e.g., an anti-TIM-3 antibody molecule.
  • Other combinations of immunomodulators with a PD-1 inhibitor e.g., one or more of PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR
  • Any of the antibody molecules known in the art or disclosed herein can be used in the aforesaid combinations of inhibitors of checkpoint molecule.
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with a PD-1 inhibitor to treat a disease, e.g., cancer.
  • the PD-1 inhibitor is selected from PDR001 (Novartis), Nivolumab (Bristol-Myers Squibb), Pembrolizumab (Merck & Co), Pidilizumab (CureTech), MEDI0680 (Medimmune), Cemiplimab (REGN2810, Regeneron), Dostarlimab (TSR-042, Tesaro), PF-06801591 (Pfizer), Tislelizumab (BGB-A317, Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), Balstilimab (AGEN2035, Agenus), Sintilimab (InnoVent), Toripalimab (Shanghai Junshi
  • the PD- 1 inhibitor is an anti-PD-1 antibody molecule as described in US 2015/0210769, published on July 30, 2015, entitled “Antibody Molecules to PD-1 and Uses Thereof,” incorporated by reference in its entirety.
  • PD-1 inhibitors the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with a PD-1 inhibitor to treat a disease, e.g., cancer.
  • the PD-1 inhibitor is selected from PDR001 (Novartis), Nivolumab (Bristol-Myers Squibb), Pembrolizumab (Merck & Co), Pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune).
  • Exemplary PD-1 Inhibitors In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule.
  • the PD-1 inhibitor is an anti-PD-1 antibody molecule as described in US 2015/0210769, published on July 30, 2015, entitled “Antibody Molecules to PD-1 and Uses Thereof,” incorporated by reference in its entirety.
  • the anti-PD-1 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 3 (e.g., from the heavy and light chain variable region sequences of BAP049-Clone-E or BAP049-Clone-B disclosed in Table 3), or encoded by a nucleotide sequence shown in Table 3.
  • CDRs complementarity determining regions
  • the CDRs are according to the Kabat definition (e.g., as set out in Table 3). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 3). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 3). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 2).
  • one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 1, or encoded by a nucleotide sequence shown in Table 1.
  • the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 12, a VLCDR2 amino acid sequence of SEQ ID NO: 13, and a VLCDR3 amino acid sequence of SEQ ID NO: 88, each disclosed in Table 1.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 26, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 27, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 28; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 32, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 33, each disclosed in Table 1.
  • the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 8. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 22. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 18.
  • the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 8 and a VL comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 8 and a VL comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 9.
  • the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 23 or 19, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 23 or 19.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 9 and a VL encoded by the nucleotide sequence of SEQ ID NO: 23 or 19.
  • the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 10.
  • the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 24. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 20. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 24.
  • the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 11, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 11.
  • the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 25 or 21, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 25 or 21.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 11 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 25 or 21.
  • the antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0210769, incorporated by reference in its entirety. Table 1. Amino acid and nucleotide sequences of exemplary anti-PD-1 antibody molecules
  • the anti-PD-1 antibody is Nivolumab (CAS Registry Number: 946414-94- 4).
  • Alternative names for Nivolumab include MDX-1106, MDX-1106-04, ONO-4538, BMS-936558 or OPDIVO®.
  • Nivolumab is a fully human IgG4 monoclonal antibody, which specifically blocks PD1.
  • Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US Pat No.8,008,449 and PCT Publication No. WO2006/121168, incorporated by reference in their entirety.
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Nivolumab, e.g., as disclosed in Table 2.
  • the anti-PD-1 antibody is Pembrolizumab.
  • Pembrolizumab (Trade name KEYTRUDA formerly Lambrolizumab, also known as Merck 3745, MK-3475 or SCH-900475) is a humanized IgG4 monoclonal antibody that binds to PD1.
  • Pembrolizumab is disclosed, e.g., in Hamid, O. et al.
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Pembrolizumab, e.g., as disclosed in Table 2.
  • the anti-PD-1 antibody is Pidilizumab.
  • Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1.
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Pidilizumab, e.g., as disclosed in Table 2.
  • Other anti-PD1 antibodies are disclosed in US Patent No. 8,609,089, US Publication No. 2010028330, and/or US Publication No.20120114649, incorporated by reference in their entirety.
  • Other anti-PD1 antibodies include AMP 514 (Amplimmune).
  • the anti-PD-1 antibody molecule is MEDI0680 (Medimmune), also known as AMP-514. MEDI0680 and other anti-PD-1 antibodies are disclosed in US 9,205,148 and WO 2012/145493, incorporated by reference in their entirety.
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of MEDI0680.
  • the anti-PD-1 antibody molecule is REGN2810 (Regeneron).
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of REGN2810. In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (Pfizer). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of PF-06801591. In one embodiment, the anti-PD-1 antibody molecule is BGB-A317 or BGB-108 (Beigene).
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BGB-A317 or BGB-108.
  • the anti-PD-1 antibody molecule is INCSHR1210 (Incyte), also known as INCSHR01210 or SHR-1210.
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of INCSHR1210.
  • the anti-PD-1 antibody molecule is TSR-042 (Tesaro), also known as ANB011.
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-042.
  • Further known anti-PD-1 antibodies include those described, e.g., in WO 2015/112800, WO 2016/092419, WO 2015/085847, WO 2014/179664, WO 2014/194302, WO 2014/209804, WO 2015/200119, US 8,735,553, US 7,488,802, US 8,927,697, US 8,993,731, and US 9,102,727, incorporated by reference in their entirety.
  • the anti-PD-1 antibody is an antibody that competes for binding with, and/or binds to the same epitope on PD-1 as, one of the anti-PD-1 antibodies described herein.
  • the PD-1 inhibitor is a peptide that inhibits the PD-1 signalling pathway, e.g., as described in US 8,907,053, incorporated by reference in its entirety.
  • the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD-1 inhibitor is AMP-224 (B7-DCIg (Amplimmune), e.g., disclosed in WO 2010/027827 and WO 2011/066342, incorporated by reference in their entirety).
  • Table 2 Amino acid sequences of other exemplary anti-PD-1 antibody molecules
  • the anti-PD-1 antibody is Tislelizumab.Tislelizumab can have a heavy chain of SEQ ID NO: 43 and a light chain of SEQ ID NO: 44.
  • the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Tislelizumab, e.g., as disclosed in Table 3.
  • the anti-PD-1 antibody is dosed at 100 mg per week. In some embodiments, tislelizumab and is dosed at 300 mg IV on day 1 of each 28 day cycle.
  • tislelizumab can be dosed at 500 mg once every four (4) weeks.
  • the anti-PD-1 antibody molecule e.g., tislelizumab, and comprises a heavy chain and/or light chain, VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following: TABLE 3: Amino acid sequences of other exemplary anti-PD-1 antibody molecules
  • the PD-1 inhibitor comprises the HCDRs and LCDRs of tislelizumab as set forth in SEQ ID NOs: 47-52.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor is administered at a flat dose of between about 100 mg to about 600 mg.
  • the PD-1 inhibitor is administered at a dose of between about 100 mg to about 500 mg.
  • the PD-1 inhibitor is administered at a dose of between about 100 mg to about 400 mg.
  • the PD-1 inhibitor is administered at a dose of between about 100 mg to about 300 mg.
  • the PD-1 inhibitor is administered at a dose of between about 100 mg to about 200 mg.
  • the PD-1 inhibitor is administered at a dose of between about 200 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200 mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200 mg to about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300 mg to about 400 mg.
  • the PD-1 inhibitor is administered at a dose of between about 400 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 400 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 500 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 600 mg to about 700 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 700 mg to about 800 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 800 mg to about 900 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 900 mg to about 1000 mg.
  • the PD-1 inhibitor (e.g., tislelizumab) is administered at a flat dose of about 100 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 700 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 800 mg.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor is administered at a dose of about 900 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 1000 mg. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered once every ten weeks. In some embodiments, the PD-1 inhibitor is administered once every nine weeks. In some embodiments, the PD-1 inhibitor is administered once every eight weeks. In some embodiments, the PD-1 inhibitor is administered once every seven weeks. In some embodiments, the PD-1 inhibitor is administered once every six weeks. In some embodiments, the PD-1 inhibitor is administered once every five weeks. In some embodiments, the PD-1 inhibitor is administered once every four weeks.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor is administered once every ten weeks. In some embodiments, the PD-1 inhibitor is administered once every nine weeks. In some embodiments, the PD-1 inhibitor is administered once every eight weeks. In some embodiments, the PD-1 inhibitor is
  • the PD-1 inhibitor is administered once every three weeks. In some embodiments, the PD-1 inhibitor is administered once every two weeks. In some embodiments, the PD-1 inhibitor is administered once every week. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered intravenously. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered over a period of about 20 minutes to 40 minutes (e.g., about 30 minutes). In some embodiments, the PD-1 inhibitor is administered over a period of about 30 minutes. In some embodiments, the PD-1 inhibitor is administered over a period of about an hour. In some embodiments, the PD-1 inhibitor is administered over a period of about two hours.
  • the PD-1 inhibitor is administered intravenously. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered over a period of about 20 minutes to 40 minutes (e.g., about 30 minutes). In some embodiments, the
  • the PD-1 inhibitor is administered over a period of about three hours. In some embodiments, the PD-1 inhibitor is administered over a period of about four hours. In some embodiments, the PD-1 inhibitor is administered over a period of about five hours. In some embodiments, the PD-1 inhibitor is administered over a period of about six hours. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered at a dose between about 300 mg to about 500 mg (e.g., about 400 mg), intravenously, once every four weeks. In some embodiments, the PD-1 inhibitor is administered at a dose between about 200 mg to about 400 mg (e.g., about 300 mg), intravenously, once every three weeks.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor is administered at a dose between about 300 mg to about 500 mg (e.g., about 400 mg), intravenously, once every four weeks. In some embodiments, the PD-1 inhibitor is administered at
  • tislelizumab is administered at a dose of 400 mg, once every four weeks. In some embodiments, tislelizumab is administered at a dose of 300 mg, once every three weeks. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered at a dose between about 300 mg to about 500 mg (e.g., about 400 mg), intravenously, over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes), once every two weeks.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor is administered at a dose between about 200 mg to about 400 mg (e.g., about 300 mg), intravenously, over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes), once every three weeks.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor is administered at a dose of about 100 mg per week. For example, if a 10-week dose is given to a patient, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 1000 mg. If a 9-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 900 mg.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor can be given at 800 mg.
  • a 7-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 700 mg.
  • a 6-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 600 mg.
  • a 5-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 500 mg.
  • a 4-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 400 mg.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor can be given at 300 mg.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor can be given at 200 mg.
  • the PD-1 inhibitor e.g., tislelizumab
  • the PD-1 inhibitor can be given at 100 mg.
  • an anti-PD-1 antibody such as tislelizumab
  • it can be administered at a dose of 200 mg as an intravenous infusion, once every three week.
  • tislelizumab can be administered at a dose of 300 mg as an intravenous infusion, once every four weeks. If an anti-PD-1 antibody, such as tislelizumab is used, it can be administered at a dose of 300 mg as an intravenous infusion, once every three week. Alternatively, tislelizumab can be administered at a dose of 400 mg as an intravenous infusion, once every four weeks.
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, of the present disclosure are used in combination with a PD-L1 inhibitor for treating a disease, e.g., cancer.
  • the PD-L1 inhibitor is selected from FAZ053 (Novartis), Atezolizumab (Genentech/Roche), Avelumab (Merck Serono and Pfizer), Durvalumab (MedImmune/AstraZeneca), or BMS-936559 (Bristol-Myers Squibb).
  • the PD-L1 inhibitor is an anti-PD-L1 antibody molecule.
  • the PD-L1 inhibitor is an anti-PD-L1 antibody molecule as disclosed in US 2016/0108123, published on April 21, 2016, entitled “Antibody Molecules to PD-L1 and Uses Thereof,” incorporated by reference in its entirety.
  • the anti-PD-L1 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 5 (e.g., from the heavy and light chain variable region sequences of BAP058-Clone O or BAP058-Clone N disclosed in Table 5), or encoded by a nucleotide sequence shown in Table 5.
  • the CDRs are according to the Kabat definition (e.g., as set out in Table 5).
  • the CDRs are according to the Chothia definition (e.g., as set out in Table 5).
  • the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 5).
  • the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTSYWMY (SEQ ID NO: 53).
  • one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 4, or encoded by a nucleotide sequence shown in Table 4.
  • the anti-PD-L1 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 54, a VHCDR2 amino acid sequence of SEQ ID NO: 55, and a VHCDR3 amino acid sequence of SEQ ID NO: 56; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 63, a VLCDR2 amino acid sequence of SEQ ID NO: 64, and a VLCDR3 amino acid sequence of SEQ ID NO: 65, each disclosed in Table 4.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-L1 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 81, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 82, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 83; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 86, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 87, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 88, each disclosed in Table 4.
  • the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 59. In one embodiment, the anti-PD-L1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 69. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 73.
  • the anti-PD-L1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 77.
  • the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 69.
  • the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 77.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 60, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 60. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 78, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 78.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 74, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 74. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 78, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 78. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 60 and a VL encoded by the nucleotide sequence of SEQ ID NO: 78.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 74 and a VL encoded by the nucleotide sequence of SEQ ID NO: 78.
  • the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 61.
  • the anti-PD-L1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 70.
  • the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 75. In one embodiment, the anti-PD-L1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 79. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 70.
  • the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a light chain comprising the amino acid sequence of SEQ ID NO: 79.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 62, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 62.
  • the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 72, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 72.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 76, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 76. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 80, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 80. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 62 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 72.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 76 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 80.
  • the antibody molecules described herein can be made by vectors, host cells, and methods described in US 2016/0108123, incorporated by reference in its entirety. Table 4. Amino acid and nucleotide sequences of exemplary anti-PD-L1 antibody molecules
  • the PD-L1 inhibitor is anti-PD-L1 antibody.
  • the anti-PD-L1 inhibitor is selected from YW243.55.S70, MPDL3280A, MEDI-4736, or MDX- 1105MSB-0010718C (also referred to as A09-246-2) disclosed in, e.g., WO 2013/0179174, and having a sequence disclosed herein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified).
  • the PD-L1 inhibitor is MDX-1105.
  • MDX-1105 also known as BMS-936559, is an anti-PD-L1 antibody described in PCT Publication No. WO 2007/005874.
  • the PD-L1 inhibitor is YW243.55.S70.
  • the YW243.55.S70 antibody is an anti-PD-L1 described in PCT Publication No. WO 2010/077634.
  • the PD-L1 inhibitor is MDPL3280A (Genentech / Roche) also known as Atezolizumabm, RG7446, RO5541267, YW243.55.S70, or TECENTRIQTM.
  • MDPL3280A is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1.
  • the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Atezolizumab, e.g., as disclosed in Table 5.
  • the PD-L2 inhibitor is AMP-224.
  • AMP-224 is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1 (B7-DCIg; Amplimmune; e.g., disclosed in PCT Publication Nos. WO2010/027827 and WO2011/066342).
  • the PD-L1 inhibitor is an anti-PD-L1 antibody molecule.
  • the anti-PD-L1 antibody molecule is Avelumab (Merck Serono and Pfizer), also known as MSB0010718C. Avelumab and other anti-PD-L1 antibodies are disclosed in WO 2013/079174, incorporated by reference in its entirety.
  • the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Avelumab, e.g., as disclosed in Table 5.
  • the anti-PD-L1 antibody molecule is Durvalumab (MedImmune/AstraZeneca), also known as MEDI4736. Durvalumab and other anti-PD-L1 antibodies are disclosed in US 8,779,108, incorporated by reference in its entirety.
  • the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Durvalumab, e.g., as disclosed in Table 5.
  • the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers Squibb), also known as MDX-1105 or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2015/081158, incorporated by reference in their entirety.
  • the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BMS-936559, e.g., as disclosed in Table 5.
  • anti-PD-L1 antibodies include those described, e.g., in WO 2015/181342, WO 2014/100079, WO 2016/000619, WO 2014/022758, WO 2014/055897, WO 2015/061668, WO 2013/079174, WO 2012/145493, WO 2015/112805, WO 2015/109124, WO 2015/195163, US 8,168,179, US 8,552,154, US 8,460,927, and US 9,175,082, incorporated by reference in their entirety.
  • the anti-PD-L1 antibody is an antibody that competes for binding with, and/or binds to the same epitope on PD-L1 as, one of the anti-PD-L1 antibodies described herein. Table 5. Amino acid sequences of other exemplary anti-PD-L1 antibody molecules
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, of the present disclosure are used in combination with a LAG-3 inhibitor to treat a disease, e.g., cancer.
  • the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), or TSR- 033 (Tesaro).
  • Exemplary LAG-3 Inhibitors In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule.
  • the LAG-3 inhibitor is an anti-LAG-3 antibody molecule as disclosed in US 2015/0259420, published on September 17, 2015, entitled “Antibody Molecules to LAG-3 and Uses Thereof,” incorporated by reference in its entirety.
  • the anti-LAG-3 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 7 (e.g., from the heavy and light chain variable region sequences of BAP050-Clone I or BAP050-Clone J disclosed in Table 7), or encoded by a nucleotide sequence shown in Table 7.
  • CDRs complementarity determining regions
  • the CDRs are according to the Kabat definition (e.g., as set out in Table 7). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 7). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 7). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GFTLTNYGMN (SEQ ID NO: 100).
  • one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 6, or encoded by a nucleotide sequence shown in Table 6.
  • amino acid substitutions e.g., conservative amino acid substitutions
  • deletions e.g., conservative amino acid substitutions
  • the anti-LAG-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 101, a VHCDR2 amino acid sequence of SEQ ID NO: 102, and a VHCDR3 amino acid sequence of SEQ ID NO: 103; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 112, a VLCDR2 amino acid sequence of SEQ ID NO: 113, and a VLCDR3 amino acid sequence of SEQ ID NO: 114, each disclosed in Table 6.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-LAG-3 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 136 or 144, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 138 or 146, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 140 or 148; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 146 or 154, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 148 or 156, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 150 or 158, each disclosed in Table 7.
  • the anti-LAG-3 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 158 or 144, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 159 or 146, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 160 or 148; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 146 or 154, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 148 or 156, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 150 or 158, each disclosed in Table 6.
  • the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 106. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 118. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 124.
  • the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 130.
  • the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 106 and a VL comprising the amino acid sequence of SEQ ID NO: 118.
  • the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 124 and a VL comprising the amino acid sequence of SEQ ID NO: 130.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 107 or 115, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 107 or 115. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 119 or 127, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 119 or 127.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 125 or 133, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 125 or 133. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 131 or 139, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 131 or 139.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 107 or 115 and a VL encoded by the nucleotide sequence of SEQ ID NO: 119 or 127. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 125 or 133 and a VL encoded by the nucleotide sequence of SEQ ID NO: 131 or 139. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 109, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 109.
  • the anti-LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 121, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 121. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 127, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 127. In one embodiment, the anti-LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 133.
  • the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 109 and a light chain comprising the amino acid sequence of SEQ ID NO: 121. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 127 and a light chain comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 110 or 124, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 110 or 124.
  • the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 122 or 130, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 122 or 130. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 128 or 136, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 128 or 136.
  • the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 134 or 142, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 134 or 142.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 110 or 124 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 122 or 130.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 128 or 136 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 134 or 142.
  • the antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0259420, incorporated by reference in its entirety. Table 6. Amino acid and nucleotide sequences of exemplary anti-LAG-3 antibody molecules
  • the LAG-3 inhibitor is an anti-LAG-3 antibody molecule.
  • the LAG-3 inhibitor is BMS-986016 (Bristol-Myers Squibb), also known as BMS986016.
  • BMS- 986016 and other anti-LAG-3 antibodies are disclosed in WO 2015/116539 and US 9,505,839, incorporated by reference in their entirety.
  • the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BMS-986016, e.g., as disclosed in Table 7.
  • the anti-LAG-3 antibody molecule is TSR-033 (Tesaro). In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-033. In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (GSK and Prima BioMed). IMP731 and other anti-LAG-3 antibodies are disclosed in WO 2008/132601 and US 9,244,059, incorporated by reference in their entirety.
  • the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of IMP731, e.g., as disclosed in Table 8.
  • the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of GSK2831781.
  • the anti-LAG-3 antibody molecule is IMP761 (Prima BioMed).
  • the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of IMP761.
  • Further known anti-LAG-3 antibodies include those described, e.g., in WO 2008/132601, WO 2010/019570, WO 2014/140180, WO 2015/116539, WO 2015/200119, WO 2016/028672, US 9,244,059, US 9,505,839, incorporated by reference in their entirety.
  • the anti-LAG-3 antibody is an antibody that competes for binding with, and/or binds to the same epitope on LAG-3 as, one of the anti-LAG-3 antibodies described herein.
  • the anti-LAG-3 inhibitor is a soluble LAG-3 protein, e.g., IMP321 (Prima BioMed), e.g., as disclosed in WO 2009/044273, incorporated by reference in its entirety. Table 7. Amino acid sequences of other exemplary anti-LAG-3 antibody molecules TIM-3 Inhibitors
  • the inhibitor of an immune checkpoint molecule is an inhibitor of TIM-3.
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, or tautomer thereof, of the present disclosure are used in combination with a TIM-3 inhibitor to treat a disease, e.g., cancer.
  • the TIM-3 inhibitor is MGB453 (Novartis) or TSR- 022 (Tesaro).
  • Exemplary TIM-3 Inhibitors In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule. In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule as disclosed in US 2015/0218274, published on August 6, 2015, entitled “Antibody Molecules to TIM-3 and Uses Thereof,” incorporated by reference in its entirety.
  • the anti-TIM-3 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 9 (e.g., from the heavy and light chain variable region sequences of ABTIM3-hum11 or ABTIM3-hum03 disclosed in Table 8), or encoded by a nucleotide sequence shown in Table 8.
  • the CDRs are according to the Kabat definition (e.g., as set out in Table 9).
  • the CDRs are according to the Chothia definition (e.g., as set out in Table 9).
  • one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 8, or encoded by a nucleotide sequence shown in Table 8.
  • amino acid substitutions e.g., conservative amino acid substitutions
  • deletions e.g., conservative amino acid substitutions
  • the anti-TIM-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 174, a VHCDR2 amino acid sequence of SEQ ID NO: 166, and a VHCDR3 amino acid sequence of SEQ ID NO: 168; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 175, a VLCDR2 amino acid sequence of SEQ ID NO: 176, and a VLCDR3 amino acid sequence of SEQ ID NO: 177, each disclosed in Table 9.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-TIM-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 174, a VHCDR2 amino acid sequence of SEQ ID NO: 185, and a VHCDR3 amino acid sequence of SEQ ID NO: 168; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 175, a VLCDR2 amino acid sequence of SEQ ID NO: 176, and a VLCDR3 amino acid sequence of SEQ ID NO: 177, each disclosed in Table 8.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 171, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 171.
  • the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 181.
  • the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 187, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 187.
  • the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 191, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 191.
  • the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 171 and a VL comprising the amino acid sequence of SEQ ID NO: 181.
  • the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 187 and a VL comprising the amino acid sequence of SEQ ID NO: 191.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 172, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 172. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 182, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 182.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 188, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 188. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 192, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 192. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 172 and a VL encoded by the nucleotide sequence of SEQ ID NO: 182.
  • the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 188 and a VL encoded by the nucleotide sequence of SEQ ID NO: 192.
  • the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 173, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 173.
  • the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 183, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 183.
  • the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 189, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 189. In one embodiment, the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 193. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 173 and a light chain comprising the amino acid sequence of SEQ ID NO: 183.
  • the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 189 and a light chain comprising the amino acid sequence of SEQ ID NO: 193.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 174, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 174.
  • the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 184, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 184.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 190, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 190. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 194, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 194. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 174 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 184.
  • the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 190 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 194.
  • the antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0218274, incorporated by reference in its entirety. Table 8. Amino acid and nucleotide sequences of exemplary anti-TIM-3 antibody molecules
  • the anti-TIM-3 antibody molecule is TSR-022 (AnaptysBio/Tesaro). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-022. In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of APE5137 or APE5121, e.g., as disclosed in Table 9.
  • the anti-TIM-3 antibody molecule is the antibody clone F38-2E2.
  • the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of F38-2E2.
  • Further known anti-TIM-3 antibodies include those described, e.g., in WO 2016/111947, WO 2016/071448, WO 2016/144803, US 8,552,156, US 8,841,418, and US 9,163,087, incorporated by reference in their entirety.
  • the anti-TIM-3 antibody is an antibody that competes for binding with, and/or binds to the same epitope on TIM-3 as, one of the anti-TIM-3 antibodies described herein.
  • Table 9. Amino acid sequences of other exemplary anti-TIM-3 antibody molecules Cytokines
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more cytokines, including but not limited to, interferon, IL-2, IL-15, IL-7, or IL21.
  • compounds of Formula (I), or a pharmaceutically acceptable salt, thereof are administered in combination with an IL-15/IL-15Ra complex.
  • the IL-15/IL-15Ra complex is selected from NIZ985 (Novartis), ATL-803 (Altor) or CYP0150 (Cytune).
  • exemplary IL-15/IL-15Ra complexes the cytokine is IL-15 complexed with a soluble form of IL-15 receptor alpha (IL-15Ra).
  • the IL-15/IL-15Ra complex may comprise IL-15 covalently or noncovalently bound to a soluble form of IL-15Ra.
  • the human IL-15 is noncovalently bonded to a soluble form of IL-15Ra.
  • the human IL-15 of the formulation comprises an amino acid sequence of SEQ ID NO: 199 in Table 10 or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 199
  • the soluble form of human IL-15Ra comprises an amino acid sequence of SEQ ID NO: 200 in Table 10, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 200, as described in WO 2014/066527, incorporated by reference in its entirety.
  • the molecules described herein can be made by vectors, host cells, and methods described in WO 2007084342, incorporated by reference in its entirety. Table 10.
  • the IL-15/IL-15Ra complex is ALT-803, an IL-15/IL-15Ra Fc fusion protein (IL-15N72D:IL-15RaSu/Fc soluble complex). ALT-803 is described in WO 2008/143794, incorporated by reference in its entirety.
  • the IL-15/IL-15Ra Fc fusion protein comprises the sequences as disclosed in Table 11.
  • the IL-15/IL-15Ra complex comprises IL-15 fused to the sushi domain of IL- 15Ra (CYP0150, Cytune).
  • the sushi domain of IL-15Ra refers to a domain beginning at the first cysteine residue after the signal peptide of IL-15Ra, and ending at the fourth cysteine residue after said signal peptide.
  • the complex of IL-15 fused to the sushi domain of IL-15Ra is described in WO 2007/04606 and WO 2012/175222, incorporated by reference in their entirety.
  • the IL-15/IL-15Ra sushi domain fusion comprises the sequences as disclosed in Table 11. Table 11.
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more agonists of toll like receptors (TLRs, e.g., TLR7, TLR8, TLR9) to treat a disease, e.g., cancer.
  • TLRs toll like receptors
  • a compound of the present disclosure can be used in combination with a TLR7 agonist or a TLR7 agonist conjugate.
  • the TLR7 agonist comprises a compound disclosed in International Application Publication No. WO2011/049677, which is hereby incorporated by reference in its entirety.
  • the TLR7 agonist comprises 3-(5-amino-2-(4-(2-(3,3-difluoro-3- phosphonopropoxy)ethoxy)-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid.
  • the TLR7 agonist comprises a compound of formula:
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more angiogenesis inhibitors to treat cancer, e.g., Bevacizumab (Avastin®), axitinib (Inlyta®); Brivanib alaninate (BMS-582664, (S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1- f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); Sorafenib (Nexavar®); Pazopanib (Votrient®); Sunitinib malate (Sutent®); Cediranib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more heat shock protein inhibitors to treat cancer, e.g., Tanespimycin (17-allylamino-17-demethoxygeldanamycin, also known as KOS-953 and 17-AAG, available from SIGMA, and described in US Patent No.
  • HDAC inhibitors include, but not limited to, Voninostat (Zolinza®); Romidepsin (Istodax®); Treichostatin A (TSA); Oxamflatin; Vorinostat (Zolinza®, Suberoylanilide hydroxamic acid); Pyroxamide (syberoyl-3-aminopyridineamide hydroxamic acid); Trapoxin A (RF-1023A); Trapoxin B (RF-10238); Cyclo[( ⁇ S,2S)- ⁇ -amino- ⁇ -oxo-2- oxiraneoctanoyl-O-methyl-D-tyrosyl-L-isoleucyl-L-prolyl] (Cyl-1); Cyclo[( ⁇ S,2S)- ⁇ -amino- ⁇ -ox
  • epigenetic modifiers include but not limited to inhibitors of EZH2 (enhancer of zeste homolog 2), EED (embryonic ectoderm development), or LSD1 (lysine-specific histone demethylase 1A or KDM1A).
  • the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more inhibitors of indoleamine-pyrrole 2,3-dioxygenase (IDO), for example, Indoximod (also known as NLG-8189), ⁇ -Cyclohexyl-5H-imidazo[5,1-a]isoindole-5-ethanol (also known as NLG919), or (4E)-4-[(3- Chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine (also known as INCB024360), to treat cancer.
  • IDO indoleamine-pyrrole 2,3-dioxygenase
  • Chimeric Antigen Receptors The present disclosure provides for the compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in combination with adoptive immunotherapy methods and reagents such as chimeric antigen receptor (CAR) immune effector cells, e.g., T cells and/or NK cells, or chimeric TCR-transduced immune effector cells, e.g., T cells.
  • CAR chimeric antigen receptor
  • T cells and/or NK cells e.g., T cells and/or NK cells
  • chimeric TCR-transduced immune effector cells e.g., T cells.
  • This section describes CAR technology generally that is useful in combination with the Compound of Formula (I), or a pharmaceutically acceptable salt thereof, and describes CAR reagents, e.g., cells and compositions, and methods.
  • aspects of the present disclosure pertain to or include an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor antigen as described herein, a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain described herein) (e.g., an intracellular signaling domain comprising a costimulatory domain (e.g., a costimulatory domain described herein) and/or a primary signaling domain (e.g., a primary signaling domain described herein).
  • an antigen binding domain e.g., antibody or antibody fragment, TCR or TCR fragment
  • TCR or TCR fragment binds to a tumor antigen as described herein
  • a transmembrane domain e.g., a transmembr
  • the present disclosure includes: host cells containing the above nucleic acids and isolated proteins encoded by such nucleic acid molecules.
  • CAR nucleic acid constructs, encoded proteins, containing vectors, host cells, pharmaceutical compositions, methods of making, and methods of administration and treatment related to the present disclosure are disclosed in detail in International Patent Application Publication Nos., WO2020/047452, WO2019/241426, WO2016/164731, WO2021/108613, and WO2020/176397which are incorporated by reference in its entirety.
  • the disclosure pertains to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain described herein) (e.g., an intracellular signaling domain comprising a costimulatory domain (e.g., a costimulatory domain described herein) and/or a primary signaling domain (e.g., a primary signaling domain described herein).
  • an antigen binding domain e.g., antibody or antibody fragment, TCR or TCR fragment
  • a tumor-supporting antigen e.g., a tumor-supporting antigen as described herein
  • the tumor-supporting antigen is an antigen present on a stromal cell or a myeloid-derived suppressor cell (MDSC).
  • the disclosure features polypeptides encoded by such nucleic acids and host cells containing such nucleic acids and/or polypeptides.
  • aspects of the disclosure pertain to isolated nucleic acid encoding a chimeric T cell receptor (TCR) comprising a TCR alpha and/or TCR beta variable domain with specificity for a cancer antigen described herein. See for example, Dembic et al., Nature, 320, 232-238 (1986), Schumacher, Nat. Rev. Immunol., 2, 512-519 (2002), Kershaw et al., Nat. Rev.
  • Such chimeric TCRs may recognize, for example, cancer antigens such as MART-1, gp-100, p53, and NY-ESO-1, MAGE A3/A6, MAGEA3, SSX2, HPV-16 E6 or HPV-16 E7.
  • cancer antigens such as MART-1, gp-100, p53, and NY-ESO-1, MAGE A3/A6, MAGEA3, SSX2, HPV-16 E6 or HPV-16 E7.
  • the disclosure features polypeptides encoded by such nucleic acids and host cells containing such nucleic acids and/or polypeptides.
  • the present disclosure provides cells, e.g., immune effector cells (e.g., T cells, NK cells), that are engineered to contain one or more CARs that direct the immune effector cells to undesired cells (e.g., cancer cells). This is achieved through an antigen binding domain on the CAR that is specific for a cancer associated antigen.
  • cancer associated antigens tumor antigens
  • MHC major histocompatibility complex
  • the tumor antigen is chosen from one or more of: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1- 4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAc ⁇ -Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase- like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycose-like
  • a CAR described herein can comprise an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor- supporting antigen as described herein).
  • the tumor-supporting antigen is an antigen present on a stromal cell or a myeloid-derived suppressor cell (MDSC).
  • Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation.
  • the CAR-expressing cells destroy the tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival.
  • the stromal cell antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin.
  • BST2 bone marrow stromal cell antigen 2
  • FAP fibroblast activation protein
  • tenascin tenascin.
  • the FAP-specific antibody is, competes for binding with, or has the same CDRs as, sibrotuzumab.
  • the MDSC antigen is chosen from one or more of: CD33, CD11b, C14, CD15, and CD66b.
  • the tumor-supporting antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CD11b, C14, CD15, and CD66b.
  • BST2 bone marrow stromal cell antigen 2
  • FAP fibroblast activation protein
  • tenascin CD33, CD11b, C14, CD15, and CD66b.
  • the antigen binding domain of the encoded CAR molecule comprises an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab’)2, a single domain antibody (SDAB), a VH or VL domain, a camelid VHH domain or a bi-functional (e.g.
  • scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers.
  • the scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition.
  • the linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site.
  • linker orientation and size see, e.g., Hollinger et al.1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. WO2006/020258 and WO2007/024715, is incorporated herein by reference.
  • An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions.
  • the linker sequence may comprise any naturally occurring amino acid.
  • the linker sequence comprises amino acids glycine and serine.
  • the linker sequence comprises sets of glycine and serine repeats such as (Gly 4 Ser)n, where n is a positive integer equal to or greater than 1 ( SEQ ID NO: 205).
  • the linker can be (Gly 4 Ser) 4 ( SEQ ID NO: 206) or (Gly 4 Ser) 3 ( SEQ ID NO: 207).
  • the antigen binding domain is a T cell receptor (“TCR”), or a fragment thereof, for example, a single chain TCR (scTCR).
  • TCR T cell receptor
  • scTCR single chain TCR
  • scTCR can be engineered that contains the V ⁇ and V ⁇ genes from a T cell clone linked by a linker (e.g., a flexible peptide).
  • a linker e.g., a flexible peptide.
  • This approach is very useful to cancer associated target that itself is intracellar, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by MHC.
  • the encoded antigen binding domain has a binding affinity KD of 10 -4 M to 10 -8 M.
  • the encoded CAR molecule comprises an antigen binding domain that has a binding affinity KD of 10 -4 M to 10 -8 M, e.g., 10 -5 M to 10 -7 M, e.g., 10 -6 M or 10 -7 M, for the target antigen.
  • the antigen binding domain has a binding affinity that is at least five- fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or 1,000-fold less than a reference antibody, e.g., an antibody described herein.
  • the encoded antigen binding domain has a binding affinity at least 5-fold less than a reference antibody (e.g., an antibody from which the antigen binding domain is derived).
  • such antibody fragments are functional in that they provide a biological response that can include, but is not limited to, activation of an immune response, inhibition of signal-transduction origination from its target antigen, inhibition of kinase activity, and the like, as will be understood by a skilled artisan.
  • the antigen binding domain of the CAR is a scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived.
  • the antigen binding domain of a CAR of the disclosure e.g., a scFv
  • entire CAR construct of the disclosure is encoded by a nucleic acid molecule whose entire sequence has been codon optimized for expression in a mammalian cell.
  • Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences.
  • a variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least US Patent Numbers 5,786,464 and 6,114,148.
  • an antigen binding domain against CD19 is an antigen binding portion, e.g., CDRs, of a CAR, antibody or antigen-binding fragment thereof described in, e.g., PCT publication WO2012/079000; PCT publication WO2014/153270; Kochenderfer, J.N. et al., J. Immunother.32 (7), 689-702 (2009); Kochenderfer, J.N., et al., Blood, 116 (20), 4099-4102 (2010); PCT publication WO2014/031687; Bejcek, Cancer Research, 55, 2346-2351, 1995; or U.S. Patent No. 7,446,190.
  • CDRs antigen binding portion
  • an antigen binding domain against mesothelin is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2015/090230.
  • an antigen binding domain against mesothelin is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in, e.g., PCT publication WO1997/025068, WO1999/028471, WO2005/014652, WO2006/099141, WO2009/045957, WO2009/068204, WO2013/142034, WO2013/040557, or WO2013/063419.
  • an antigen binding domain against mesothelin is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO/2015/090230.
  • an antigen binding domain against CD123 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2014/130635.
  • an antigen binding domain against CD123 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in, e.g., PCT publication WO2014/138805, WO2014/138819, WO2013/173820, WO2014/144622, WO2001/66139, WO2010/126066, WO2014/144622, or US2009/0252742.
  • an antigen binding domain against CD123 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO/2017/028896.
  • an antigen binding domain against EGFRvIII is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment or CAR described in, e.g., WO/2014/130657.
  • an antigen binding domain against CD22 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2016/164731; Haso et al., Blood, 121(7): 1165-1174 (2013); Wayne et al., Clin Cancer Res 16(6): 1894-1903 (2010); Kato et al., Leuk Res 37(1):83- 88 (2013); Creative BioMart (creativebiomart.net): MOM-18047-S(P).
  • an antigen binding domain against CS-1 is an antigen binding portion, e.g., CDRs, of Elotuzumab (BMS), see e.g., Tai et al., 2008, Blood 112(4):1329-37; Tai et al., 2007, Blood.110(5):1656-63.
  • an antigen binding domain against CLL-1 is an antigen binding portion, e.g., CDRs, of an antibody available from R&D, ebiosciences, Abcam, for example, PE-CLL1-hu Cat# 353604 (BioLegend); and PE-CLL1 (CLEC12A) Cat# 562566 (BD).
  • an antigen binding domain against CLL-1 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO/2017/014535.
  • an antigen binding domain against CD33 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Bross et al., Clin Cancer Res 7(6):1490-1496 (2001) (Gemtuzumab Ozogamicin, hP67.6),Caron et al., Cancer Res 52(24):6761-6767 (1992) (Lintuzumab, HuM195), Lapusan et al., Invest New Drugs 30(3):1121-1131 (2012) (AVE9633), Aigner et al., Leukemia 27(5): 1107-1115 (2013) (AMG330, CD33 BiTE), Dutour et al., Adv hematol 2012:683065 (2012), and
  • an antigen binding domain against CD33 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO/2017/014576.
  • an antigen binding domain against GD2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Mujoo et al., Cancer Res.
  • an antigen binding domain against GD2 is an antigen binding portion of an antibody selected from mAb 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see e.g., WO2012033885, WO2013040371, WO2013192294, WO2013061273, WO2013123061, WO2013074916, and WO201385552.
  • an antigen binding domain against GD2 is an antigen binding portion of an antibody described in US Publication No.: 20100150910 or PCT Publication No.: WO 2011160119.
  • an antigen binding domain against BCMA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2012163805, WO200112812, WO2003062401, WO/2017/014565, and WO2019/241426.
  • an antigen binding domain against BCMA is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO2019/241426.
  • an antigen binding domain against Tn antigen is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US8,440,798, Brooks et al., PNAS 107(22):10056- 10061 (2010), and Stone et al., OncoImmunology 1(6):863-873(2012).
  • an antigen binding portion e.g., CDRs
  • an antigen binding domain against PSMA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Parker et al., Protein Expr Purif 89(2):136-145 (2013), US 20110268656 (J591 ScFv); Frigerio et al, European J Cancer 49(9):2223-2232 (2013) (scFvD2B); WO 2006125481 (mAbs 3/A12, 3/E7 and 3/F11) and single chain antibody fragments (scFv A5 and D7).
  • CDRs antigen binding portion
  • an antigen binding domain against ROR1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hudecek et al., Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847; and US20130101607.
  • an antigen binding domain against FLT3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2011076922, US5777084, EP0754230, US20090297529, and several commercial catalog antibodies (R&D, ebiosciences, Abcam).
  • an antigen binding domain against TAG72 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hombach et al., Gastroenterology 113(4):1163-1170 (1997); and Abcam ab691.
  • an antigen binding domain against FAP is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Ostermann et al., Clinical Cancer Research 14:4584- 4592 (2008) (FAP5), US Pat.
  • an antigen binding domain against CD38 is an antigen binding portion, e.g., CDRs, of daratumumab (see, e.g., Groen et al., Blood 116(21):1261-1262 (2010); MOR202 (see, e.g., US8,263,746); or antibodies described in US8,362,211.
  • an antigen binding domain against CD44v6 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Casucci et al., Blood 122(20):3461-3472 (2013).
  • an antigen binding domain against CEA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Chmielewski et al., Gastoenterology 143(4):1095-1107 (2012).
  • an antigen binding domain against EPCAM is an antigen binding portion, e.g., CDRS, of an antibody selected from MT110, EpCAM-CD3 bispecific Ab (see, e.g., clinicaltrials.gov/ct2/show/NCT00635596); Edrecolomab; 3622W94; ING-1; and adecatumumab (MT201).
  • an antigen binding domain against PRSS21 is an antigen binding portion, e.g., CDRs, of an antibody described in US Patent No.: 8,080,650.
  • an antigen binding domain against B7H3 is an antigen binding portion, e.g., CDRs, of an antibody MGA271 (Macrogenics).
  • an antigen binding domain against KIT is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7915391, US 20120288506, and several commercial catalog antibodies.
  • an antigen binding domain against IL-13Ra2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2008/146911, WO2004087758, several commercial catalog antibodies, and WO2004087758.
  • an antigen binding domain against CD30 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7090843 B1, and EP0805871.
  • an antigen binding domain against GD3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7253263; US 8,207,308; US 20120276046; EP1013761; WO2005035577; and US6437098.
  • an antigen binding domain against CD171 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hong et al., J Immunother 37(2):93-104 (2014).
  • an antigen binding domain against IL-11Ra is an antigen binding portion, e.g., CDRs, of an antibody available from Abcam (cat# ab55262) or Novus Biologicals (cat# EPR5446).
  • an antigen binding domain again IL-11Ra is a peptide, see, e.g., Huang et al., Cancer Res 72(1):271-281 (2012).
  • an antigen binding domain against PSCA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Morgenroth et al., Prostate 67(10):1121-1131 (2007) (scFv 7F5); Nejatollahi et al., J of Oncology 2013(2013), article ID 839831 (scFv C5-II); and US Pat Publication No.20090311181.
  • CDRs antigen binding portion
  • an antigen binding domain against VEGFR2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Chinnasamy et al., J Clin Invest 120(11):3953-3968 (2010).
  • an antigen binding domain against LewisY is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Kelly et al., Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab (scFvs)); Dolezal et al., Protein Engineering 16(1):47-56 (2003) (NC10 scFv).
  • an antigen binding domain against CD24 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Maliar et al., Gastroenterology 143(5):1375-1384 (2012).
  • an antigen binding domain against PDGFR-beta is an antigen binding portion, e.g., CDRs, of an antibody Abcam ab32570.
  • an antigen binding domain against SSEA-4 is an antigen binding portion, e.g., CDRs, of antibody MC813 (Cell Signaling), or other commercially available antibodies.
  • an antigen binding domain against CD20 is an antigen binding portion, e.g., CDRs, of the antibody Rituximab, Ofatumumab, Ocrelizumab, Veltuzumab, or GA101; or antibodies described in WO2016/164731.
  • an antigen binding domain against Folate receptor alpha is an antigen binding portion, e.g., CDRs, of the antibody IMGN853, or an antibody described in US20120009181; US4851332, LK26: US5952484.
  • an antigen binding domain against ERBB2 (Her2/neu) is an antigen binding portion, e.g., CDRs, of the antibody trastuzumab, or pertuzumab.
  • an antigen binding domain against MUC1 is an antigen binding portion, e.g., CDRs, of the antibody SAR566658.
  • the antigen binding domain against EGFR is antigen binding portion, e.g., CDRs, of the antibody cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.
  • an antigen binding domain against NCAM is an antigen binding portion, e.g., CDRs, of the antibody clone 2-2B: MAB5324 (EMD Millipore).
  • an antigen binding domain against Ephrin B2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Abengozar et al., Blood 119(19):4565-4576 (2012).
  • an antigen binding domain against IGF-I receptor is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US8344112 B2; EP2322550 A1; WO 2006/138315, or PCT/US2006/022995.
  • an antigen binding domain against CAIX is an antigen binding portion, e.g., CDRs, of the antibody clone 303123 (R&D Systems).
  • an antigen binding domain against LMP2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7,410,640, or US20050129701.
  • an antigen binding domain against gp100 is an antigen binding portion, e.g., CDRs, of the antibody HMB45, NKIbetaB, or an antibody described in WO2013165940, or US20130295007
  • an antigen binding domain against tyrosinase is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US5843674; or US19950504048.
  • an antigen binding domain against EphA2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Yu et al., Mol Ther 22(1):102-111 (2014).
  • an antigen binding domain against GD3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7253263; US 8,207,308; US 20120276046; EP1013761 A3; 20120276046; WO2005035577; or US6437098.
  • an antigen binding domain against fucosyl GM1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US20100297138; or WO2007/067992.
  • an antigen binding domain against sLe is an antigen binding portion, e.g., CDRs, of the antibody G193 (for lewis Y), see Scott AM et al, Cancer Res 60: 3254-61 (2000), also as described in Neeson et al, J Immunol May 2013190 (Meeting Abstract Supplement) 177.10.
  • an antigen binding domain against GM3 is an antigen binding portion, e.g., CDRs, of the antibody CA 2523449 (mAb 14F7).
  • an antigen binding domain against HMWMAA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Kmiecik et al., Oncoimmunology 3(1):e27185 (2014) (PMID: 24575382) (mAb9.2.27); US6528481; WO2010033866; or US 20140004124.
  • an antigen binding domain against o-acetyl-GD2 is an antigen binding portion, e.g., CDRs, of the antibody 8B6.
  • an antigen binding domain against TEM1/CD248 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Marty et al., Cancer Lett 235(2):298-308 (2006); Zhao et al., J Immunol Methods 363(2):221-232 (2011).
  • an antigen binding domain against CLDN6 is an antigen binding portion, e.g., CDRs, of the antibody IMAB027 (Ganymed Pharmaceuticals), see e.g., clinicaltrial.gov/show/NCT02054351.
  • an antigen binding domain against TSHR is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US8,603,466; US8,501,415; or US8,309,693.
  • an antigen binding domain against GPRC5D is an antigen binding portion, e.g., CDRs, of the antibody FAB6300A (R&D Systems); or LS-A4180 (Lifespan Biosciences).
  • an antigen binding domain against CD97 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US6,846,911;de Groot et al., J Immunol 183(6):4127- 4134 (2009); or an antibody from R&D:MAB3734.
  • an antigen binding domain against ALK is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010).
  • an antigen binding domain against polysialic acid is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Nagae et al., J Biol Chem 288(47):33784- 33796 (2013).
  • an antigen binding domain against PLAC1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Ghods et al., Biotechnol Appl Biochem 2013 doi:10.1002/bab.1177.
  • an antigen binding domain against GloboH is an antigen binding portion of the antibody VK9; or an antibody described in, e.g., Kudryashov V et al, Glycoconj J.15(3):243-9 (1998), Lou et al., Proc Natl Acad Sci USA 111(7):2482-2487 (2014) ; MBr1: Bremer E-G et al. J Biol Chem 259:14773–14777 (1984).
  • an antigen binding domain against NY-BR-1 is an antigen binding portion, e.g., CDRs of an antibody described in, e.g., Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007).
  • an antigen binding domain against WT-1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Dao et al., Sci Transl Med 5(176):176ra33 (2013); or WO2012/135854.
  • an antigen binding domain against MAGE-A1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv).
  • an antigen binding domain against sperm protein 17 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Song et al., Target Oncol 2013 Aug 14 (PMID: 23943313); Song et al., Med Oncol 29(4):2923-2931 (2012).
  • an antigen binding domain against Tie 2 is an antigen binding portion, e.g., CDRs, of the antibody AB33 (Cell Signaling Technology).
  • an antigen binding domain against MAD-CT-2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., PMID: 2450952; US7635753.
  • an antigen binding domain against Fos-related antigen 1 is an antigen binding portion, e.g., CDRs, of the antibody 12F9 (Novus Biologicals).
  • an antigen binding domain against MelanA/MART1 is an antigen binding portion, e.g., CDRs, of an antibody described in, EP2514766 A2; or US 7,749,719.
  • an antigen binding domain against sarcoma translocation breakpoints is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Luo et al, EMBO Mol. Med. 4(6):453-461 (2012).
  • an antigen binding domain against TRP-2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Wang et al, J Exp Med.184(6):2207-16 (1996).
  • an antigen binding domain against CYP1B1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Maecker et al, Blood 102 (9): 3287-3294 (2003).
  • an antigen binding domain against RAGE-1 is an antigen binding portion, e.g., CDRs, of the antibody MAB5328 (EMD Millipore).
  • an antigen binding domain against human telomerase reverse transcriptase is an antigen binding portion, e.g., CDRs, of the antibody cat no: LS-B95-100 (Lifespan Biosciences)
  • an antigen binding domain against intestinal carboxyl esterase is an antigen binding portion, e.g., CDRs, of the antibody 4F12: cat no: LS-B6190-50 (Lifespan Biosciences).
  • an antigen binding domain against mut hsp70-2 is an antigen binding portion, e.g., CDRs, of the antibody Lifespan Biosciences: monoclonal: cat no: LS-C133261-100 (Lifespan Biosciences).
  • an antigen binding domain against CD79a is an antigen binding portion, e.g., CDRs, of the antibody Anti-CD79a antibody [HM47/A9] (ab3121), available from Abcam; antibody CD79A Antibody #3351 available from Cell Signalling Technology; or antibody HPA017748 - Anti- CD79A antibody produced in rabbit, available from Sigma Aldrich.
  • an antigen binding portion e.g., CDRs, of the antibody Anti-CD79a antibody [HM47/A9] (ab3121), available from Abcam; antibody CD79A Antibody #3351 available from Cell Signalling Technology; or antibody HPA017748 - Anti- CD79A antibody produced in rabbit, available from Sigma Aldrich.
  • an antigen binding domain against CD79b is an antigen binding portion, e.g., CDRs, of the antibody polatuzumab vedotin, anti-CD79b described in Dornan et al., “Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma” Blood. 2009 Sep 24;114(13):2721-9. doi: 10.1182/blood-2009-02- 205500.
  • an antigen binding portion e.g., CDRs
  • an antigen binding domain against CD72 is an antigen binding portion, e.g., CDRs, of the antibody J3-109 described in Myers, and Uckun, “An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia.” Leuk Lymphoma.
  • an antigen binding domain against LAIR1 is an antigen binding portion, e.g., CDRs, of the antibody ANT-301 LAIR1 antibody, available from ProSpec; or anti-human CD305 (LAIR1) Antibody, available from BioLegend.
  • an antigen binding domain against FCAR is an antigen binding portion, e.g., CDRs, of the antibody CD89/FCARAntibody (Catalog#10414-H08H), available from Sino Biological Inc.
  • an antigen binding domain against LILRA2 is an antigen binding portion, e.g., CDRs, of the antibody LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or Mouse Anti-LILRA2 antibody, Monoclonal (2D7), available from Lifespan Biosciences..
  • an antigen binding domain against CD300LF is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CMRF35-like molecule 1 antibody, Monoclonal[UP-D2], available from BioLegend, or Rat Anti-CMRF35-like molecule 1 antibody, Monoclonal[234903], available from R&D Systems.
  • CDRs antigen binding portion
  • an antigen binding domain against CLEC12A is an antigen binding portion, e.g., CDRs, of the antibody Bispecific T cell Engager (BiTE) scFv-antibody and ADC described in Noordhuis et al., “Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug- Conjugates and Bispecific CLL-1xCD3 BiTE Antibody” 53 rd ASH Annual Meeting and Exposition, December 10-13, 2011, and MCLA-117 (Merus).
  • BiTE Bispecific T cell Engager
  • an antigen binding domain against BST2 is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CD317 antibody, Monoclonal[3H4], available from Antibodies-Online or Mouse Anti-CD317 antibody, Monoclonal [696739], available from R&D Systems.
  • an antigen binding domain against EMR2 is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CD312 antibody, Monoclonal [LS-B8033] available from Lifespan Biosciences, or Mouse Anti-CD312 antibody, Monoclonal [494025] available from R&D Systems.
  • an antigen binding domain against LY75 is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-Lymphocyte antigen 75 antibody, Monoclonal [HD30] available from EMD Millipore or Mouse Anti-Lymphocyte antigen 75 antibody, Monoclonal[A15797] available from Life Technologies.
  • an antigen binding domain against GPC3 is an antigen binding portion, e.g., CDRs, of the antibody hGC33 described in Nakano K, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization.
  • an antigen binding domain against FCRL5 is an antigen binding portion, e.g., CDRs, of the anti-FcRL5 antibody described in Elkins et al., “FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma” Mol Cancer Ther. 2012 Oct;11(10):2222-32.
  • an antigen binding domain against FCRL5 is an antigen binding portion, e.g., CDRs, of the anti-FcRL5 antibody described in, for example, WO2001/038490, WO/2005/117986, WO2006/039238, WO2006/076691, WO2010/114940, WO2010/120561, or WO2014/210064.
  • an antigen binding domain against IGLL1 is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-Immunoglobulin lambda-like polypeptide 1 antibody, Monoclonal [AT1G4] available from Lifespan Biosciences, Mouse Anti-Immunoglobulin lambda- like polypeptide 1 antibody, Monoclonal [HSL11] available from BioLegend.
  • CDRs an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-Immunoglobulin lambda-like polypeptide 1 antibody, Monoclonal [AT1G4] available from Lifespan Biosciences, Mouse Anti-Immunoglobulin lambda- like polypeptide 1 antibody, Monoclonal [HSL11] available from BioLegend.
  • the antigen binding domain comprises one, two three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, from an antibody listed above, and/or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above.
  • the antigen binding domain comprises a heavy chain variable region and/or a variable light chain region of an antibody listed above.
  • the antigen binding domain comprises a humanized antibody or an antibody fragment.
  • a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof.
  • the antigen binding domain is humanized.
  • the antigen-binding domain of a CAR e.g., a CAR expressed by a cell of the disclosure, binds to CD19.
  • CD19 is found on B cells throughout differentiation of the lineage from the pro/pre-B cell stage through the terminally differentiated plasma cell stage.
  • the antigen binding domain is a murine scFv domain that binds to human CD19, e.g., the antigen binding domain of CTL019 (e.g., SEQ ID NO: 208).
  • the antigen binding domain is a humanized antibody or antibody fragment, e.g., scFv domain, derived from the murine CTL019 scFv.
  • the antigen binding domain is a human antibody or antibody fragment that binds to human CD19.
  • Exemplary scFv domains (and their sequences, e.g., CDRs, VL and VH sequences) that bind to CD19 are provided in Table 12.
  • the scFv domain sequences provided in Table 12 include a light chain variable region (VL) and a heavy chain variable region (VH).
  • VL and VH are attached by a linker comprising the sequence GGGGSGGGGSGGGGS ( SEQ ID NO: 5794), e.g., in the following orientation: VL-linker-VH. Table 12. Antigen Binding domains that bind CD19
  • the antigen binding domain comprises an anti-CD19 antibody, or fragment thereof, e.g., an scFv.
  • the antigen binding domain comprises a variable heavy chain and a variable light chain listed in Table 15.
  • the linker sequence joining the variable heavy and variable light chains can be any of the linker sequences described herein, or alternatively, can be GSTSGSGKPGSGEGSTKG ( SEQ ID NO: 229).
  • the light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker- light chain variable region. Table 15.
  • the CD19 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a CD19 binding domain described herein, e.g., provided in Table 12 or 13, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a CD19 binding domain described herein, e.g., provided in Table 12 or 14.
  • LC CDR1 light chain complementary determining region 1
  • HC CDR2 light chain complementary determining region 2
  • HC CDR3 light chain complementary determining region 3
  • the CD19 binding domain comprises one, two, or all of LC CDR1, LC CDR2, and LC CDR3 of any amino acid sequences as provided in Table 14, incorporated herein by reference; and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of any amino acid sequences as provided in Table 13.
  • Any known CD19 CAR e.g., the CD19 antigen binding domain of any known CD19 CAR, in the art can be used in accordance with the instant disclosure to construct a CAR.
  • an antigen binding domain against CD19 is an antigen binding portion, e.g., CDRs, of a CAR, antibody or antigen- binding fragment thereof described in, e.g., PCT publication WO2012/079000; PCT publication WO2014/153270; Kochenderfer, J.N. et al., J. Immunother.32 (7), 689-702 (2009); Kochenderfer, J.N., et al., Blood, 116 (20), 4099-4102 (2010); PCT publication WO2014/031687; Bejcek, Cancer Research, 55, 2346-2351, 1995; or U.S. Patent No.7,446,190.
  • CDRs antigen binding portion, e.g., CDRs, of a CAR, antibody or antigen- binding fragment thereof described in, e.g., PCT publication WO2012/079000; PCT publication WO2014/153270; Kochenderfer, J.N. et al., J. Immunother.32 (7)
  • the antigen-binding domain of CAR binds to BCMA.
  • BCMA is found preferentially expressed in mature B lymphocytes.
  • the antigen binding domain is a murine scFv domain that binds to human BCMA.
  • the antigen binding domain is a humanized antibody or antibody fragment, e.g., scFv domain, that binds human BCMA.
  • the antigen binding domain is a human antibody or antibody fragment that binds to human BCMA.
  • exemplary BCMA CAR constructs are generated using the VH and VL sequences from PCT Publication WO2012/0163805 (the contents of which are hereby incorporated by reference in its entirety).
  • additional exemplary BCMA CAR constructs are generated using the VH and VL sequences from PCT Publication WO2016/014565 (the contents of which are hereby incorporated by reference in its entirety).
  • additional exemplary BCMA CAR constructs are generated using the VH and VL sequences from PCT Publication WO2014/122144 (the contents of which are hereby incorporated by reference in its entirety).
  • additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the VH and VL sequences from PCT Publication WO2016/014789 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the VH and VL sequences from PCT Publication WO2014/089335 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the VH and VL sequences from PCT Publication WO2014/140248 (the contents of which are hereby incorporated by reference in its entirety).
  • a CAR e.g., a CAR expressed by the cell of the disclosure, comprises a CAR molecule comprising an antigen binding domain that binds to a B cell antigen, e.g., as described herein, such as CD19 or BCMA.
  • the CAR comprises a CAR molecule comprising a CD19 antigen binding domain (e.g., a murine, human or humanized antibody or antibody fragment that specifically binds to CD19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain).
  • a CD19 antigen binding domain e.g., a murine, human or humanized antibody or antibody fragment that specifically binds to CD19
  • a transmembrane domain e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain.
  • an intracellular signaling domain e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain.
  • Exemplary CAR molecules described herein are provided in Table 16.
  • the CAR molecules in Table 16 comprise a CD19 antigen binding domain, e.g., an amino
  • a CAR e.g., a CAR expressed by the cell of the disclosure, comprises a CAR molecule comprising an antigen binding domain that binds to BCMA, e.g., comprises a BCMA antigen binding domain (e.g., a murine, human or humanized antibody or antibody fragment that specifically binds to BCMA, e.g., human BCMA), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain).
  • BCMA antigen binding domain e.g., a murine, human or humanized antibody or antibody fragment that specifically binds to BCMA, e.g., human BCMA
  • a transmembrane domain e.g., a transmembrane domain
  • an intracellular signaling domain e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain.
  • a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region).
  • the transmembrane domain is one that is associated with one of the other domains of the CAR e.g., in one embodiment, the transmembrane domain may be from the same protein that the signaling domain, costimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell.
  • the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target.
  • a transmembrane domain of particular use in this disclosure may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD22
  • the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine can be found at each end of a recombinant transmembrane domain.
  • a short oligo- or polypeptide linker between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 250).
  • the linker is encoded by a nucleotide sequence of In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.
  • Signaling domains In embodiments of the disclosure having an intracellular signaling domain, such a domain can contain, e.g., one or more of a primary signaling domain and/or a costimulatory signaling domain.
  • the intracellular signaling domain comprises a sequence encoding a primary signaling domain.
  • the intracellular signaling domain comprises a costimulatory signaling domain.
  • the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain.
  • the intracellular signaling sequences within the cytoplasmic portion of the CAR of the disclosure may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid e.g., an alanine, a glycine, can be used as a suitable linker.
  • the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains.
  • the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein.
  • the intracellular signaling domain comprises two costimulatory signaling domains.
  • the linker molecule is a glycine residue.
  • the linker is an alanine residue.
  • Primary Signaling domains A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM containing primary intracellular signaling domains that are of particular use in the disclosure include those of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.
  • a CAR of the disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta.
  • the encoded intracellular signaling domain comprises a costimulatory signaling domain.
  • the intracellular signaling domain can comprise a primary signaling domain and a costimulatory signaling domain.
  • the encoded costimulatory signaling domain comprises a functional signaling domain of a protein chosen from one or more of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49
  • the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD27.
  • the signaling domain of CD27 comprises an amino acid sequence of Vectors
  • the disclosure pertains to a vector comprising a nucleic acid sequence encoding a CAR described herein.
  • the vector is chosen from a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.
  • the vector is a lentivirus vector.
  • the vectors may be used to deliver nucleic acid directly to the cell, e.g., the immune effector cell, e.g., the T cell, e.g., the allogeneic T cell, independent of the CRISPR system.
  • the present disclosure also provides vectors in which a DNA of the present disclosure is inserted.
  • Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco- retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes.
  • a retroviral vector may also be, e.g., a gammaretroviral vector.
  • a gammaretroviral vector may include, e.g., a promoter, a packaging signal ( ⁇ ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and a transgene of interest, e.g., a gene encoding a CAR.
  • a gammaretroviral vector may lack viral structural gens such as gag, pol, and env.
  • Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom.
  • MMV Murine Leukemia Virus
  • SFFV Spleen-Focus Forming Virus
  • MPSV Myeloproliferative Sarcoma Virus
  • Other gammaretroviral vectors are described, e.g., in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses.2011 Jun; 3(6): 677–713.
  • the vector comprising the nucleic acid encoding the desired CAR of the disclosure is an adenoviral vector (A5/35).
  • nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al.2009Nature Reviews Immunology 9.10: 704-716, is incorporated herein by reference.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • RNA CAR in vitro transcribed RNA CAR
  • the present disclosure also includes a CAR encoding RNA construct that can be directly transfected into a cell.
  • a method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequence (“UTR”), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length.
  • RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the CAR.
  • non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject.
  • the non-viral method includes the use of a transposon (also called a transposable element).
  • a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome.
  • a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition.
  • cells e.g., T or NK cells
  • a nuclease e.g., Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system, or engineered meganuclease re-engineered homing endonucleases.
  • cells of the disclosure e.g., T or NK cells, e.g., allogeneic T cells, e.g., described herein, (e.g., that express a CAR described herein) are generated by contacting the cells with (a) a composition comprising one or more gRNA molecules, e.g., as described herein, and one or more Cas molecules, e.g., a Cas9 molecule, e.g., as described herein, and (b) nucleic acid comprising sequence encoding a CAR, e.g., described herein (such as a template nucleic acid molecule as described herein).
  • a composition comprising one or more gRNA molecules, e.g., as described herein, and one or more Cas molecules, e.g., a Cas9 molecule, e.g., as described herein
  • nucleic acid comprising sequence encoding a CAR, e.g., described herein (such
  • composition of (a), above will induce a break at or near the genomic DNA targeted by the targeting domain of the gRNA molecule(s), and the nucleic acid of (b) will incorporate, e.g., partially or wholly, into the genome at or near said break, such that upon integration, the encoded CAR molecule is expressed.
  • expression of the CAR will be controlled by promoters or other regulatory elements endogenous to the genome (e.g., the promoter controlling expression from the gene in which the nucleic acid of (b) was inserted).
  • the nucleic acid of (b) further comprises a promoter and/or other regulatory elements, e.g., as described herein, e.g., an EF1-alpha promoter, operably linked to the sequence encoding the CAR, such that upon integration, expression of the CAR is controlled by that promoter and/or other regulatory elements.
  • a promoter and/or other regulatory elements e.g., as described herein, e.g., an EF1-alpha promoter
  • Additional features of the disclosure relating to use of CRISPR/Cas9 systems, e.g., as described herein, to direct incorporation of nucleic acid sequence encoding a CAR, e.g., as described herein, are described elsewhere in this application, e.g., in the section relating to gene insertion and homologous recombination.
  • the composition of a) above is a composition comprising RNPs comprising the one or more gRNA molecules.
  • RNPs comprising gRNAs targeting unique target sequences are introduced into the cell simultaneously, e.g., as a mixture of RNPs comprising the one or more gRNAs.
  • RNPs comprising gRNAs targeting unique target sequences are introduced into the cell sequentially.
  • use of a non-viral method of delivery permits reprogramming of cells, e.g., T or NK cells, and direct infusion of the cells into a subject.
  • non-viral vectors include but are not limited to the ease and relatively low cost of producing sufficient amounts required to meet a patient population, stability during storage, and lack of immunogenicity.
  • Promoters In one embodiment, the vector further comprises a promoter.
  • the promoter is chosen from an EF-1 promoter, a CMV IE gene promoter, an EF-1 ⁇ promoter, an ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter.
  • the promoter is an EF-1 promoter.
  • an immune effector cell e.g., a population of cells, e.g., a population of immune effector cells
  • immune effector cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and, optionally, to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions.
  • a semi-automated “flow-through” centrifuge for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL TM gradient or by counterflow centrifugal elutriation.
  • the methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells, that are a T regulatory cell-depleted population, CD25+ depleted cells, using, e.g., a negative selection technique, e.g., described herein.
  • the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.
  • T regulatory cells, e.g., CD25+ T cells are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2.
  • the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead.
  • the anti-CD25 antibody, or fragment thereof is conjugated to a substrate as described herein.
  • the T regulatory cells e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from Miltenyi TM .
  • the ratio of cells to CD25 depletion reagent is 1e7 cells to 20 uL, or 1e7 cells to15 uL, or 1e7 cells to 10 uL, or 1e7 cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL.
  • T regulatory cells e.g., CD25+ depletion
  • greater than 500 million cells/ml is used.
  • a concentration of cells of 600, 700, 800, or 900 million cells/ml is used.
  • the population of immune effector cells to be depleted includes about 6 x 10 9 CD25+ T cells.
  • the population of immune effector cells to be depleted include about 1 x 10 9 to 1x 10 10 CD25+ T cell, and any integer value in between.
  • the resulting population T regulatory depleted cells has 2 x 10 9 T regulatory cells, e.g., CD25+ cells, or less (e.g., 1 x 10 9 , 5 x 10 8 , 1 x 10 8 , 5 x 10 7 , 1 x 10 7 , or less CD25+ cells).
  • the T regulatory cells, e.g., CD25+ cells are removed from the population using the CliniMAC system with a depletion tubing set, such as, e.g., tubing 162-01.
  • the CliniMAC system is run on a depletion setting such as, e.g., DEPLETION2.1.
  • a depletion setting such as, e.g., DEPLETION2.1.
  • decreasing the level of negative regulators of immune cells e.g., decreasing the number of unwanted immune cells, e.g., T REG cells
  • T REG cells e.g., T REG cells
  • methods of depleting T REG cells are known in the art. Methods of decreasing T REG cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25-depletion, and combinations thereof.
  • the manufacturing methods comprise reducing the number of (e.g., depleting) T REG cells prior to manufacturing of the CAR-expressing cell.
  • manufacturing methods comprise contacting the sample, e.g., the apheresis sample, with an anti- GITR antibody and/or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand), e.g., to deplete T REG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product.
  • a subject is pre-treated with one or more therapies that reduce T REG cells prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment.
  • methods of decreasing T REG cells include, but are not limited to, administration to the subject of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof, can occur before, during or after an infusion of the CAR-expressing cell product.
  • a subject is pre-treated with cyclophosphamide prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR- expressing cell treatment.
  • a subject is pre-treated with an anti-GITR antibody prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment.
  • the population of cells to be removed are neither the regulatory T cells or tumor cells, but cells that otherwise negatively affect the expansion and/or function of CART cells, e.g., cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potentially immune suppressive cells.
  • such cells are envisioned to be removed concurrently with regulatory T cells and/or tumor cells, or following said depletion, or in another order.
  • the methods described herein can include more than one selection step, e.g., more than one depletion step.
  • Enrichment of a T cell population by negative selection can be accomplished, e.g., with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail can include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • the methods described herein can further include removing cells from the population which express a tumor antigen, e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 or CD11b, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted, and tumor antigen depleted cells that are suitable for expression of a CAR, e.g., a CAR described herein.
  • a tumor antigen e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 or CD11b
  • tumor antigen expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells.
  • T regulatory e.g., CD25+ cells.
  • an anti-CD25 antibody, or fragment thereof, and an anti-tumor antigen antibody, or fragment thereof can be attached to the same substrate, e.g., bead, which can be used to remove the cells or an anti-CD25 antibody, or fragment thereof, or the anti-tumor antigen antibody, or fragment thereof, can be attached to separate beads, a mixture of which can be used to remove the cells.
  • the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the tumor antigen expressing cells is sequential, and can occur, e.g., in either order.
  • a check point inhibitor e.g., a check point inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells
  • check point inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA and LAIR1.
  • check point inhibitor expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells.
  • an anti-CD25 antibody, or fragment thereof, and an anti-check point inhibitor antibody, or fragment thereof can be attached to the same bead which can be used to remove the cells, or an anti-CD25 antibody, or fragment thereof, and the anti-check point inhibitor antibody, or fragment there, can be attached to separate beads, a mixture of which can be used to remove the cells.
  • the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the check point inhibitor expressing cells is sequential, and can occur, e.g., in either order. Methods described herein can include a positive selection step.
  • T cells can isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer and all integer values there between.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 24 hours, e.g., 24 hours.
  • TIL tumor infiltrating lymphocytes
  • T cell population can be selected that expresses one or more of IFN- ⁇ , TNF ⁇ , IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines.
  • Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO 2013/126712.
  • the concentration of cells and surface can be varied.
  • it may be desirable to significantly decrease the volume in which beads and cells are mixed together e.g., increase the concentration of cells, to ensure maximum contact of cells and beads.
  • a concentration of 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml, or 5 billion/ml is used.
  • a concentration of 1 billion cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used.
  • concentrations of 125 or 150 million cells/ml can be used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
  • use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain.
  • using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations.
  • the concentration of cells used is 5 x 10 6 /ml. In other aspects, the concentration used can be from about 1 x 10 5 /ml to 1 x 10 6 /ml, and any integer value in between.
  • the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 o C or at room temperature.
  • T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.
  • collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed.
  • the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in immune effector cell therapy for any number of diseases or conditions that would benefit from immune effector cell therapy, such as those described herein.
  • a blood sample or an apheresis is taken from a generally healthy subject.
  • a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
  • the T cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.
  • agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3
  • T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • the immune effector cells expressing a CAR molecule e.g., a CAR molecule described herein, are obtained from a subject that has received a low, immune enhancing dose of an mTOR inhibitor.
  • the population of immune effector cells, e.g., T cells, to be engineered to express a CAR are harvested after a sufficient time, or after sufficient dosing of the low, immune enhancing, dose of an mTOR inhibitor, such that the level of PD1 negative immune effector cells, e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g., T cells/ PD1 positive immune effector cells, e.g., T cells, in the subject or harvested from the subject has been, at least transiently, increased.
  • population of immune effector cells e.g., T cells, which have, or will be engineered to express a CAR
  • a T cell population is diaglycerol kinase (DGK)-deficient.
  • DGK-deficient cells include cells that do not express DGK RNA or protein or have reduced or inhibited DGK activity.
  • DGK-deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent DGK expression.
  • RNA-interfering agents e.g., siRNA, shRNA, miRNA
  • DGK- deficient cells can be generated by treatment with DGK inhibitors described herein.
  • a T cell population is Ikaros-deficient.
  • Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity
  • Ikaros-deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression.
  • Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, e.g., lenalidomide.
  • a T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity.
  • DGK and Ikaros- deficient cells can be generated by any of the methods described herein.
  • the NK cells are obtained from the subject.
  • the NK cells are an NK cell line, e.g., NK-92 cell line (Conkwest).
  • the cells of the disclosure are induced pluripotent stem cells (“iPSCs”) or embryonic stem cells (ESCs), or are T cells generated from (e.g., differentiated from) said iPSC and/or ESC.
  • iPSCs can be generated, for example, by methods known in the art, from peripheral blood T lymphocytes, e.g., peripheral blood T lymphocytes isolated from a healthy volunteer.
  • T lymphocytes e.g., peripheral blood T lymphocytes isolated from a healthy volunteer.
  • T lymphocytes e.g., peripheral blood T lymphocytes isolated from a healthy volunteer.
  • T lymphocytes e.g., peripheral blood T lymphocytes isolated from a healthy volunteer.
  • T lymphocytes e.g., peripheral blood T lymphocytes isolated from a healthy volunteer.
  • T lymphocytes e.g., peripheral blood T lymphocytes isolated from a healthy volunteer.
  • T lymphocytes e.g., peripheral blood T lymphocytes isolated from a healthy volunteer.
  • CARTs disclosed herein can be manufactured ex vivo by any known methods in the art. For example, methods described in WO2012/079000, or WO2020/047452 (both incorporated herein by reference). CARTs disclosed herein can also be manufactured in vivo by any known methods in the art. For example, methods described in WO2020/176397 (incorporated herein by reference).
  • An immune effector cell e.g., T cell or NK cell
  • the methods disclosed herein may manufacture immune effector cells engineered to express one or more CARs in less than 24 hours.
  • the methods provided herein preserve the undifferentiated phenotype of T cells, such as naive T cells, during the manufacturing process. These CAR-expressing cells with an undifferentiated phenotype may persist longer and/or expand better in vivo after infusion.
  • CART cells produced by the manufacturing methods provided herein comprise a higher percentage of stem cell memory T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq.
  • CART cells produced by the manufacturing methods provided herein comprise a higher percentage of effector T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq.
  • CART cells produced by the manufacturing methods provided herein better preserve the sternness of T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq.
  • CART cells produced by the manufacturing methods provided herein show a lower level of hypoxia, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq.
  • CART cells produced by the manufacturing methods provided herein show a lower level of autophagy, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq.
  • the immune effector cells are engineered to comprise a nucleic acid molecule encoding one or more CARs disclosed herein.
  • the methods disclosed herein do not involve using a bead, such as Dynabeads® (for example, CD3/CD28 Dynabeads®), and do not involve a de-beading step.
  • the CART cells manufactured by the methods disclosed herein may be administered to a subject with minimal ex vivo expansion, for example, less than 1 day, less than 12 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or no ex vivo expansion. Accordingly, the methods described herein provide a fast manufacturing process of making improved CAR-expressing cell products for use in treating a disease in a subject.
  • the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) comprising: (i) contacting a population of cells (for example, T cells, for example, T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells; (ii) contacting the population of cells (for example, T cells) with a nucleic acid molecule(s) (for example, a DNA or RNA molecule) encoding the CAR(s), thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and (iii) harvesting the population of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration, wherein: (a) step (ii) is performed together with step (i) or no
  • the nucleic acid molecule in step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is on a viral vector, for example, a viral vector chosen from a lentivirus vector, an adenoviral vector, or a retrovirus vector. In some embodiments, the nucleic acid molecule in step (ii) is on a non- viral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not on any vector.
  • step (ii) comprises transducing the population of cells (for example, T cells) a viral vector(s) comprising a nucleic acid molecule encoding the CAR(s).
  • the population of cells for example, T cells
  • the population of cells is collected from an apheresis sample (for example, a leukapheresis sample) from a subject.
  • the apheresis sample for example, a leukapheresis sample
  • T cells for example, CD4+ T cells and/or CD8+ T cells
  • T cells are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device).
  • the selected T cells are then seeded for CART manufacturing using the activation process described herein.
  • the selected T cells undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.
  • the apheresis sample (for example, a leukapheresis sample) is collected from the subject and shipped as a fresh product (for example, a product that is not frozen) to a cell manufacturing facility.
  • T cells for example, CD4+ T cells and/or CD 8+ T cells
  • the selected T cells are then seeded for CART manufacturing using the activation process described herein.
  • the selected T cells undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.
  • the apheresis sample (for example, a leukapheresis sample) is collected from the subject.
  • T cells (for example, CD4+ T cells and/or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device).
  • the selected T cells are then shipped as a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility.
  • the selected T cells (for example, CD4+ T cells and/or CD8+ T cells) are later thawed and seeded for CART manufacturing using the activation process described herein.
  • cells for example, T cells
  • the cells are washed and formulated for storage or administration.
  • brief CD3 and CD28 stimulation may promote efficient transduction of self-renewing T cells.
  • the activation process provided herein does not involve prolonged ex vivo expansion. Similar to the cytokine process, the activation process provided herein also preserves undifferentiated T cells during CART manufacturing.
  • the population of cells is contacted with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells.
  • the agent that stimulates a CD3/TCR complex is an agent that stimulates CD3.
  • the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof.
  • the agent that stimulates a costimulatory molecule is an agent that stimulates CD28.
  • the agent that stimulates a CD3/TCR complex is chosen from an antibody (for example, a single-domain antibody (for example, a heavy chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (for example, a naturally-existing, recombinant, or chimeric ligand).
  • the agent that stimulates a CD3/TCR complex is an antibody.
  • the agent that stimulates a CD3/TCR complex is an anti-CD3 antibody.
  • the agent that stimulates a costimulatory molecule is chosen from an antibody (for example, a single-domain antibody (for example, a heavy chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (for example, a naturally-existing, recombinant, or chimeric ligand).
  • the agent that stimulates a costimulatory molecule is an antibody.
  • the agent that stimulates a costimulatory molecule is an anti-CD28 antibody.
  • the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead.
  • the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix.
  • the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix.
  • the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransActTM.
  • the matrix comprises or consists of a polymeric, for example, biodegradable or biocompatible inert material, for example, which is non-toxic to cells.
  • the matrix is composed of hydrophilic polymer chains, which obtain maximal mobility in aqueous solution due to hydration of the chains.
  • the mobile matrix may be of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions.
  • a polysaccharide may include for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectins, xanthan, guar gum or alginate.
  • the mobile matrix is a polymer of dextran.
  • the population of cells is contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs).
  • the population of cells is transduced with a DNA molecule (e.g., one or more DNA molecules) encoding a CAR (e.g., one or more CARs).
  • a DNA molecule e.g., one or more DNA molecules
  • a CAR e.g., one or more CARs
  • each of the vectors containing nucleic acid molecules encoding the CAR can be added to the reaction mixture (e.g., containing a cell population) at a different multiplicity of infection (MOI).
  • MOI multiplicity of infection
  • MOIs for the vectors containing nucleic acid molecules which encode distinct CAR molecules may affect the final composition of the cellular population.
  • different MOIs can be used to maximize the percent of preferred mono CART cells and dual CART cells, while resulting in fewer undesired mono CART cells and untransduced cells.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs simultaneously with contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 20 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 19 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 18 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 17 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 16 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 15 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 14 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 14 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 13 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 12 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 11 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 10 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 9 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 8 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 7 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 6 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 5 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 4 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 3 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 2 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 1 hour after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 30 minutes after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration.
  • the population of cells is harvested for storage or administration no later than 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 26 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • the population of cells is harvested for storage or administration no later than 25 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 24 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • the population of cells is harvested for storage or administration no later than 23 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 22 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is not expanded ex vivo.
  • the population of cells is expanded by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • the population of cells is expanded by no more than 5%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • the population of cells is expanded by no more than 10%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 15%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • the population of cells is expanded by no more than 20%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 25%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • the population of cells is expanded by no more than 30%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 35%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • the population of cells is expanded by no more than 40%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • the population of cells is expanded by no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above.
  • the activation process is conducted in serum free cell media.
  • the activation process is conducted in cell media comprising one or more cytokines chosen from: IL-2, IL-15 (for example, hetIL-15 (IL15/sIL-15Ra)), or IL-6 (for example, IL-6/sIL- 6Ra).
  • cytokines chosen from: IL-2, IL-15 (for example, hetIL-15 (IL15/sIL-15Ra)), or IL-6 (for example, IL-6/sIL- 6Ra).
  • hetIL-15 comprises the amino acid sequence of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSITCPPPMSVEHADIWVK SYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVT TAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTP SQT TAKNWELTASASHQPPGVYPQG (SEQ ID NO: 254).
  • the activation process is conducted in cell media comprising a LSD1 inhibitor. In some embodiments, the activation process is conducted in cell media comprising a MALT1 inhibitor.
  • the serum free cell media comprises a serum replacement. In some embodiments, the serum replacement is CTSTM Immune Cell Serum Replacement (ICSR).
  • the level of ICSR can be, for example, up to 5%, for example, about 1%, 2%, 3%, 4%, or 5%.
  • using cell media for example, Rapid Media shown in Table 21 or Table 25, comprising ICSR, for example, 2% ICSR, may improve cell viability during a manufacture process described herein.
  • the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) comprising: (a) providing an apheresis sample (for example, a fresh or cryopreserved leukapheresis sample) collected from a subject; (b) selecting T cells from the apheresis sample (for example, using negative selection, positive selection, or selection without beads); (c) seeding isolated T cells at, for example, 1 x 10 6 to 1 x 10 7 cells/mL; (d) contacting T cells with an agent that stimulates T cells, for example, an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells (for example, contacting T cells with anti-CD3 and/or anti- CD28 antibody, for example, contacting T cells with TransAct); (e) contacting T cells with a nucleic acid molecule(s) (for example, a
  • step (f) is performed no later than 30 hours after the beginning of step (d) or (e), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the beginning of step (d) or (e).
  • a population of cells for example, immune effector cells, for example, T cells or NK cells
  • the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) (1) is the same as, (2) differs, for example, by no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, from, or (3) is increased, for example, by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, as compared to, the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein).
  • the population of cells at the end of the manufacturing process shows a higher percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% higher), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).
  • naive T cells for example, CD45RA+ CD45RO- CCR7+ T cells
  • the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) is not less than 20, 25, 30, 35, 40, 45, 50, 55, or 60%.
  • the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) (1) is the same as, (2) differs, for example, by no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% from, or (3) is decreased, for example, by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, as compared to, the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein).
  • the population of cells at the end of the manufacturing process shows a lower percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% lower), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).
  • central memory T cells for example, CD95+ central memory T cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% lower)
  • CD95+ central memory T cells for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% lower
  • the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the end of the manufacturing process is no more than 40, 45, 50, 55, 60, 65, 70, 75, or 80%.
  • the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) after being administered in vivo, persists longer or expands at a higher level (for example, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).
  • a higher level for example, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher
  • the population of cells has been enriched for IL6R-expressing cells (for example, cells that are positive for IL6Ra and/or I L6 Kb) prior to the beginning of the manufacturing process (for example, prior to the beginning of the cytokine process or the activation process described herein).
  • the population of cells comprises, for example, no less than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% of IL6R-expressing cells (for example, cells that are positive for IL6Ra and/or I L6 Kb) at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein).
  • protecting groups for sensitive or reactive groups may be employed where necessary in accordance with general principles of chemistry.
  • Protecting groups are manipulated according to standard methods of organic synthesis (T.W. Green and P.G.M. Wuts (1999) Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons). These groups are removed at a convenient stage of compound synthesis using methods that are readily apparent to those skilled in the art. Temperatures are given in degrees Celsius. As used herein, unless specified otherwise, the term “room temperature” or “ambient temperature” means a temperature of from 15°C to 30°C, such as of from 20°C to 30°C, such as of from 20°C to 25°C.
  • the sample was dissolved in a suitable solvent such as MeCN, DMSO, or MeOH and was injected directly into the column using an automated sample handler.
  • a suitable solvent such as MeCN, DMSO, or MeOH
  • the analysis is performed on Waters Acquity UPLC system (Column: Waters Acquity UPLC BEH C181.7 ⁇ m, 2.1 x 30mm; Flow rate: 1 mL/min; 55°C (column temperature); Solvent A: 0.05% formic acid in water, Solvent B: 0.04% formic acid in MeOH; gradient 95% Solvent A from 0 to 0.10 min; 95% Solvent A to 20% Solvent A from 0.10 to 0.50 min; 20% Solvent A to 5% Solvent A from 0.50 to 0.60 min; hold at 5% Solvent A from 0.6 min to 0.8 min; 5% Solvent A to 95% Solvent A from 0.80 to 0.90 min; and hold 95% Solvent A from 0.90 to 1.15 min.
  • starting materials are either commercially available or are prepared by known methods.
  • Compounds in formula (1-4a – 1-4b) according to the invention can be prepared stepwise starting with the synthesis depicted in scheme 1.
  • Key intermediate 1-2a-b can be prepared via cyclization (step 1.a) of the corresponding 5-amino pyrazole bearing either a 4-cyano (1-2a) or 4- ethyl ester (1-2b) functionality and (E)-3-(dimethylamino)acrylonitrile.
  • step 1.b Bromination of amino- pyrazolopyrimidines (1-2a-b) with N-bromosuccinimide in dichloromethane (step 1.b) yields intermediates 1-3a-b and subsequent –Boc protection (step 1.c) provide the final compounds of formula 1-4a and 1-4b.
  • Compounds in formula (2-3a-b) according to the invention can be prepared stepwise starting with the synthesis depicted in scheme 2.
  • Key intermediate 2-2a-b can be prepared via cyclization of amino-pyrazole 2-1 and 2-bromomalonaldehyde (step 2.a). Coupling of the resulting pyrazolopyrimidines 2-2a-b and 4,4,4,4,5,5,5,5-Octamethyl-2,2-bi-(1,3,2-dioxaborolane using a palladium catalyst and potassium acetate (step 2.b) to provide boronic acids of formula 2-3a – 2- 3b.
  • Compounds in formula (3-3) according to the invention can be prepared stepwise starting with the synthesis depicted in scheme 3.
  • Key intermediate 3-2 can be prepared via bromination of 3-1 with N-bromosuccinimide in acetonitrile (step 3.a). Coupling of the resulting naphthyl compounds 3-2 and 4,4,4,4,5,5,5,5-Octamethyl-2,2-bi-(1,3,2-dioxaborolane) using a palladium catalyst and potassium acetate (step 3.b) to provide boronic acids of formula 3-3.
  • Compounds in formula (III) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 10.
  • Key intermediate 10-1 can be prepared Suzuki reaction (step 10.a) of the corresponding pyrazolopyrimidine bromide 1-4b and appropriate biaryl boronic acid (3-3), which are either commercially available or synthesized as described in schemes 3.
  • the resulting biaryl pyrazolopyrimidines 10-1 were treated with hydrochloric acid to give esters 10-2 that upon hydrolysis with NaOH provide the final compounds of formula III.
  • Compounds in formula (III) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 11.
  • Key intermediate 11-1 can be prepared via cyclization (step 11.a) of 5-amino-1H-pyrazole-4-carbonitrile and the corresponding enol intermediate 8-1, which are synthesized as described in scheme 8, in toluene with pTSA.
  • the resulting esters (11- 1) were hydrolyzed (step 11.b) to provide the final compounds of formula (III). Synthesis of Intermediates Intermediate 1-4a.
  • reaction mixture was cooled at room temperature and quenched with saturated NaHCO 3 solution (100 mL), the solid precipitated, filtered, washed with n-pentane and diethyl ether, dried under vacuum to afford 7- aminopyrazolo[1,5-a]pyrimidine-3-carbonitrile (1-2a) (10.2g, 64.1 mmol, 62.5%) as yellow solid.
  • ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-bromopyrazolo[1,5- a]pyrimidine-3-carboxylate Step 1.a. ethyl 7-aminopyrazolo[1,5-a]pyrimidine-3-carboxylate (1-2b) Mixture of ethyl 5-amino-1H-pyrazole-4-carboxylate (1-1) (10.0g, 65.41 mmol) and (E)-3- (dimethylamino)acrylonitrile (9.2g, 96.77 mmol) taken in acetic acid (70.0 mL) and HCl in Ethanol (70.0 mL) at room temperature.
  • reaction mixture irradiated under microwave at 110°C for 8h.
  • the completion of the reaction was monitored by TLC, using mobile phase 80% EtOAc in hexane. Cooled the reaction mixture to room temperature and evaporated to dryness under vacuum. Diluted with saturated NaHCO 3 solution and extracted the product with ethyl acetate (40 mL x2). The combined organic layer was washed with brine solution and dried (Na 2 SO 4 ) and concentrated under reduced pressure.
  • the progress of the reaction was monitored by TLC, using mobile phase 50% EtOAc in hexane.
  • the reaction mixture was diluted with water (25 mL) and extracted the crude product with DCM.
  • the combined organic layer was washed with brine solution (10mL x 3) and dried with anhy. Na 2 SO 4 and concentrated the organic layer under reduced pressure to afford the crude.
  • the progress of the reaction checked by TLC with mobile phase 30% EtOAc in hexane.
  • the reaction mass was poured into cold ice water (400 mL) and added saturated NaHCO 3 solution (100 mL) to this aqueous portion and extracted the product with EtOAc (800 mL) and washed with 0.5N HCl (100 mL) and organic layer dried over anhy. Na 2 SO 4 and concentrated under vacuum to get the crude.
  • Step 3 1-bromo-6-methoxynaphthalene (3-2h) To a stirred solution of 4-bromo-7-methoxy-1,2-dihydronaphthalene (From step 2, 1.8 g 7.5 mmol) in toluene (18 mL) was added DDQ (1.87 g,8.25 mmol) at room temperature and refluxed the reaction mass at 110°C for 12h. The completion of the reaction was monitored by TLC using mobile phase: 10 % EtOAc in hexane. The reaction mixture was quenched with dil. 10% H 2 SO 4 , water (50 mL) and extracted with dichloromethane (50 mL x 3).
  • the reaction mixture was quenched with water (10.0 mL) and extracted with ethyl acetate (2*20 mL). The organic layer was washed with brine solution, dried over anhy. Na 2 SO 4 and then concentrated under vacuum to get the crude.
  • the crude was purified by combi flash using (4g Silicycle cartridge), eluting the product at neat hexane to afford 1-bromo-2,6- dimethoxynaphthalene (3-2i) (3.10 g, 11.61 mmol, 72.94%) as white colored solid.
  • Step 3.4-bromonaphthalen-2-ol To a stirred solution of 4-bromo-1-(diazen-1-ium-2-yl)naphthalen-2-olate (From step 2, 2.48g, 11.21 mmol) in EtOH (24.8mL) at 0° C, was added sodium borohydride (0.445g, 11.17 mmol) in portion wise at same temperature and the reaction mixture was allowed to stir at 0°C for 30 min. The progress of the reaction was monitored by TLC, using mobile phase: 15 % EtOAc in hexane. The reaction mixture was partitioned between ice cold water and extracted the product with ethyl acetate. The organic layer was washed with brine solution and dried with anhy.
  • Step 4.1-bromo-3-methoxynaphthalene (3.2j) To a stirred solution of 4-bromonaphthalen-2-ol (From step 3, 0.60g, 2.68mmol) in THF (12mL) was added MeI (0.334mL, 5.378mmol). Then added sodium hydride (0.083g, 3.496mmol) in portion wise at 0°C and the reaction mixture was stirred at room temperature for 6h. The progress of the reaction was monitored by TLC, using mobile phase: 20 % EtOAc in hexane. The reaction mixture was portioned between ice cold water and extracted the product with ethyl acetate. The organic layer was washed with brine solution and dried with anhy.
  • 1-bromo-3-methoxynaphthalene (3-2j) (0.62g, 2.95mmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (0.895g, 5.904 mmol) in dioxane (6.20 mL)
  • potassium acetate(0.434g, 4.428mml) was added and purged the reaction mixture with argon for 30 minutes .
  • PdCl 2 (dppf)DCM(0.482g, 0.590mmol) to reaction mixture and purged again with argon for 5 minutes.
  • the reaction mixture was stirred at 100°C for 12h. The progress of the reaction was monitored by TLC, using mobile phase: 10 % EtOAc in hexane. Cooled the reaction mixture to room temperature and was concentrated under reduced pressure to afford the crude.
  • the crude was purified by using (4 g Silicycle cartridge) Buchi, 8-12% EtOAc in hexane as eluent to afford 2-(3-methoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (3-3j) (0.56g, 1.97mmol, 75.04%) as yellow liquid.
  • Step 2.1-bromo-2-methoxy-6-methylnaphthalene (3-2k) To a stirred suspension of 2-methoxy-6-methylnaphthalene (From step 1, 1.00 g, 5.81 mmol) in MeCN (30 mL), was added NBS (1.14 g, 6.39 mmol) and stirred the reaction mixture at room temperature for 2h.
  • 3,4-dihydronaphthalen-1-yl trifluoromethanesulfonate (From step 1, 0.500 g ,1.79 mmol) in Dioxane (5.0 mL) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2- dioxaborolane (0.68 g, 2.69 mmol), potassium acetate (0.266 g, 2.69 mmol) and the reaction mixture was purged with argon for 15 min.
  • N-(4-bromo-2-methylnaphthalen-1-yl)acetamide To a stirred solution of N-(2-methylnaphthalen-1-yl)acetamide (From step 1, 10.8g, 54.27 mmol) in acetic acid (162.0 mL) was added bromine (3.35 mL, 65.12 mmol) at room temperature dropwise with 4h duration. The reaction mixture heated at 55°C for 6h. The completion of the reaction was monitored by TLC, using mobile phase: 40% EtOAc in hexane. Reaction mixture was poured into ice water, the solid precipitated.
  • Step 4.1 bromo-3-methylnaphthalene
  • 4-bromo-2-methylnaphthalen-1-amine from step 3, 9.2g,38.98mmol
  • concentrated HCl 10mL
  • acetic acid 73mL
  • sodium nitrate solution 3.22g, 46.77 mmol
  • water 25.0mL
  • H 3 PO 2 73.0 mL
  • the reaction mixture stirred at room temperature for 12h, followed by 100°C for 1h.
  • the completion of the reaction was monitored by TLC using mobile phase: 10% EtOAc in hexane.
  • the mixture was then heated to 85°C. over 16hrs. Filtered through celite washing with Ethyl Acetate to remove Palladium residues then diluted with water (200 mL). The aqueous was extracted with Ethyl Acetate (x2) and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO 4 ), filtered and evaporated to dryness.
  • the mixture was then heated to 85°C. Overnight. Filtered through celite washing with Ethyl Acetate, to remove Pd residues then diluted with water (200 mL). The aqueous was washed with Ethyl Acetate and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO 4 ), filtered and evaporated to dryness.
  • PdCl 2 (dppf).CH 2 Cl 2 adduct (91 mg, 0.112 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was then heated to 85°C. Overnight. Filtered through celite to remove Pd residues then diluted with water (200 mL) and Water and Ethyl Acetate were added and the mixture was separated. The aqueous was washed with Ethyl Acetate and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness.
  • PdCl2(dppf).CH2Cl2 adduct (91 mg, 0.112 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was then heated to 85°C. overnight. Filtered through celite to remove Pd residues then diluted with water (200 mL) and Water and Ethyl Acetate were added and the mixture was separated. The aqueous was washed with Ethyl Acetate and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness.
  • PdCl 2 (dppf).CH 2 Cl 2 adduct (91 mg, 0.112 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was then heated to 85°C. overnight. Filtered through celite to remove Pd residues then diluted with water (200 mL) and Water and Ethyl Acetate were added and the mixture was separated. The aqueous was washed with Ethyl Acetate and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness.
  • Step 2.1-bromo-6-isopropyl-2-methoxynaphthalene (3-2x) NBS (471 mg, 2.64 mmol) was added to a solution of 2-isopropyl-6-methoxynaphthalene (From step 1, 481 mg, 2.404 mmol) in Acetonitrile (Volume: 8 mL) and the reaction was stirred at rt. After 1 h, the reaction was concentrated under reduced pressure. The residue was taken up in EtOAc, washed with sat Na2S2O3(aq) and the layers were separated.
  • Desired fractions were combined, concentrated and further dired under high vac to provide 2-(3- isopropylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3y) (1.278 g, 4.31 mmol, 80 %, major required isomer and contains minor regio isomer).). It was taken as is for further steps and isolated the desired product in the final step.
  • Desired fractions were combined and concentrated under high vac to provide 2-(2- ethylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3z) (3.78 g, 10.49 mmol, 78 %, major required isomer and contains minor regio isomer). It was taken as it is for further steps and isolated the desired product in the final step.
  • the reaction mixture was quenched with saturated NH 4 Cl solution (10 mL) and extracted to ethyl acetate (2*10 mL). The organic layer was separated, dried over anhy. sodium sulfate filtered and concentrated under reduced pressure to get the crude.
  • the crude product was purified by combi-flash using 4g cartridge and product eluted with eluted with 0-20% ethyl acetate in heptane to afford 1-((1- bromonaphthalen-2-yl)methyl)pyrrolidin-2-one (3-2aa) (1.01 g, 3.33 mmol, 99%) as a viscous oil.
  • N, N-dimethyl formamide (3.45 mL, 44.55 mmol) dropwise and the reaction continued at same temperature for 3h.
  • the progress of the reaction was monitored by TLC, using mobile phase 100% hexane. Quenched the reaction mixture with saturated ammonium chloride solution (50.0 mL) and extracted with ethyl acetate (2 x 25 mL). The combined organic layer was washed with brine solution (20.0 mL), dried over anhy. Na 2 SO 4 and concentrated the organic layer to afford 3-bromo-5-ethyl-2-fluorobenzaldehyde (4.40 g) as brown gummy liquid.
  • Step 4.4,4,5,5-tetramethyl-2-(5-methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (4-6b)
  • 7-bromo-5-methylbenzo[b]thiophene (4-5b) (0.3 g, 1.32 mmol) in dioxane (3.0 mL)
  • 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane (0.67 g, 2.64 mmol)
  • potassium acetate (0.38 g, 3.96 mmol
  • N, N-dimethyl formamide (3.68 mL, 47.61 mmol) was added dropwise, and the reaction was continued at same temperature for 2h. The completion of the reaction was monitored by TLC using mobile phase: 5% EtOAc in hexane.
  • the reaction mixture was quenched with saturated ammonium chloride solution (10.0 mL) and extracted the product with ethyl acetate (2x20 mL). The organic layer was washed with brine solution (1x10.0 mL) and dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude.
  • the reaction mixture was partitioned between water (50 mL) and EtOAc (25 mL), and the aqueous layer was extracted with EtOAc (20 mL x 2). Combined organic phases were washed with saturated brine solution, dried over Na 2 SO 4 and the solvent was removed under reduced pressure to furnish the desired compound in its crude form.
  • the crude was purified by combi flash using 12 g cartridge using 100% hexane as eluent to afford ethyl 7-bromo-6-methylbenzo[b]thiophene-2-carboxylate (1.40 g, 4.67 mmol, 67.96 %) as pale-yellow solid.
  • Rection mixture was degassed with argon gas for 20 min. Pd(dppf)Cl 2 .
  • DCM (0.179 g, 0.220 mmol) was added to reaction mixture at room temperature. The reaction mixture was heated at 80°C for 16h. The completion of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. Reaction mass was cooled and added brine solution (10 mL), then extracted with EtOAc (2x10mL) and dried over Na 2 SO 4 and concentrated under vacuum.
  • the reaction mixture was extracted with water and Ethyl acetate (50 mL x 3) twice.
  • the organic layer was washed with brine solution (30 mL x 3) and dried with Na 2 SO 4 and concentrated under reduced pressure to get the crude.
  • the crude was purified by Chromatography using 40g silicycle column, eluting the product at neat hexane to afford ethyl 7-bromo-3-(dimethylamino)benzo[b]thiophene-2-carboxylate (2.10 g, 6.40 mmol, 96.03%) as yellow liquid.
  • N,O-dimethylhydroxylamine 2.44 g ,25.11 mmol
  • the progress of the reaction was monitored by TLC, using mobile phase 30% EtOAc in hexane.
  • the reaction mixture was diluted with ice water (200 mL) and extracted the product with ethyl acetate (100mL x 3).
  • the combined organic layer was washed with brine solution, dried over anhydrous sodium sulphate and concentrated the organic layer.
  • 3-bromo-2-fluoro-N-methoxy-N-methylbenzamide From step 1, 4.4 g ,16.78 mmol
  • Methyl Magnesium bromide (3M in THF, 16.86 mL,50.36 mmol) dropwise at -78°C and stirred the reaction mixture for 6h.
  • the progress of the reaction was monitored by TLC, using mobile phase 10% EtOAc in hexane.
  • Step 5.7-bromo-3-methylbenzo[b]thiophene (4-5e) To a solution of 7-bromo-3-methylbenzo[b]thiophene-2-carboxylic acid (From step 4, 1.2 g,4.42 mmol) in Quinoline (14.0 mL), was added copper powder (421.90 mg ,6.63 mmol) and the reaction mixture was heated to 130 o C for 12h. The progress of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. The reaction mixture was diluted with water (100 mL) and extracted to ethyl acetate (2 x 100 mL).
  • Step 4.7-bromo-3-ethylbenzo[b]thiophene (4-5f) To a stirred solution of 7-bromo-3-(dimethylamino)benzo[b]thiophene-2-carboxylic acid (From step 3, 1.0 g, 3.506 mmol) in dimethylacetamide (8.0 mL) was added DBU (1.67 mL, 11.22 mmol) in microwave vial and the reaction mixture was irradiated in microwave at 200°C at 12 bar pressure for 70 min. The completion of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Cooled the reaction mixture to room temperature and evaporated under vacuum to get the crude.
  • Step 5.2-(3-ethylbenzo[b]thiophen-7-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (4-6f)
  • 7-bromo-3-ethylbenzo[b]thiophene (4-5f) (0.70g, 2.902 mmol) in 1,4-dioxane (7.0 mL)
  • 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane 0.696 g, 4.354 mmol
  • potassium acetate 0.427 g, 4.354 mmol
  • Step 2.7-bromo-6-fluorobenzo[b]thiophene-2-carboxylic acid To a vial containing ethyl 7-bromo-6-fluorobenzo[b]thiophene-2-carboxylate (From step 1, 1200 mg, 3.96 mmol) was added THF (10 mL, Ratio: 2), MeOH (5.00 mL, Ratio: 1.000) and then NaOH (4N) (2.97 mL, 11.88 mmol). The mixture was agitated at 60-Deg-C for 1 hr, at which time LCMS showed completion of the reaction. The reaction mixture was cooled to room temperature and concentrated under reduced pressure.
  • Step 3.7-bromo-6-fluorobenzo[b]thiophene To a suspension of 7-bromo-6-fluorobenzo[b]thiophene-2-carboxylic acid (From step 2, 1000 mg, 2.54 mmol), Ag2CO3 (210 mg, 0.763 mmol) in NMP (12 mL) in a 100 mL RB flask was added Acetic acid (0.044 mL, 0.763 mmol) and heated at 130 °C to reflux for 16 hours with air condenser under N2. The reaction mixture was filtered through celite pad, diluted with EtOAc and water. Aq.
  • N,N- dimethyl formamide (4.29 g, 58.47 mmol) dropwise, and the reaction continued at same temperature for 1.5h. Progress of reaction was monitored by TLC using mobile phase 30% EtOAc in hexane. The reaction mixture was quenched with saturated water and acetic acid (7 mL) and extracted the product with ethyl acetate (100 mL). The organic layer was washed with brine solution (1 x 50.0 mL) and dried over anhydrous sodium sulphate and concentrated under reduced pressure.
  • Step 2.4-bromo-6-methoxybenzo[b]thiophene-2-carboxylate To a solution of 2-bromo-6-fluoro-4-methoxybenzaldehyde (From step 1, 0.5 g, 2.14 mmol) in DMF (5.0 mL) were added potassium carbonate (0.450 g, 3.21 mmol) and ethyl 2- mercaptoacetate (0.380 g, 3.21 mmol). The reaction mixture was heated to 70 o C for 16h. The progress of the reaction was monitored by TLC, using 10% EtOAc in hexane.
  • the reaction mixture was partitioned between water (50 mL) and EtOAc (25 mL), and the aqueous layer was extracted with EtOAc (20 mL x 2). The combined organic phases were washed with saturated brine solution, dried over anhy. Na 2 SO 4 and concentrated under reduced pressure to furnish the desired compound.
  • the crude was purified by combi flash using (4 g, Silicycle cartridge column) 10% EtOAc hexane as eluent to afford ethyl 4-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (0.360 g, 1.14mmol, 54%) as off-white solid.
  • Step 3.4-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid To a solution of 4-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (From step 2, 0.350 g, 1.11mmol) in THF (3.5 mL) was added 4N NaOH (3.5 mL) and the reaction mixture was heated to 70 o C for 6h.
  • 4-bromo-6-methoxybenzo[b]thiophene (5-4a) (0.20 g, 0.820 mmol) in dioxane (2.0 mL, 10 vol) were added 4,4,4′,4′,5,5,5′,5′-Octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.410 g, 1.62 mmol), potassium acetate (0.240g, 2.4 mmol).
  • the reaction mixture was purged with argon for 30 min.
  • the reaction mixture was partitioned between water (50 mL) and EtOAc (25 mL) and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic phases were washed with saturated brine solution, dried over anhydrous Na 2 SO 4 and the solvent was removed under reduced pressure to furnish the crude.
  • the crude was purified by combi flash using 24 g cartridge and eluted the product at 8% ethyl acetate in hexane to afford ethyl 4-bromo-5-methoxybenzo[b]thiophene-2-carboxylate (1.2 g, 3.80 mmol, 25.53 %).
  • reaction mixture was diluted with water (10 mL) and extracted to ethyl acetate (2 x 15 mL). The organic layer was separated, washed with 2N HCl (2 x 10 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to get crude.
  • N,N-dimethyl formamide (4.6 g, 63.48 mmol) dropwise, and the reaction continued at same temperature for 2h. Progress of reaction was monitored by TLC using mobile phase 30% EtOAc in hexane. The reaction mixture was quenched with water(100mL) and acetic acid (7.0 ml) and extracted the product with ethyl acetate (2x100 ml). The organic layer was washed with brine solution (1x50.0 ml) and dried over anhydrous sodium sulphate and concentrated under reduced pressure to get the crude.
  • Step 3 4-bromo-6-methylbenzo[b]thiophene-2-carboxylic acid To a solution of ethyl 4-bromo-6-methylbenzo[b]thiophene-2-carboxylate (From step 2, 3.20 g, 10.69 mmol) in THF (32.0 ml) was added 4N NaOH (32.0 ml) and the reaction mixture was heated to 70 o C for 16h.
  • reaction mixture was partitioned between water (50 mL) and EtOAc (3x100 mL), and the aqueous layer was extracted with EtOAc. The combined organic phases were washed with saturated brine solution, dried over anhy. Na 2 SO 4 and concentrated under reduced pressure to furnish the desired compound in its crude form.
  • Step 2 4-bromo-5-methylbenzo[b]thiophene-2-carboxylic acid
  • ethyl 4-bromo-5-methylbenzo[b]thiophene-2-carboxylate (From step 1, 3.00 g, 10.02 mmol) in THF (30 mL) was added 4N NaOH (36.0 mL) and the reaction mixture was heated to 70 o C for 4h. The completion of the reaction was monitored by TLC, using mobile phase: 50 % EtOAc in hexane. Cooled the reaction mass to room temperature. The reaction mixture was concentrated to remove the volatiles and acidified using conc. HCl at 0 o C.
  • reaction was monitored by TLC, using mobile phase 30% EtOAc in hexane.
  • the reaction mixture was partitioned between saturated ammonium chloride solution (10 mL) and EtOAc (2*10 mL), and the aqueous layer was extracted with EtOAc (2*10 mL). Combined organic phases were washed once with saturated brine solution, dried over Na2SO4 and the solvent was removed under reduced pressure to get the crude.
  • tert-butyl(6-bromo-3-cyanopyrazolo[1,5-a]pyrimidine-7-yl) (tert- butoxycarbonyl)carbamate (1-4a, 0.5g, 1.14 mmol) and (2-methoxynaphthalen-1-yl)boronic acid (0.27g, 1.36 mmol) in THF (2.5 mL) were added 2% TPGS (5 mL, 10V) and triethylamine (0.62 mL, 4.56 mmol) and purged with argon for 10 min.
  • PdCl 2 (dppf).CH 2 Cl 2 adduct (306 mg, 0.375 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was stirred at 110 o C for 48 hrs. Filtered through celite washing with ethyl acetate and evaporated to dryness. The residue was chromatographed on the ISCO 120g cartridge, eluting with 0-20% Ethyl Acetate in heptane.
  • reaction mixture was purged again with argon for 25 min and heated up to 50°C for 12 h.
  • the progress of the reaction was monitored by TLC, using mobile phase:30 % EtOAc in Hexane.
  • the reaction mixture was diluted with water (10 mL) and extracted to ethyl acetate (2 x 15 mL). The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure to get the crude.
  • Step 1 2-(benzo[b]thiophen-3-yl) acetamide To a stirred solution of 2-(benzo[b]thiophen-3-yl)acetic acid (3.0 g, 15.60 mmol) in DMF (30.0 mL), were added EDCI. HCl (4.49g, 23.41 mmol), HOBt.H 2 O (3.58g, 23.41mmol), NH 4 Cl(2.50g, 46.82 mmol), DMAP(0.19g, 1.56mmol) and DIPEA (10.87mL, 62.43mmol) and the reaction mixture was allowed to stir at room temperature for 16 h. The completion of the reaction was monitored by TLC, using mobile phase 5% MeOH in DCM.
  • Example I - 3 6-(5-methylbenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5- a]pyrimidin-7-amine Step 1. 2-(5-methylbenzo[b]thiophen-4-yl)acetonitrile To a stirred solution of 4-bromo-5-methylbenzo[b]thiophene (5-5d) (1.800 g, 7.92 mmol) in THF (20.0 mL, 10 vol) were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (1.85 g, 9.51 mmol), followed by TPGS-750-M (20.0 mL, 10.0 vol) and triethylamine (4.41 mL, 31.70 mmol).
  • reaction mixture was purged with argon for 30 minutes. After 30 minutes, added [1,1′-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (0.774 g, 1.18 mmol), and heated up to 50°C for 1h, followed by further heating to 80°C for 12 h. The progress of the reaction was monitored by TLC, using 30 % EtOAc in hexane. Reaction mass was cooled, and brine solution added (10 mL), then extracted with EtOAc (2 x 10mL) and dried over Na 2 SO 4 and concentrated under vacuum.
  • reaction mixture Concentrated the reaction mixture and added toluene (2.5 mL, 10 vol), acetic acid (2.5 mL, 10.0 vol) and 5-amino-1H-pyrazole-4-carbonitrile (0.144 g, 1.34 mmol). Then the reaction mixture was stirred at 130°C for 12h. The completion of the reaction was monitored by TLC, using mobile phase: 30% EtOAc in hexane. Reaction mixture was basified with saturated NaHCO 3 solution and extracted the product with EtOAc (20 x 3 mL). The combined organic layer was washed with brine solution and dried over anhy. Na 2 SO 4 and concentrated under vacuum to get crude.
  • Example I - 4 6-(8-chloronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7- amine
  • Step 1 2-(8-chloronaphthalen-1-yl)acetonitrile
  • To a stirred solution of 1-bromo-8-chloronaphthalene (0.50 g, 2.070 mmol) and 4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (0.40 g, 2.070 mmol) in THF(2.5 mL) were added 2% TPGS-750-M (5.0 mL) and Et 3 N (1.16 mL, 8.250 mmol) at room temperature and the reaction mixture was degassed with argon gas for 20 minute.
  • the reaction mixture was heated at 100°C for 12h. The color of the reaction mixture changes from white to brown. The progress of the reaction was monitored by TLC, using mobile phase:5% MeOH in DCM. Cooled the reaction mixture to room temperature and the reaction mixture was poured to ice cold water, the solid precipitated. Filtered the solid, dried under vacuum, washed with pentane to get the crude.
  • Example II - 1 3-(7-amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)- 1,2,4-oxadiazol-5(4H)-one
  • pyridine 0.020 mL, 0.250 mmol
  • 2-ethylhexyl carbonochloridate 0.040 mL, 0.208 mmol
  • Desired fractions were combined and lyophilized to provide with desired product 3-(7- amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)-one (I-3) (7.5 mg, 0.019 mmol, 9.35 % yield).
  • Example III - 1 7-amino-6-(2-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylic acid
  • Step 1 ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(2-methoxynaphthalen-1- yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (17-1)
  • ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-bromopyrazolo[1,5- a]pyrimidine-3-carboxylate (1-4b) (0.600 gm,1.236 mml) in THF (6.0 ml) was added (2- methoxynaphthalen-1-yl)boronic acid (0.274 gm,1.359 mmol), 2% TPGS-750-M (aq) solution (4.5 ml) and purged with argon for 30 min.
  • Example III - 2 7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylic acid
  • Step 1 ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carboxylate (18-1)
  • ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-bromopyrazolo[1,5- a]pyrimidine-3-carboxylate (1-4b) (0.600 gm,1.236 mml) in THF (6.0 mL)
  • 4b 4,4,5,5- tetramethyl-2-(3-methylnaphthalen-1-yl)-1,3,2-dioxaborolane
  • 2% TPGS-750-M (aq) solution 4.5
  • reaction mixture was stirred at room temperature for 2h. The progress of the reaction was monitored by TLC, using mobile phase:50 % EtOAc in hexane. The reaction mixture was evaporated in rotavapor under nitrogen. The crude was triturated with n-pentane to afford ethyl 7-amino-6-(3-methylnaphthalen- 1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate(18-2) (0.280g, 0.808 mmol, 98.19%) as pale-yellow solid.
  • the reaction mixture was evaporated under vacuum to get residue.
  • Example 1 HTRF assay to measure inhibition of TET2 enzymatic activity A homogeneous time resolved energy transfer system was used to develop high- throughput and quantitative assays to measure changes in TET2-induced 5hmC levels in response to compounds. Assays were developed to assess the ability of compounds to inhibit recombinant human TET2 and TET3.
  • wash buffer supplemented with 0.4 mg of His-TEV protease (produced in-house) was added to the resins and the mixture was placed in a tube and incubated overnight at 4oC for on-beads cleavage of GST tag.
  • His-TEV protease produced in-house
  • the mixture was placed in a gravity column and drained, then washed three times with 2.5 ml of wash buffer each time. Flow through and washes (containing His-TEV cleaved sample) were collected and mixed with 0.25 ml of equilibrated Talon resins (Takara Bio) for 1 hour at 4oC to bind His-TEV protease.
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • TET3 688-1019-(GS)x3-1501-1582
  • the cells were thawed and resuspended in lysis buffer (800mL per cells from 12L culture) 50mM HEPES (pH 8.0), 300mM NaCl, 1mM TCEP, 100ug/ml lysozyme, 200ug/ml DNase, 2mM MgCl2, supplemented with protease inhibitor cocktail (Roche cOmplete EDTA-free protease inhibitor tablets, 1 tablet per 50 mL of buffer), then lysed by passing through a microfluidizer (M-110L, Microfluidics) once at 15k psi on ice.
  • M-110L Microfluidics
  • the lysate was cleared by centrifugation in a JA25.50 rotor at 50,000xG for 1 hour.
  • the clarified lysate was mixed with 40ml of Talon beads (Takara Bio) equilibrated in equilibration buffer 50mM HEPES (pH 8.0), 300mM NaCl, 1mM TCEP, and 20mM Imidazole and rocked at 4 o C for 1 hour.
  • the bound beads were washed with five column volumes of equilibration buffer using a gravity column.
  • the bound material (containing His-TEV- human TET3 (688-1019-(GS)3-1501-1582) was eluted with five column volumes of elution buffer 50mM HEPES (pH 8.0), 300mM NaCl, 1mM TCEP, and 300mM Imidazole.
  • elution buffer 50mM HEPES pH 8.0
  • 300mM NaCl 300mM NaCl
  • 1mM TCEP 300mM Imidazole.
  • TEV protease was added to the Talon eluate at a ratio of 1mg TEV to 50mg fusion protein.
  • the mixture was transferred to a dialysis tubing with 3500 Da molecular weight cutoff, then dialyzed against 4L of S-0 buffer 50mM Hepes (pH8.0), 1mM TCEP at 4 o C overnight.
  • the dialyzed mixture was filtered using a 0.22 mm filter and loaded onto a HiTrap Q (GE Healthcare) equilibrated in S- 0 buffer. Following capture, the bound protein was eluted by a linear gradient elution using the same buffer supplemented with 1M NaCl (3mL/min, over 20 column volumes).
  • Fractions containing human TET3 (688-1019-(GS)x3-1501-1582) were pooled and loaded onto a HiLoad 26/600 Superdex 200 column (GE Healthcare) equilibrated in 25 mM HEPES (pH 8.0), 150 mM NaCl, 1 mM TCEP.
  • the reaction was quenched by adding solution containing EDTA (final concentration 66.6 ⁇ M), and scavenger DNA (5’- CTTAGTGCCTCGTTCGCTTGCTCCGGTCT-3’, final concentration 125 ⁇ M) and DNA was denatured at 95 degrees C for 5 minutes and allowed to cool to room temperature. Conversion of 5mC to 5hmC was measured by adding HTRF detection mix containing Europium coupled anti-5hmC antibody (final concentration 0.5 nM in PBS 0.5%BSA) and streptavidin labeled with XL665 (10 nM final concentration). After 1.5-hour incubation, fluorescence was read on a Pherastar reader. Substrate solution and assay buffer controls were also read.
  • Example 2 Cellular assay system to quantify effect of TET2 inhibitor compounds on TET2 enzymatic activity To assess the activity of TET2 inhibitor compounds in cells we utilized a HeLa cell line engineered to overexpress TET2 catalytic domain when treated with doxycycline.
  • the cell line was generated using parental HeLa cells (ATCC CCL2) infected with lentivirus expressing the TET2C delta construct (human TET21130-1459 (GS)31844-1925-HA) cloned into the Lenti-X Tet-One Inducible Expression System (Puro) (Takara Bio Cat #634847).
  • HeLa cells were seeded at 150,000 per well in 6 well cell culture plates.
  • Lentivirus expressing the TET2 construct was added with polybrene, final concentration 10 ⁇ g/ml. Cells were then split into T 75 flasks and puromycin was added at a final concentration of 1 ⁇ g/ml.
  • pCR148 clone 12D was identified as expressing high levels of TET2 after doxycycline treatment, as measured by Western blot using anti-HA antibody (Cell Signaling Technologies). Cells were expanded in culture and frozen down in liquid nitrogen for use in cellular assay to assess TET2 inhibitor compounds. To assess the cellular activity of TET2 inhibitor compounds, on day 0, HeLa cells (pCR148 clone 12D) were seeded in 384 well plates (black, clear bottom GreinerBio cat #781091), 1000 cells per well in 30 ul of culture medium (DMEM/10%FBS/Penicillin/Streptomycin).
  • AC50 values were calculated using internal software (Helios). Values were calculated for both 5hmC detection and DAPI nuclei count (to assess viability of the cells). Representative inhibition curves are depicted in Figure 2. AC50 and qualified AC50 values for each compound were calculated using internal software and are depicted in Table 27.
  • Example 3 Primary human T cell assay for assessment of compounds Given the effects of TET2 genetic disruption on CART cells observed in Patient 10, including enhanced memory cell and T cell stemness phenotypes, we treated primary, in vitro activated human T cells from normal healthy donors with TET2 inhibitors and assessed the impact on T cell activation, exhaustion, and memory phenotypes.
  • T cells isolated were stimulated with anti-CD3/anti-CD28 coated beads for 4 days, then re-stimulated for an additional 3 days.
  • Flow cytometry was performed to assess the ability of compounds to modulate expression of TIGIT, FOXP3, and TCF7 by stimulated T cells.
  • PBMCs were isolated from blood taken from normal healthy donors and collected into CPT tubes (BD #02-685-125). Tubes were centrifuged at 1800xG for 20 minutes at room temperature and inverted several times to mix cells. Supernatants were collected and washed once in sterile PBS.
  • CD3+ T cells were enriched using Miltenyi Pan T cells isolation kit (#130-096-535) following the manufacturer’s instructions. Isolated T cells were resuspended in RPMI/10% FBS and added to 96-well round bottomed tissue culture plates, 80,000 cells per well cells per well in 200 ⁇ l media. Cells were stimulated with anti- CD3/28 Dynabeads (Thermo Fisher # 11132D, 1:1 bead to cell ratio) and incubated overnight at 37 degrees C, 5% CO2. On day 1, compounds were added to cells at doses 3, 1, or 0.3 ⁇ M. DMSO was added to control cells. Cells were incubated an additional 3 days at 37 degrees C, 5% CO2.
  • FACS buffer PBS/0.5%BSA
  • fixation/permeabilization buffer Ebioscience #00-05523
  • 200 ⁇ l per sample 45 minutes at 4 degrees Celsius
  • Cells were then washed twice with cold FACS buffer and stained with fluorescently labeled anti-FOXP3 and anti-TCF7 antibodies in fixation/permeabilization buffer for 45 minutes at room temperature.
  • Cells were then washed twice with 200 ⁇ l of fixation/permeabilization buffer, resuspended in 200 ⁇ l FACS buffer, and read on an LSR Fortessa flow cytometer.
  • TIGIT is a marker of exhausted T cells
  • FOXP3 is a marker of regulatory T cells and is known to be regulated by TET2 via demethylation of cis-regulatory elements in the Foxp3 locus (Yue, X., (2016), J Exp Med. 213: 377-97).

Abstract

The present invention relates to novel pyrazolopyrimidine compounds that are TET2 inhibitors, processes for their preparation, pharmaceutical compositions, and medicaments containing them, and their use in diseases and disorders mediated by a TET2 inhibitor.

Description

PYRAZOLOPYRIMIDINE DERIVATIVES AND USES THEREOF AS TET2 INHIBITORS FIELD OF THE INVENTION The present disclosure relates to pyrazolopyrimidine compounds and compositions and their use for the treatment of proliferative diseases or disorders where the inhibition of TET2 can ameliorate a disease or disorder. BACKGROUND OF THE INVENTION Ten-Eleven Translocation 2 (TET2) is one of three members of the TET family of dioxygenases found in mammals. TET2 contains a C-terminal catalytic domain that catalyzes the oxidation of methylated DNA (5-methyl cytosine, 5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidizes this to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). These reactions initiate the process of DNA demethylation, which is a known mechanism of transcriptional regulation (Schubeler, D., (2015), Nature.517:321-26). TET2 is a 2002 amino acid protein that consists of an N-terminal domain, cysteine-rich region, and C-terminal catalytic domain. Unlike TET1 and TET3, TET2 does not contain a CXXC domain that targets the protein to CpG sequences in DNA. This region of TET2 separated from the protein due to chromosomal inversion and evolved as a separate gene (IDAX) which is thought to interact with TET2 and regulate binding to DNA sequences (Jio, C., (2020), J Biosci.45:21). TET2 utilizes alpha-keto glutarate, reduced iron, and molecular oxygen, as well as vitamin C as a cofactor, to oxidize 5mC, 5hmC, and 5fC and produce carbon dioxide and succinate as byproducts of the reaction. Along with TET3, TET2 is expressed ubiquitously, while TET1 expression is more restricted to embryonic cells (Pastor, W.A., (2013), Nat Rev Mol Cell Biol.14:341-56). In mice, TET2 genetic ablation is associated with enhanced survival of hematopoetic stem cells and biased differentiation of myeloid cells, as opposed to that of other lineages such as T, B, and erythroid cells (Ko, M., (2011), Proc Nat Acad Sci USA.108:145666-71). In humans, germline loss of function of TET2 in 3 children was associated with immunodeficiency and lymphoma, with altered T cell development and loss of class-switch recombination in B cells (Spegarova, J., (2020), Blood. 136: 1055-66.) TET2 mutations are commonly found in patients with blood malignancies and in clonal hematopoiesis of indeterminate potential (CHIP), however in mouse models deletion of TET2 alone leads to an incomplete oncogenic phenotype, with deletion of both TET2 and TET3 necessary for driving fully penetrant malignancies (An, J., (2015), Nat Commun.6: 10071). Genetic perturbation of TET2 has been shown to be associated with enhanced development of memory CD8+ cell responses in mice (Carty, S., (2018), J. of Immunol.200: 82-91). Disruption of TET2 in a single CD19- targeting CART cell in a CLL patient, via integration of the CART lentivirus into intron 9, led to eradication of the tumor cells and long term remission/cure of the cancer (Fraietta, J., (2018), Nature.558: 307-12). Due to a missense mutation in the other TET2 allele it is reasonable to assume an additive loss of TET2 protein expression and function in this cell clone, which expanded to 94% of the CART cells at the peak of response. Compared to CART cells from other complete responders, these cells consisted of a high proportion of central memory cells, which have been associated with more durable and effective responses of T cells in adoptive therapy models of cancer (Gattinoni, L., (2011), Nat Med.17: 1290-97). Genetic knockdown of TET2 in CART cells from normal healthy donors enhanced in vitro expansion when stimulated with CD19 expressing tumor cells, thus phenocopying the results observed in vivo with this patient’s CART cells. A TET2-specific enzymatic inhibitor has the potential to enhance anti-tumor T cell therapies by enhancing memory and stemness of the T cells, leading to more durable and effective responses. SUMMARY OF THE INVENTION There remains a need for new treatments and therapies for proliferative diseases or disorders where the inhibition of TET2 can ameliorate a disease or disorder. The invention provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, which compounds are TET2 inhibitors. The invention further provides methods of treating, preventing, or ameliorating proliferative diseases, comprising administering to a subject in need thereof an effective amount of an TET2 inhibitor. Various embodiments of the invention are described herein. Within certain aspects, provided herein is a compound of Formula AA:
or a pharmaceutically acceptable salt thereof. Within certain aspects, provided herein is a compound of formula (I) or a pharmaceutically acceptable salt thereof: (I). BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the Inhibition of 5hmC formation by recombinant human TET2 protein by select compounds Figure 2 illustrates the Inhibition of 5hmC production in a TET2 inducible overexpressing HeLa cell line (HeLa pCR148 clone 12D) by select compounds Figure 3 illustrates decreased expression of TIGIT by activated T cells in the presence of Compound Ex. I-5 Figure 4 illustrates the decreased expression of FOXP3 by activated T cells in the presence of Compound Ex. I-5 Figure 5 illustrates the enhanced expression of TCF7 by activated T cells in the presence of Compound Ex. I-5 Figure 6 illustrates the reduced FOXP3 expression by activated T cells in the presence of Compound Ex. I-83 Figure 7 illustrates the reduced FOXP3 expression by activated T cells in the presence of Compound Ex. II-10 Figure 8 illustrates the reduced FOXP3 expression by activated T cells in the presence of Compound Ex. I-84 DETAILED DESCRIPTION OF THE INVENTION The invention therefore provides a compound of Formula AA: or a pharmaceutically acceptable salt thereof, wherein Ring A is selected from a 6-10 membered aryl, 6-10 membered heteroaryl, and 6-10 membered partially saturated carbocyclyl, wherein the aryl, heteroaryl and carbocyclyl are each independently unsubstituted or substituted with 0 to 5 substituents represented by R2, R3, R4, R5, R6, R7, or R8; R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(CH2)1-6R11, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O which is unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6 alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S,which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; E is S or CR3; U is C or CR4; W is C or CR7; m is 0 or 1; and n is 0 or 1. In one embodiment is a compound of Formula I:
or a pharmaceutically acceptable salt thereof , wherein is a single or a double bond; R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(CH2)1-6R11, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O which is unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6 alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S,which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; E is S or CR3; U is C or CR4; W is C or CR7; m is 0 or 1; and n is 0 or 1. In one embodiment is a compound of Formula II (II) or a pharmaceutically acceptable salt thereof, wherein: ------- is a single or a double bond; R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(CH2)1-6R11, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O which is unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, or N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy (C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; W is C or CR7; and m is 0 or 1. Another embodiment is a compound of Formula IIa or a pharmaceutically acceptable salt thereof, wherein: R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, -OH, halogen, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(C1-6alkyl)R11, or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which is unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which are unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; and G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5. Another embodiment is a compound of Formula IIb or a pharmaceutically acceptable salt thereof, wherein: R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(C1-6alkyl)R11, or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy (C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which are unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; and G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5. Another embodiment is a compound of Formula III
or a pharmaceutically acceptable salt thereof, wherein: ------- is a single or a double bond; R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(C1-6alkyl)R11, or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl; haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which are unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; E is S or CR3; U is C or CR4; and n is 0 or 1. Another embodiment is a compound of Formula IIIa or a pharmaceutically acceptable salt thereof, wherein: R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(C1-6alkyl)R11, or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which are unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, O, OH, COOH, O(C1-6alkyl), or CN; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; and E is S or CR3. In another embodiment, Ring A is selected from naphthyl, benzothiophenyl, tetrahydronaphthyl, or indane. In another embodiment, R1 is H or NH2. In another embodiment, R2 is H or CH3. In another embodiment,R9 is H or NH2. In another embodiment, R10 is COOH,
In another embodiment, A is CR8.
In another embodiment, A is S, and , G is N, NR5, or CR5.
In another embodiment, G is CR5.
In another embodiment, G is S, and A is N, NR8, or CR8.
17. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein E is CR3.
18. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein E is S.
Specifica compounds include:
6-(benzo[b]thiophen-3-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(5-methoxybenzo[b]thiophen-4-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine; 6-(5- methylbenzo[b]thiophen-4-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(8-chloronaphthalen-1 -y l)-3-( 1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(naphthalen-1-yl)-3-(2H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(2-methoxynaphthalen-1 -y l)-3-( 1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(6-methoxybenzo[b]thiophen-4-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(5-methylbenzo[b]thiophen-7-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(7-methylnaphthalen-1 -y l)-3-( 1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(4-fluoronaphthalen-1-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(6-methylbenzo[b]thiophen-7-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(3-methoxynaphthalen-1 -y l)-3-( 1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(3-methylnaphthalen-1 -y l)-3-( 1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(6-methylbenzo[b]thiophen-4-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(3-methylbenzo[b]thiophen-7-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine;
6-(benzo[b]thiophen-4-yl)-3-(1 H-tetrazol-5-yl)pyrazolo[1 ,5-a]pyrimidin-7-amine; 6-(3-ethylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(7-methoxy-6-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-isopropylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(7-ethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(5,6,7,8-tetrahydronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-ethoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 4-(7-amino-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-ol; 6-(2,6-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6,7-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-(tert-butyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(7-(tert-butyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2,6-dimethoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(3,4-dihydronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(6-methylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(6-fluoro-2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; (1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)methanol; 1-(1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)-N,N- dimethylmethanamine; 6-(2,7-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(6-methylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 7-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)-N,N-dimethylbenzo[b]thiophen-3-amine; 6-(7-isopropylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2,6-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 2-(4-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)-3-methylnaphthalen-2-yl)-1,3,4- oxadiazole; 6-(3-((1H-pyrazol-1-yl)methyl)-2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5- a]pyrimidine; 6-(6-(tert-butyl)naphthalen-2-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(5-fluoro-2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(3-(difluoromethyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 2-((1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)oxy)acetic acid; 3-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)benzo[b]thiophene-2-carboxylic acid; 6-(3-(1H-tetrazol-5-yl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 4-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-ol; 6-(3-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-methylbenzo[b]thiophen-3-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-(methoxymethyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; (4-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)methanol; 1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-ol; 6-(4-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 1-((1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)methyl)pyrrolidin-2-one; 6-(5-fluoronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-methoxy-6-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2,7-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2-methoxy-7-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2,3-dihydro-1H-inden-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(3-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(5-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(3-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2-(1H-tetrazol-5-yl)benzo[b]thiophen-3-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2- amine; 6-(2-(difluoromethyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(5-ethylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methoxy-7-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-fluoro-2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2,7-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(8-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(benzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 7-(7-amino-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)-6-methylbenzo[b]thiophene-2- carboxylic acid; 6-(6-fluorobenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-ethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-isopropyl-2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methoxy-6-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-ethyl-2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-isopropylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 3-(7-amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)- one; 3-(7-amino-6-(5-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(6-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(3-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)- one; 3-(7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)- one; 3-(7-amino-6-(6-methylbenzo[b]thiophen-4-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(5-methylbenzo[b]thiophen-4-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(2-(difluoromethyl)naphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(4-methylbenzo[b]thiophen-3-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)-one; 7-amino-6-(2-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(5-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(6-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(5-methylbenzo[b]thiophen-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(7-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(8-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid, or a pharmaceutically acceptable salt thereof. One embodiment is a pharmaceutical composition comprising a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. In another embodiment, is the pharmaceutical composition, further comprising at least one additional pharmaceutical agent. In another embodiment, the pharmaceutical composition is for use in the treatment of a disease or disorder that is affected by the inhibition of TET2. Another emobidment is a method of inhibiting TET2 comprising administering to the patient in need thereof a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof. Another emobidment is a method of reducing the proliferation of a cell, the method comprising contacting the cell with a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof, and inhibition TET2. Another emobidment is a method of treating cancer comprising administering to the patient in need thereof a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof. Another emobidment is a method wherein the cancer is selected from non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS). Another emobidment is a method wherein the non-small cell lung cancer (NSCLC) is selected from adenocarcinoma, squamous cell carcinoma, large cell carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, and sarcomatoid carcinoma. Another embodiment is the compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or disorder that is affected by the inhibition of TET2. Another embodiment is the use of a compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or disorder that is affected by the inhibition of TET2. In a further embodiment, the disease or disorder is selected from non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS). Another embodiment is the compound of Formula (AA), (I), (II), or (III), or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for treating a disease or disorder that is affected by the inhibition of TET2. In a further embodiment, the use of compound of Formula (AA), (I), (II), or (III), wherein the disease or disorder is selected from non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T- cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS). Unless specified otherwise, the term “compounds of the present disclosure” or “compound of the present disclosure” refers to compounds of formula (I) subformulae thereof, and exemplified compounds, and salts thereof, as well as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties. DEFINITIONS As used herein, the terms "Halogen", “halide”, or, alternatively, “halo” refer to bromo, chloro, fluoro or iodo. As used herein, the term “C1-6alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molecule by a single bond. The term “C1-4alkyl” is to be construed accordingly. Examples of C1-6alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl and 1,1-dimethylethyl (t- butyl). As used herein, the term “C3-8cycloalkyl” refers to a monocyclic or polycyclic radical that contains only carbons and hydrogen, having from three to eight ring atoms, and can be saturated or partially unsaturated. Examples of C3-8cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentyenyl, cyclohexyl, cycloheptyl, and cyclooctyl. As used herein, the term "hydroxyC1-6alkyl” refers to a C1-6alkyl radical as defined above, wherein one of the hydrogen atoms of the C1-6alkyl radical is replaced by OH. Examples of hydroxyC1-6alkyl include, but are not limited to, hydroxy-methyl, 2-hydroxy-ethyl, 2-hydroxy- propyl, 3-hydroxy-propyl and 5-hydroxy-pentyl. As used herein, the term "haloC1-6alkyl" refers to C1-6alkyl radical, as defined above, substituted by one or more halo radicals, as defined above. Examples of halo C1-6alkyl include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,3- dibromopropan-2-yl 3-bromo-2-fluoropropyl and 1,4,4-trifluorobutan-2-yl. As used herein, the term “Aryl” refers to an aromatic hydrocarbon ring system. Aryl groups are monocyclic ring systems or bicyclic ring systems. Monocyclic aryl ring refers to phenyl. Bicyclic aryl rings refer to naphthyl. Aryl groups may be optionally substituted with one or more substituents as defined in formula (I). The term “oxo” refers to O. By way of example, substitution of a CH2 a group with oxo gives a C=O group. As used herein, the terms “the ring A” or “A” are used interchangeably to denote in formula AA. As used herein, the term “Heterocyclic” or “heterocyclyl” refers to a 3 to 8 membered saturated or partially unsaturated monocyclic or bicyclic ring containing from 1 to 5 heteroatoms. Heterocyclic ring systems are not aromatic. Heterocyclic groups containing more than one heteroatom may contain different heteroatoms. Heterocyclic includes ring systems wherein a carbon atom is oxidized forming a cyclic ketone or lactam group. Heterocyclic also includes ring systems wherein a sulfur atom is oxidized to form SO or SO2. Heterocyclic groups may be optionally substituted with one or more substituents as defined in formula (I). Heterocyclic groups are monocyclic, spiro, or fused or bridged bicyclic ring systems. Monocyclic heterocyclic have 3 to 7 ring atoms, unless otherwise defined. Examples of monocyclic heterocyclic groups include tetrahydrofuranyl, dihydrofuranyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, piperazinyl, piperidinyl, 1,3-dioxolanyl, imidazolidinyl, imidazolinyl, pyrrolinyl, pyrrolidinyl, tetrahydropyranyl, dihydropyranyl, oxathiolanyl, dithiolanyl, 1,3-dioxanyl, 1,3-dithianyl, oxathianyl, thiomorpholinyl and the like. Fused heterocyclic ring systems have from 8 to 11 ring atoms and include groups wherein a heterocyclic ring is fused to a phenyl or monocyclic heteroaryl ring. Examples of fused heterocyclic rings include 3,4-dihydroquinolin-2(1H)-onyl and the like. As used herein, the term “Heteroaryl” refers to an aromatic ring system containing from 1 to 5 heteroatoms. Heteroaryl groups containing more than one heteroatom may contain different heteroatoms. Heteroaryl groups may be optionally substituted with one or more substituents as defined in formula (I). Heteroaryl groups are monocyclic ring systems or are fused bicyclic ring systems. Monocyclic heteroaryl rings have from 5 to 6 ring atoms. Bicyclic heteroaryl rings have from 8 to 10 member atoms. Heteroaryl includes, but is not limited to, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, furanyl, furanzanyl, thienyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, trazinyl, tetrazinyl, tetrzolyl, indonyl, isoindolyl, indolizinyl, indazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, benzimidazolyl, benzopyranyl, benzopyranyl, benzoxazolyl, benzoisoxazolyl, benzofuranyl, benzothiazolyl, benzothienyl, and naphthyridinyl. It is understood that the actual electronic structure of some chemical entities, e.g. aromatic ring substituents, cannot be adequately represented by only one canonical form, e.g. Lewis structure. While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or “localization” of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as being one or more of the predominant resonance forms for a particular species. As used herein, the terms “salt” or “salts” refers to an acid addition or base addition salt of a compound of the present invention. “Salts” include in particular “pharmaceutical acceptable salts”. The term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. When both a basic group and an acid group are present in the same molecule, the compounds of the present invention may also form internal salts, e.g., zwitterionic molecules. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine. In another aspect, the present invention provides compounds of the present invention in acetate, ascorbate, adipate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, caprate, chloride/hydrochloride, chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, mucate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate, propionate, sebacate, stearate, succinate, sulfosalicylate, sulfate, tartrate, tosylate trifenatate, trifluoroacetate or xinafoate salt form. Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. lsotopically labeled compounds have structures depicted by the formulae given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Isotopes that can be incorporated into compounds of the disclosure include, for example, isotopes of hydrogen. For example, Formula (II) is deuterated as shown in the compound of formula (IIc): (IIc) or a pharmaceutically acceptable salt thereof, wherein R1, through R10 are defined as in Formula (II), RD1 through RD10 are independently H or D. Further, incorporation of certain isotopes, particularly deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index or tolerability. It is understood that deuterium in this context is regarded as a substituent of a compound of the present disclosure. The concentration of deuterium may be defined by the isotopic enrichment factor. The term "isotopic enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of this disclosure is denoted as being deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation). It should be understood that the term “isotopic enrichment factor” can be applied to any isotope in the same manner as described for deuterium. Other examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as 3H, 11C, 13C, 14C, 15N, 18F , 35S, 36Cl, 123I, 124I, 125I respectively. Accordingly it should be understood that the disclosure includes compounds that incorporate one or more of any of the aforementioned isotopes, including for example, radioactive isotopes, such as 3H and 14C, or those into which non- radioactive isotopes, such as 2H and 13C are present. Such isotopically labelled compounds are useful in metabolic studies (with 14C), reaction kinetic studies (with, for example 2H or 3H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an 18F or labeled compound may be particularly desirable for PET or SPECT studies. Isotopically-labeled compounds of the present disclosure can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed. As used herein, the term “pharmaceutical composition” refers to a compound of the disclosure, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier, in a form suitable for oral or parenteral administration. As used herein, the term "pharmaceutically acceptable carrier" refers to a substance useful in the preparation or use of a pharmaceutical composition and includes, for example, suitable diluents, solvents, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffering agents, emulsifiers, absorption delaying agents, salts, drug stabilizers, binders, excipients, disintegration agents, lubricants, wetting agents, sweetening agents, flavoring agents, dyes, and combinations thereof, as would be known to those skilled in the art (see, for example, Remington The Science and Practice of Pharmacy, 22nd Ed. Pharmaceutical Press, 2013, pp.1049-1070). The term "a therapeutically effective amount" of a compound of the present disclosure refers to an amount of the compound of the present disclosure that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme, receptor, ion channel, or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present disclosure that, when administered to a subject, is effective to (1) at least partially alleviate, prevent and/or ameliorate a condition, or a disorder or a disease (i) mediated by TET2, or (ii) associated with TET2 activity, or (iii) characterized by activity (normal or abnormal) of TET2; or (2) reduce or inhibit the activity of TET2; or (3) reduce or inhibit the expression of TET2. In another embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present disclosure that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of TET2; or at least partially reducing or inhibiting the expression of TET2. The meaning of the term “a therapeutically effective amount” as illustrated in the above embodiment for TET2 also applies by the same means to any other relevant proteins/peptides/enzymes/receptors/ion channels. As used herein, the term “subject” refers to primates (e.g., humans, male or female), dogs, rabbits, guinea pigs, pigs, rats and mice. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human. As used herein, the term “inhibit”, "inhibition" or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process. As used herein, the term “treat”, “treating" or "treatment" of any disease or disorder refers to alleviating or ameliorating the disease or disorder (i.e., slowing or arresting the development of the disease or at least one of the clinical symptoms thereof); or alleviating or ameliorating at least one physical parameter or biomarker associated with the disease or disorder, including those which may not be discernible to the patient. As used herein, the term “prevent”, “preventing" or “prevention” of any disease or disorder refers to the prophylactic treatment of the disease or disorder; or delaying the onset or progression of the disease or disorder As used herein, a subject is “in need of” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment. As used herein, the term "a,” "an,” "the” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. Any asymmetric atom (e.g., carbon or the like) of the compound(s) of the present disclosure can be present in racemic or enantiomerically enriched, for example the (R)-, (S)- or (R,S)- configuration. In certain embodiments, each asymmetric atom has at least 50 % enantiomeric excess, at least 60 % enantiomeric excess, at least 70 % enantiomeric excess, at least 80 % enantiomeric excess, at least 90 % enantiomeric excess, at least 95 % enantiomeric excess, or at least 99 % enantiomeric excess in the (R)- or (S)- configuration. Substituents at atoms with unsaturated double bonds may, if possible, be present in cis- (Z)- or trans- (E)- form. Accordingly, as used herein a compound of the present disclosure can be in the form of one of the possible stereoisomers, rotamers, atropisomers, tautomers or mixtures thereof, for example, as substantially pure geometric (cis or trans) stereoisomers, diastereomers, optical isomers (antipodes), racemates or mixtures thereof. Any resulting mixtures of stereoisomers can be separated on the basis of the physicochemical differences of the constituents, into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and/or fractional crystallization. Any resulting racemates of compounds of the present disclosure or of intermediates can be resolved into the optical antipodes by known methods, e.g., by separation of the diastereomeric salts thereof, obtained with an optically active acid or base, and liberating the optically active acidic or basic compound. In particular, a basic moiety may thus be employed to resolve the compounds of the present disclosure into their optical antipodes, e.g., by fractional crystallization of a salt formed with an optically active acid, e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di-O,O'-p-toluoyl tartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. Racemic compounds of the present disclosure or racemic intermediates can also be resolved by chiral chromatography, e.g., high pressure liquid chromatography (HPLC) using a chiral adsorbent. The disclosure further includes any variant of the present processes, in which an intermediate obtainable at any stage thereof is used as starting material and the remaining steps are carried out, or in which the starting materials are formed in situ under the reaction conditions, or in which the reaction components are used in the form of their salts or optically pure material. Compounds of the present disclosure and intermediates can also be converted into each other according to methods generally known to those skilled in the art. Pharmaceutical Compositions In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration (e.g., by injection, infusion, transdermal or topical administration), and rectal administration. Topical administration may also pertain to inhalation or intranasal application. The pharmaceutical compositions of the present disclosure can be made up in a solid form (including, without limitation, capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including, without limitation, solutions, suspensions or emulsions). Tablets may be either film coated or enteric coated according to methods known in the art. Typically, the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more of: a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and e) absorbents, colorants, flavors and sweeteners. Methods of Use The compounds of the present invention in free form or in pharmaceutically acceptable salt form, exhibit valuable pharmacological properties, e.g., TET2 inhibition, e.g., as indicated in vitro and in vivo tests as provided in the next sections, and are therefore indicated for therapy or for use as research chemicals, e.g., as tool compounds. Compounds of the present invention may be useful in the treatment of an indication selected from: non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS). Thus, as a further aspect, the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt thereof in therapy. In a further embodiment, the therapy is selected from a disease which may be treated by inhibition of TET2. In another embodiment, the disease is selected from the afore-mentioned list. Thus, as a further aspect, the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament. In a further embodiment, the medicament is for treatment of a disease which may be treated by inhibition of TET2. In another embodiment, the disease is selected from the afore-mentioned list. In one embodiment, there is provided the compound of Formula (I) for use in the treatment of non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS). The pharmaceutical composition or combination of the present disclosure can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg, or about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or about 1-50 mg of active ingredients. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease. The above-cited dosage properties are demonstrable in vitro and in vivo tests using advantageously mammals, e.g., mice, rats, dogs, monkeys or isolated organs, tissues and preparations thereof. The compounds of the present disclosure can be applied in vitro in the form of solutions, e.g., aqueous solutions, and in vivo either internally, parenterally, advantageously intravenously, e.g., as a suspension or in aqueous solution. The dosage in vitro may range between about 10-3 molar and 10-9 molar concentrations. A therapeutically effective amount in vivo may range depending on the route of administration, between about 0.1-500 mg/kg, or between about 1-100 mg/kg. Combinations “Combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present disclosure and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a coope-rative, e.g., synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one therapeutic agent and includes both fixed and non- fixed combinations of the therapeutic agents. The term “fixed combination” means that the therapeutic agents, e.g., a compound of the present disclosure and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the therapeutic agents, e.g., a compound of the present disclosure and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more therapeutic agents. The term “pharmaceutical combination” as used herein refers to either a fixed combination in one dosage unit form, or non-fixed combination or a kit of parts for the combined administration where two or more therapeutic agents may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect. The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses co-administration in multiple, or in separate containers (e.g., tablets, capsules, powders, and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to a desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein. The compound of the present disclosure may be administered either simultaneously with, or before or after, one or more other therapeutic agent. The compound of the present disclosure may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents. A therapeutic agent is, for example, a chemical compound, peptide, antibody, antibody fragment or nucleic acid, which is therapeutically active or enhances the therapeutic activity when administered to a patient in combination with a compound of the present disclosure. In one embodiment, the disclosure provides a product comprising a compound of the present disclosure and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a disease or condition mediated by inhibition of TET2. Products provided as a combined preparation include a composition comprising the compound of the present disclosure and the other therapeutic agent(s) together in the same pharmaceutical composition, or the compound of the present disclosure and the other therapeutic agent(s) in separate form, e.g. in the form of a kit. In one embodiment, the disclosure provides a pharmaceutical composition comprising a compound of the present disclosure and another therapeutic agent(s). Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, as described above. In one embodiment, the disclosure provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of the present disclosure. In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like. The kit of the disclosure may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit of the disclosure typically comprises directions for administration. In the combination therapies of the disclosure, the compound of the present disclosure and the other therapeutic agent may be manufactured and/or formulated by the same or different manufacturers. Moreover, the compound of the present disclosure and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the compound of the present disclosure and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of the compound of the present disclosure and the other therapeutic agent. Accordingly, the disclosure provides the use of a compound of the present disclosure for treating a disease or condition mediated by inhibition of TET2, wherein the medicament is prepared for administration with another therapeutic agent. The disclosure also provides the use of another therapeutic agent for treating a disease or condition mediated by inhibition of TET2, wherein the medicament is administered with a compound of the present disclosure. The disclosure also provides a compound of the present disclosure for use in a method of treating a disease or condition mediated by inhibition of TET2, wherein the compound of the present disclosure is prepared for administration with another therapeutic agent. The disclosure also provides another therapeutic agent for use in a method of treating a disease or condition mediated by inhibition of TET2, wherein the other therapeutic agent is prepared for administration with a compound of the present disclosure. The disclosure also provides a compound of the present disclosure for use in a method of treating a disease or condition mediated by inhibition of TET2, wherein the compound of the present disclosure is administered with another therapeutic agent. The disclosure also provides another therapeutic agent for use in a method of treating a disease or condition mediated inhibition of TET2, wherein the other therapeutic agent is administered with a compound of the present disclosure. The disclosure also provides the use of a compound of the present disclosure for treating a disease or condition mediated by inhibition of TET2, wherein the patient has previously (e.g., within 24 hours) been treated with another therapeutic agent. The disclosure also provides the use of another therapeutic agent for treating a disease or condition mediated by inhibition of TET2, wherein the patient has previously (e.g., within 24 hours) been treated with compound of the present disclosure. In one embodiment, the other therapeutic agent is selected from: anti-cancer agents, anti-nausea agents (or anti-emetics), a chemotherapy, pain relievers, cytoprotective agents, and combinations thereof. In some embodiments, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof of the present disclosure are administered in combination with one or more second agent(s) selected from a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a cytokine, an A2A antagonist, a GITR agonist, a TIM-3 inhibitor, a STING agonist, and a TLR7 agonist, to treat a disease, e.g., cancer. In another embodiment, one or more chemotherapeutic agents are used in combination with the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, for treating a disease, e.g., cancer, wherein said chemotherapeutic agents include, but are not limited to, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5- fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®), epirubicin (Ellence®), oxaliplatin (Eloxatin®), exemestane (Aromasin®), letrozole (Femara®), and fulvestrant (Faslodex®). In other embodiments, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more other anti-HER2 antibodies, e.g., trastuzumab, pertuzumab, margetuximab, or HT-19 described above, or with other anti-HER2 conjugates, e.g., ado-trastuzumab emtansine (also known as Kadcyla®, or T- DM1). In other embodiments, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more tyrosine kinase inhibitors, including but not limited to, EGFR inhibitors, Her3 inhibitors, IGFR inhibitors, and Met inhibitors, for treating a disease, e.g., cancer. For example, tyrosine kinase inhibitors include but are not limited to, Erlotinib hydrochloride (Tarceva®); Linifanib (N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N'-(2-fluoro-5-methylphenyl)urea, also known as ABT 869, available from Genentech); Sunitinib malate (Sutent®); Bosutinib (4- [(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1- yl)propoxy]quinoline-3-carbonitrile, also known as SKI-606, and described in US Patent No. 6,780,996); Dasatinib (Sprycel®); Pazopanib (Votrient®); Sorafenib (Nexavar®); Zactima (ZD6474); and Imatinib or Imatinib mesylate (Gilvec® and Gleevec®). Epidermal growth factor receptor (EGFR) inhibitors include but are not limited to, Erlotinib hydrochloride (Tarceva®), Gefitinib (Iressa®); N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3''S'')- tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®); (3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1- f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); Canertinib dihydrochloride (CI-1033); 6- [4-[(4-Ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]- 7H-Pyrrolo[2,3-d]pyrimidin-4- amine (AEE788, CAS 497839-62-0); Mubritinib (TAK165); Pelitinib (EKB569); Afatinib (Gilotrif®); Neratinib (HKI-272); N-[4-[[1-[(3-Fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5- methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester (BMS599626); N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2- methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4-quinazolinamine (XL647, CAS 781613-23-8); and 4- [4-[[(1R)-1-Phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol (PKI166, CAS187724-61- 4). EGFR antibodies include but are not limited to, Cetuximab (Erbitux®); Panitumumab (Vectibix®); Matuzumab (EMD-72000); Nimotuzumab (hR3); Zalutumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1). Other HER2 inhibitors include but are not limited to, Neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4- [(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2- enamide, and described PCT Publication No. WO 05/028443); Lapatinib or Lapatinib ditosylate (Tykerb®); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5- yl)methyl)piperidin-3-ol (BMS690514); (2E)-N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3S)- tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide (BIBW-2992, CAS 850140-72-6); N-[4-[[1-[(3-Fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1- f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester (BMS 599626, CAS 714971- 09-2); Canertinib dihydrochloride (PD183805 or CI-1033); and N-(3,4-Dichloro-2-fluorophenyl)-6- methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4- quinazolinamine (XL647, CAS 781613-23-8). HER3 inhibitors include but are not limited to, LJM716, MM-121, AMG-888, RG7116, REGN- 1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A. MET inhibitors include but are not limited to, Cabozantinib (XL184, CAS 849217-68-1); Foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); Tivantinib (ARQ197, CAS 1000873-98-2); 1- (2-Hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2- phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458); Cryzotinib (Xalkori®, PF-02341066); (3Z)-5-(2,3-Dihydro-1H-indol-1-ylsulfonyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]- 1H-pyrrol-2-yl}methylene)-1,3-dihydro-2H-indol-2-one (SU11271); (3Z)-N-(3-Chlorophenyl)-3- ({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N-methyl-2- oxoindoline-5-sulfonamide (SU11274); (3Z)-N-(3-Chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpholin- 4-ylpropyl)-1H-pyrrol-2-yl]methylene}-N-methyl-2-oxoindoline-5-sulfonamide (SU11606); 6- [Difluoro[6-(1-methyl-1Hpyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]-quinoline (JNJ38877605, CAS 943540-75-8); 2-[4-[1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin- 6-yl]-1H-pyrazol-1-yl]ethanol (PF0421225, CAS 956905-27-4); N-((2R)-1,4-Dioxan-2-ylmethyl)- N-methyl-N'-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7- yl]sulfamide (MK2461, CAS 917879-39-1); 6-[[6-(1-Methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3- b]pyridazin 3-yl]thio]-quinoline (SGX523, CAS 1022150-57-7); and (3Z)-5-[[(2,6- Dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4-[[(2R)-2-(1-pyrrolidinylmethyl)-1- pyrrolidinyl]carbonyl]-1H-pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one (PHA665752, CAS 477575-56-7). IGFR inhibitors include but are not limited to, BMS-754807, XL-228, OSI-906, GSK0904529A, A- 928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. See e.g., Yee, JNCI, 104; 975 (2012) for review. In another embodiment, the compounds of Formula (I) of the present disclosure are used in combination with one or more proliferation signalling pathway inhibitors, including but not limited to, MEK inhibitors, BRAF inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTOR inhibitors, and CDK inhibitors, for treating a disease, e.g., cancer. For example, mitogen-activated protein kinase (MEK) inhibitors include but are not limited to, XL- 518 (also known as GDC-0973, CAS No.1029872-29-4, available from ACC Corp.); 2-[(2-Chloro- 4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (also known as CI-1040 or PD184352 and described in PCT Publication No. WO2000035436); N-[(2R)-2,3- Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]- benzamide (also known as PD0325901 and described in PCT Publication No. WO2002006213); 2,3-Bis[amino[(2- aminophenyl)thio]methylene]-butanedinitrile (also known as U0126 and described in US Patent No. 2,779,780); N-[3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)-2,3- dihydroxypropyl]- cyclopropanesulfonamide (also known as RDEA119 or BAY869766 and described in PCT Publication No. WO2007014011); (3S,4R,5Z,8S,9S,11E)-14-(Ethylamino)- 8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)- dione] (also known as E6201 and described in PCT Publication No. WO2003076424); 2’-Amino- 3’-methoxyflavone (also known as PD98059 available from Biaffin GmbH & Co., KG, Germany); Vemurafenib (PLX-4032, CAS 918504-65-1); (R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4- iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (TAK-733, CAS 1035555- 63-5); Pimasertib (AS-703026, CAS 1204531-26-9); and Trametinib dimethyl sulfoxide (GSK- 1120212, CAS 1204531-25-80). BRAF inhibitors include, but are not limited to, Vemurafenib (or Zelboraf®), GDC-0879, PLX-4720 (available from Symansis), Dabrafenib (or GSK2118436), LGX 818, CEP-32496, UI-152, RAF 265, Regorafenib (BAY 73-4506), CCT239065, or Sorafenib (or Sorafenib Tosylate, or Nexavar®), or Ipilimumab (or MDX-010, MDX-101, or Yervoy). Phosphoinositide 3-kinase (PI3K) inhibitors include, but are not limited to, 4-[2-(1H-Indazol-4-yl)- 6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC0941, RG7321, GNE0941, Pictrelisib, or Pictilisib; and described in PCT Publication Nos. WO 09/036082 and WO 09/055730); Tozasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)- 5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS 958852-01- 2); (1E,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-1-[(di-2-propenylamino)methylene]- 4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a- dimethylcyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione (PX866, CAS 502632-66-8); 8- Phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6); (S)-N1-(4-methyl-5- (2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide (also known as BYL719 or Alpelisib); 2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6- dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)-1H-pyrazol-1-yl)-2-methylpropanamide (also known as GDC0032, RG7604, or Taselisib). mTOR inhibitors include but are not limited to, Temsirolimus (Torisel®); Ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30- dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4- azatricyclo[30.3.1.04,9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); Everolimus (Afinitor® or RAD001); Rapamycin (AY22989, Sirolimus®); Simapimod (CAS 164301-51-3); (5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin- 7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]- 6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4- yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-, inner salt (SF1126, CAS 936487-67-1). CDK inhibitors include but are not limited to, Palbociclib (also known as PD-0332991, Ibrance®, 6-Acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin- 7(8H)-one). In yet another embodiment, the compounds of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, of the present disclosure are used in combination with one or more pro-apoptotics, including but not limited to, IAP inhibitors, BCL2 inhibitors, MCL1 inhibitors, TRAIL agents, CHK inhibitors, for treating a disease, e.g., cancer. For examples, IAP inhibitors include but are not limited to, LCL161, GDC-0917, AEG-35156, AT406, and TL32711. Other examples of IAP inhibitors include but are not limited to those disclosed in WO04/005284, WO 04/007529, WO05/097791, WO 05/069894, WO 05/069888, WO 05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO 06/010118, WO 06/017295, and WO08/134679, all of which are incorporated herein by reference. BCL-2 inhibitors include but are not limited to, 4-[4-[[2-(4-Chlorophenyl)-5,5-dimethyl-1- cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(4-morpholinyl)-1- [(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide (also known as ABT-263 and described in PCT Publication No. WO 09/155386); Tetrocarcin A; Antimycin; Gossypol ((-)BL-193); Obatoclax; Ethyl-2-amino-6-cyclopentyl-4-(1-cyano-2-ethoxy-2- oxoethyl)-4Hchromone-3-carboxylate (HA14 –1); Oblimersen (G3139, Genasense®); Bak BH3 peptide; (-)-Gossypol acetic acid (AT-101); 4-[4-[(4'-Chloro[1,1'-biphenyl]-2-yl)methyl]-1- piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3- nitrophenyl]sulfonyl]-benzamide (ABT-737, CAS 852808-04-9); and Navitoclax (ABT-263, CAS 923564-51-6). Proapoptotic receptor agonists (PARAs) including DR4 (TRAILR1) and DR5 (TRAILR2), including but are not limited to, Dulanermin (AMG-951, RhApo2L/TRAIL); Mapatumumab (HRS-ETR1, CAS 658052-09-6); Lexatumumab (HGS-ETR2, CAS 845816-02-6); Apomab (Apomab®); Conatumumab (AMG655, CAS 896731-82-1); and Tigatuzumab(CS1008, CAS 946415-34-5, available from Daiichi Sankyo). Checkpoint Kinase (CHK) inhibitors include but are not limited to, 7-Hydroxystaurosporine (UCN- 01); 6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(3R)-3-piperidinylpyrazolo[1,5-a]pyrimidin-7-amine (SCH900776, CAS 891494-63-6); 5-(3-Fluorophenyl)-3-ureidothiophene-2-carboxylic acid N- [(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4-[((3S)-1-Azabicyclo[2.2.2]oct-3- yl)amino]-3-(1H-benzimidazol-2-yl)-6-chloroquinolin-2(1H)-one (CHIR 124, CAS 405168-58-3); 7-Aminodactinomycin (7-AAD), Isogranulatimide, debromohymenialdisine; N-[5-Bromo-4-methyl- 2-[(2S)-2-morpholinylmethoxy]-phenyl]-N'-(5-methyl-2-pyrazinyl)urea (LY2603618, CAS 911222- 45-2); Sulforaphane (CAS 4478-93-7, 4-Methylsulfinylbutyl isothiocyanate); 9,10,11,12- Tetrahydro- 9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-1,3(2H)- dione (SB-218078, CAS 135897-06-2); and TAT-S216A (YGRKKRRQRRRLYRSPAMPENL (SEQ ID NO: 1)), and CBP501 ((d-Bpa)sws(d-Phe-F5)(d-Cha)rrrqrr). In a further embodiment, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more immunomodulators (e.g., one or more of an activator of a costimulatory molecule or an inhibitor of an immune checkpoint molecule), for treating a disease, e.g., cancer.. In certain embodiments, the immunomodulator is an activator of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from an agonist (e.g., an agonistic antibody or antigen-binding fragment thereof, or a soluble fusion) of OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand. In certain embodiments, the immunomodulator is an inhibitor of an immune checkpoint molecule. In one embodiment, the immunomodulator is an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFRbeta. In one embodiment, the inhibitor of an immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3 or CTLA4, or any combination thereof. The term “inhibition” or “inhibitor” includes a reduction in a certain parameter, e.g., an activity, of a given molecule, e.g., an immune checkpoint inhibitor. For example, inhibition of an activity, e.g., a PD-1 or PD-L1 activity, of at least 5%, 10%, 20%, 30%, 40%, 50% or more is included by this term. Thus, inhibition need not be 100%. Inhibition of an inhibitory molecule can be performed at the DNA, RNA or protein level. In some embodiments, an inhibitory nucleic acid (e.g., a dsRNA, siRNA or shRNA), can be used to inhibit expression of an inhibitory molecule. In other embodiments, the inhibitor of an inhibitory signal is a polypeptide e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof, that binds to the inhibitory molecule; e.g., an antibody or fragment thereof (also referred to herein as “an antibody molecule”) that binds to PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR beta, or a combination thereof. In one embodiment, the antibody molecule is a full antibody or fragment thereof (e.g., a Fab, F(ab')2, Fv, or a single chain Fv fragment (scFv)). In yet other embodiments, the antibody molecule has a heavy chain constant region (Fc) selected from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, selected from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, more particularly, the heavy chain constant region of IgG1 or IgG4 (e.g., human IgG1 or IgG4). In one embodiment, the heavy chain constant region is human IgG1 or human IgG4. In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). In certain embodiments, the antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity to PD-1 or PD-L1 and a second binding specificity, e.g., a second binding specificity to TIM-3, LAG-3, or PD-L2. In one embodiment, the bispecific antibody molecule binds to PD-1 or PD-L1 and TIM-3. In another embodiment, the bispecific antibody molecule binds to PD-1 or PD- L1 and LAG-3. In another embodiment, the bispecific antibody molecule binds to PD-1 and PD- L1. In yet another embodiment, the bispecific antibody molecule binds to PD-1 and PD-L2. In another embodiment, the bispecific antibody molecule binds to TIM-3 and LAG-3. Any combination of the aforesaid molecules can be made in a multispecific antibody molecule, e.g., a trispecific antibody that includes a first binding specificity to PD-1 or PD-1, and a second and third binding specificities to two or more of TIM-3, LAG-3, or PD-L2. In certain embodiments, the immunomodulator is an inhibitor of PD-1, e.g., human PD-1. In another embodiment, the immunomodulator is an inhibitor of PD-L1, e.g., human PD-L1. In one embodiment, the inhibitor of PD-1 or PD-L1 is an antibody molecule to PD-1 or PD-L1. The PD-1 or PD-L1 inhibitor can be administered alone, or in combination with other immunomodulators, e.g., in combination with an inhibitor of LAG-3, TIM-3 or CTLA4. In an exemplary embodiment, the inhibitor of PD-1 or PD-L1, e.g., the anti-PD-1 or PD-L1 antibody molecule, is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule. In another embodiment, the inhibitor of PD-1 or PD-L1, e.g., the anti-PD-1 or PD-L1 antibody molecule, is administered in combination with a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule. In yet other embodiments, the inhibitor of PD-1 or PD-L1, e.g., the anti-PD-1 antibody molecule, is administered in combination with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule, and a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule. Other combinations of immunomodulators with a PD-1 inhibitor (e.g., one or more of PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR) are also within the present disclosure. Any of the antibody molecules known in the art or disclosed herein can be used in the aforesaid combinations of inhibitors of checkpoint molecule. PD-1 inhibitors In some embodiments, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with a PD-1 inhibitor to treat a disease, e.g., cancer. In some embodiments, the PD-1 inhibitor is selected from PDR001 (Novartis), Nivolumab (Bristol-Myers Squibb), Pembrolizumab (Merck & Co), Pidilizumab (CureTech), MEDI0680 (Medimmune), Cemiplimab (REGN2810, Regeneron), Dostarlimab (TSR-042, Tesaro), PF-06801591 (Pfizer), Tislelizumab (BGB-A317, Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), Balstilimab (AGEN2035, Agenus), Sintilimab (InnoVent), Toripalimab (Shanghai Junshi Bioscience), Camrelizumab (Jiangsu Hengrui Medicine Co.), and AMP-224 (Amplimmune), Penpulimab (Akeso Biopharma Inc), Zimberelimab (Arcus Biosciences Inc), Prolgolimab (Biocad Ltd), in particular PDR001 or Tislelizumab. In a further embodiment, the PD- 1 inhibitor is an anti-PD-1 antibody molecule as described in US 2015/0210769, published on July 30, 2015, entitled “Antibody Molecules to PD-1 and Uses Thereof,” incorporated by reference in its entirety. PD-1 inhibitors In some embodiments, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with a PD-1 inhibitor to treat a disease, e.g., cancer. In some embodiments, the PD-1 inhibitor is selected from PDR001 (Novartis), Nivolumab (Bristol-Myers Squibb), Pembrolizumab (Merck & Co), Pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune). Exemplary PD-1 Inhibitors In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule as described in US 2015/0210769, published on July 30, 2015, entitled “Antibody Molecules to PD-1 and Uses Thereof,” incorporated by reference in its entirety. In one embodiment, the anti-PD-1 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 3 (e.g., from the heavy and light chain variable region sequences of BAP049-Clone-E or BAP049-Clone-B disclosed in Table 3), or encoded by a nucleotide sequence shown in Table 3. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 3). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 3). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 3). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 2). In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 1, or encoded by a nucleotide sequence shown in Table 1. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 3, a VHCDR2 amino acid sequence of SEQ ID NO: 4, and a VHCDR3 amino acid sequence of SEQ ID NO: 5; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 12, a VLCDR2 amino acid sequence of SEQ ID NO: 13, and a VLCDR3 amino acid sequence of SEQ ID NO: 88, each disclosed in Table 1. In one embodiment, the antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 26, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 27, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 28; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 32, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 33, each disclosed in Table 1. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 8. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 22. In one embodiment, the anti-PD-1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 18. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 8 and a VL comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 8 and a VL comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 9. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 23 or 19, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 23 or 19. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 9 and a VL encoded by the nucleotide sequence of SEQ ID NO: 23 or 19. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 10. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 24. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 20. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 24. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 11, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 11. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 25 or 21, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 25 or 21. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 11 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 25 or 21. The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0210769, incorporated by reference in its entirety. Table 1. Amino acid and nucleotide sequences of exemplary anti-PD-1 antibody molecules
Figure imgf000046_0001
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Figure imgf000051_0001
Figure imgf000052_0001
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Figure imgf000054_0001
Other Exemplary PD-1 Inhibitors In some embodiments, the anti-PD-1 antibody is Nivolumab (CAS Registry Number: 946414-94- 4). Alternative names for Nivolumab include MDX-1106, MDX-1106-04, ONO-4538, BMS-936558 or OPDIVO®. Nivolumab is a fully human IgG4 monoclonal antibody, which specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US Pat No.8,008,449 and PCT Publication No. WO2006/121168, incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Nivolumab, e.g., as disclosed in Table 2. In other embodiments, the anti-PD-1 antibody is Pembrolizumab. Pembrolizumab (Trade name KEYTRUDA formerly Lambrolizumab, also known as Merck 3745, MK-3475 or SCH-900475) is a humanized IgG4 monoclonal antibody that binds to PD1. Pembrolizumab is disclosed, e.g., in Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134–44, PCT Publication No. WO2009/114335, and US Patent No. 8,354,509, incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Pembrolizumab, e.g., as disclosed in Table 2. In some embodiments, the anti-PD-1 antibody is Pidilizumab. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in PCT Publication No. WO2009/101611, incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Pidilizumab, e.g., as disclosed in Table 2. Other anti-PD1 antibodies are disclosed in US Patent No. 8,609,089, US Publication No. 2010028330, and/or US Publication No.20120114649, incorporated by reference in their entirety. Other anti-PD1 antibodies include AMP 514 (Amplimmune). In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (Medimmune), also known as AMP-514. MEDI0680 and other anti-PD-1 antibodies are disclosed in US 9,205,148 and WO 2012/145493, incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of MEDI0680. In one embodiment, the anti-PD-1 antibody molecule is REGN2810 (Regeneron). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of REGN2810. In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (Pfizer). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of PF-06801591. In one embodiment, the anti-PD-1 antibody molecule is BGB-A317 or BGB-108 (Beigene). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BGB-A317 or BGB-108. In one embodiment, the anti-PD-1 antibody molecule is INCSHR1210 (Incyte), also known as INCSHR01210 or SHR-1210. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of INCSHR1210. In one embodiment, the anti-PD-1 antibody molecule is TSR-042 (Tesaro), also known as ANB011. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-042. Further known anti-PD-1 antibodies include those described, e.g., in WO 2015/112800, WO 2016/092419, WO 2015/085847, WO 2014/179664, WO 2014/194302, WO 2014/209804, WO 2015/200119, US 8,735,553, US 7,488,802, US 8,927,697, US 8,993,731, and US 9,102,727, incorporated by reference in their entirety. In one embodiment, the anti-PD-1 antibody is an antibody that competes for binding with, and/or binds to the same epitope on PD-1 as, one of the anti-PD-1 antibodies described herein. In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signalling pathway, e.g., as described in US 8,907,053, incorporated by reference in its entirety. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg (Amplimmune), e.g., disclosed in WO 2010/027827 and WO 2011/066342, incorporated by reference in their entirety). Table 2. Amino acid sequences of other exemplary anti-PD-1 antibody molecules
Figure imgf000057_0001
Figure imgf000058_0001
In some embodiments, the anti-PD-1 antibody is Tislelizumab.Tislelizumab can have a heavy chain of SEQ ID NO: 43 and a light chain of SEQ ID NO: 44. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Tislelizumab, e.g., as disclosed in Table 3. In some embodiments, the anti-PD-1 antibody is dosed at 100 mg per week. In some embodiments, tislelizumab and is dosed at 300 mg IV on day 1 of each 28 day cycle. In some embodiments, tislelizumab can be dosed at 500 mg once every four (4) weeks. In another embodiment, the anti-PD-1 antibody molecule, e.g., tislelizumab, and comprises a heavy chain and/or light chain, VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following: TABLE 3: Amino acid sequences of other exemplary anti-PD-1 antibody molecules
Figure imgf000059_0001
Figure imgf000060_0001
In some embodiments, the PD-1 inhibitor comprises the HCDRs and LCDRs of tislelizumab as set forth in SEQ ID NOs: 47-52. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered at a flat dose of between about 100 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 100 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 100 mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 100 mg to about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 100 mg to about 200 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200 mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200 mg to about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300 mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 400 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 400 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 500 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 600 mg to about 700 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 700 mg to about 800 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 800 mg to about 900 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 900 mg to about 1000 mg. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered at a flat dose of about 100 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 700 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 800 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 900 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 1000 mg. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered once every ten weeks. In some embodiments, the PD-1 inhibitor is administered once every nine weeks. In some embodiments, the PD-1 inhibitor is administered once every eight weeks. In some embodiments, the PD-1 inhibitor is administered once every seven weeks. In some embodiments, the PD-1 inhibitor is administered once every six weeks. In some embodiments, the PD-1 inhibitor is administered once every five weeks. In some embodiments, the PD-1 inhibitor is administered once every four weeks. In some embodiments, the PD-1 inhibitor is administered once every three weeks. In some embodiments, the PD-1 inhibitor is administered once every two weeks. In some embodiments, the PD-1 inhibitor is administered once every week. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered intravenously. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered over a period of about 20 minutes to 40 minutes (e.g., about 30 minutes). In some embodiments, the PD-1 inhibitor is administered over a period of about 30 minutes. In some embodiments, the PD-1 inhibitor is administered over a period of about an hour. In some embodiments, the PD-1 inhibitor is administered over a period of about two hours. In some embodiments, the PD-1 inhibitor is administered over a period of about three hours. In some embodiments, the PD-1 inhibitor is administered over a period of about four hours. In some embodiments, the PD-1 inhibitor is administered over a period of about five hours. In some embodiments, the PD-1 inhibitor is administered over a period of about six hours. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered at a dose between about 300 mg to about 500 mg (e.g., about 400 mg), intravenously, once every four weeks. In some embodiments, the PD-1 inhibitor is administered at a dose between about 200 mg to about 400 mg (e.g., about 300 mg), intravenously, once every three weeks. In some embodiments, tislelizumab is administered at a dose of 400 mg, once every four weeks. In some embodiments, tislelizumab is administered at a dose of 300 mg, once every three weeks. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered at a dose between about 300 mg to about 500 mg (e.g., about 400 mg), intravenously, over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes), once every two weeks. In some embodiments, the PD-1 inhibitor is administered at a dose between about 200 mg to about 400 mg (e.g., about 300 mg), intravenously, over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes), once every three weeks. In some embodiments, the PD-1 inhibitor (e.g., tislelizumab) is administered at a dose of about 100 mg per week. For example, if a 10-week dose is given to a patient, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 1000 mg. If a 9-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 900 mg. If an 8-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 800 mg. If a 7-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 700 mg. If a 6-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 600 mg. If a 5-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 500 mg. If a 4-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 400 mg. If a 3-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 300 mg. If a 2-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 200 mg. If a 1-week dose is given, then the PD-1 inhibitor (e.g., tislelizumab) can be given at 100 mg. For example, if an anti-PD-1 antibody, such as tislelizumab is used, it can be administered at a dose of 200 mg as an intravenous infusion, once every three week. Alternatively, tislelizumab can be administered at a dose of 300 mg as an intravenous infusion, once every four weeks. If an anti-PD-1 antibody, such as tislelizumab is used, it can be administered at a dose of 300 mg as an intravenous infusion, once every three week. Alternatively, tislelizumab can be administered at a dose of 400 mg as an intravenous infusion, once every four weeks. PD-L1 Inhibitors In some embodiments, the compounds of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, of the present disclosure are used in combination with a PD-L1 inhibitor for treating a disease, e.g., cancer. In some embodiments, the PD-L1 inhibitor is selected from FAZ053 (Novartis), Atezolizumab (Genentech/Roche), Avelumab (Merck Serono and Pfizer), Durvalumab (MedImmune/AstraZeneca), or BMS-936559 (Bristol-Myers Squibb). Exemplary PD-L1 Inhibitors In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule as disclosed in US 2016/0108123, published on April 21, 2016, entitled “Antibody Molecules to PD-L1 and Uses Thereof,” incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 5 (e.g., from the heavy and light chain variable region sequences of BAP058-Clone O or BAP058-Clone N disclosed in Table 5), or encoded by a nucleotide sequence shown in Table 5. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 5). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 5). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 5). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GYTFTSYWMY (SEQ ID NO: 53). In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 4, or encoded by a nucleotide sequence shown in Table 4. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 54, a VHCDR2 amino acid sequence of SEQ ID NO: 55, and a VHCDR3 amino acid sequence of SEQ ID NO: 56; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 63, a VLCDR2 amino acid sequence of SEQ ID NO: 64, and a VLCDR3 amino acid sequence of SEQ ID NO: 65, each disclosed in Table 4. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 81, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 82, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 83; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 86, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 87, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 88, each disclosed in Table 4. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 59, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 59. In one embodiment, the anti-PD-L1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 69. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 73. In one embodiment, the anti-PD-L1 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 77. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 69. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 77. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 60, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 60. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 78, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 78. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 74, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 74. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 78, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 78. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 60 and a VL encoded by the nucleotide sequence of SEQ ID NO: 78. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 74 and a VL encoded by the nucleotide sequence of SEQ ID NO: 78. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 61. In one embodiment, the anti-PD-L1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 70. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 75. In one embodiment, the anti-PD-L1 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 79. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 70. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a light chain comprising the amino acid sequence of SEQ ID NO: 79. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 62, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 62. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 72, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 72. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 76, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 76. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 80, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 80. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 62 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 72. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 76 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 80. The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2016/0108123, incorporated by reference in its entirety. Table 4. Amino acid and nucleotide sequences of exemplary anti-PD-L1 antibody molecules
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Other Exemplary PD-L1 Inhibitors In some embodiments, the PD-L1 inhibitor is anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 inhibitor is selected from YW243.55.S70, MPDL3280A, MEDI-4736, or MDX- 1105MSB-0010718C (also referred to as A09-246-2) disclosed in, e.g., WO 2013/0179174, and having a sequence disclosed herein (or a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence specified). In one embodiment, the PD-L1 inhibitor is MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in PCT Publication No. WO 2007/005874. In one embodiment, the PD-L1 inhibitor is YW243.55.S70. The YW243.55.S70 antibody is an anti-PD-L1 described in PCT Publication No. WO 2010/077634. In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche) also known as Atezolizumabm, RG7446, RO5541267, YW243.55.S70, or TECENTRIQ™. MDPL3280A is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in U.S. Patent No.: 7,943,743 and U.S Publication No.: 20120039906 incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Atezolizumab, e.g., as disclosed in Table 5. In other embodiments, the PD-L2 inhibitor is AMP-224. AMP-224 is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1 (B7-DCIg; Amplimmune; e.g., disclosed in PCT Publication Nos. WO2010/027827 and WO2011/066342). In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the anti-PD-L1 antibody molecule is Avelumab (Merck Serono and Pfizer), also known as MSB0010718C. Avelumab and other anti-PD-L1 antibodies are disclosed in WO 2013/079174, incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Avelumab, e.g., as disclosed in Table 5. In one embodiment, the anti-PD-L1 antibody molecule is Durvalumab (MedImmune/AstraZeneca), also known as MEDI4736. Durvalumab and other anti-PD-L1 antibodies are disclosed in US 8,779,108, incorporated by reference in its entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Durvalumab, e.g., as disclosed in Table 5. In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers Squibb), also known as MDX-1105 or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2015/081158, incorporated by reference in their entirety. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BMS-936559, e.g., as disclosed in Table 5. Further known anti-PD-L1 antibodies include those described, e.g., in WO 2015/181342, WO 2014/100079, WO 2016/000619, WO 2014/022758, WO 2014/055897, WO 2015/061668, WO 2013/079174, WO 2012/145493, WO 2015/112805, WO 2015/109124, WO 2015/195163, US 8,168,179, US 8,552,154, US 8,460,927, and US 9,175,082, incorporated by reference in their entirety. In one embodiment, the anti-PD-L1 antibody is an antibody that competes for binding with, and/or binds to the same epitope on PD-L1 as, one of the anti-PD-L1 antibodies described herein. Table 5. Amino acid sequences of other exemplary anti-PD-L1 antibody molecules
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
LAG-3 Inhibitors In some embodiments, the compounds of Formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, of the present disclosure are used in combination with a LAG-3 inhibitor to treat a disease, e.g., cancer. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), or TSR- 033 (Tesaro). Exemplary LAG-3 Inhibitors In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule as disclosed in US 2015/0259420, published on September 17, 2015, entitled “Antibody Molecules to LAG-3 and Uses Thereof,” incorporated by reference in its entirety. In one embodiment, the anti-LAG-3 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 7 (e.g., from the heavy and light chain variable region sequences of BAP050-Clone I or BAP050-Clone J disclosed in Table 7), or encoded by a nucleotide sequence shown in Table 7. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 7). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 7). In some embodiments, the CDRs are according to the combined CDR definitions of both Kabat and Chothia (e.g., as set out in Table 7). In one embodiment, the combination of Kabat and Chothia CDR of VH CDR1 comprises the amino acid sequence GFTLTNYGMN (SEQ ID NO: 100). In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 6, or encoded by a nucleotide sequence shown in Table 6. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 101, a VHCDR2 amino acid sequence of SEQ ID NO: 102, and a VHCDR3 amino acid sequence of SEQ ID NO: 103; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 112, a VLCDR2 amino acid sequence of SEQ ID NO: 113, and a VLCDR3 amino acid sequence of SEQ ID NO: 114, each disclosed in Table 6. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 136 or 144, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 138 or 146, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 140 or 148; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 146 or 154, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 148 or 156, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 150 or 158, each disclosed in Table 7. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising a VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 158 or 144, a VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 159 or 146, and a VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 160 or 148; and a VL comprising a VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 146 or 154, a VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 148 or 156, and a VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 150 or 158, each disclosed in Table 6. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 106. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 118. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 124. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 130. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 106 and a VL comprising the amino acid sequence of SEQ ID NO: 118. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 124 and a VL comprising the amino acid sequence of SEQ ID NO: 130. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 107 or 115, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 107 or 115. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 119 or 127, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 119 or 127. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 125 or 133, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 125 or 133. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 131 or 139, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 131 or 139. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 107 or 115 and a VL encoded by the nucleotide sequence of SEQ ID NO: 119 or 127. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 125 or 133 and a VL encoded by the nucleotide sequence of SEQ ID NO: 131 or 139. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 109, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 109. In one embodiment, the anti-LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 121, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 121. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 127, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 127. In one embodiment, the anti-LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 133. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 109 and a light chain comprising the amino acid sequence of SEQ ID NO: 121. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 127 and a light chain comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 110 or 124, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 110 or 124. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 122 or 130, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 122 or 130. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 128 or 136, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 128 or 136. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 134 or 142, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 134 or 142. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 110 or 124 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 122 or 130. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 128 or 136 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 134 or 142. The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0259420, incorporated by reference in its entirety. Table 6. Amino acid and nucleotide sequences of exemplary anti-LAG-3 antibody molecules
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Other Exemplary LAG-3 Inhibitors In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is BMS-986016 (Bristol-Myers Squibb), also known as BMS986016. BMS- 986016 and other anti-LAG-3 antibodies are disclosed in WO 2015/116539 and US 9,505,839, incorporated by reference in their entirety. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BMS-986016, e.g., as disclosed in Table 7. In one embodiment, the anti-LAG-3 antibody molecule is TSR-033 (Tesaro). In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-033. In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (GSK and Prima BioMed). IMP731 and other anti-LAG-3 antibodies are disclosed in WO 2008/132601 and US 9,244,059, incorporated by reference in their entirety. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of IMP731, e.g., as disclosed in Table 8. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of GSK2831781. In one embodiment, the anti-LAG-3 antibody molecule is IMP761 (Prima BioMed). In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of IMP761. Further known anti-LAG-3 antibodies include those described, e.g., in WO 2008/132601, WO 2010/019570, WO 2014/140180, WO 2015/116539, WO 2015/200119, WO 2016/028672, US 9,244,059, US 9,505,839, incorporated by reference in their entirety. In one embodiment, the anti-LAG-3 antibody is an antibody that competes for binding with, and/or binds to the same epitope on LAG-3 as, one of the anti-LAG-3 antibodies described herein. In one embodiment, the anti-LAG-3 inhibitor is a soluble LAG-3 protein, e.g., IMP321 (Prima BioMed), e.g., as disclosed in WO 2009/044273, incorporated by reference in its entirety. Table 7. Amino acid sequences of other exemplary anti-LAG-3 antibody molecules
Figure imgf000098_0001
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TIM-3 Inhibitors In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIM-3. In some embodiments, the compounds of Formula (I), or a pharmaceutically acceptable salt, or tautomer thereof, of the present disclosure are used in combination with a TIM-3 inhibitor to treat a disease, e.g., cancer. In some embodiments, the TIM-3 inhibitor is MGB453 (Novartis) or TSR- 022 (Tesaro). Exemplary TIM-3 Inhibitors In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule. In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule as disclosed in US 2015/0218274, published on August 6, 2015, entitled “Antibody Molecules to TIM-3 and Uses Thereof,” incorporated by reference in its entirety. In one embodiment, the anti-TIM-3 antibody molecule comprises at least one, two, three, four, five or six complementarity determining regions (CDRs) (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 9 (e.g., from the heavy and light chain variable region sequences of ABTIM3-hum11 or ABTIM3-hum03 disclosed in Table 8), or encoded by a nucleotide sequence shown in Table 8. In some embodiments, the CDRs are according to the Kabat definition (e.g., as set out in Table 9). In some embodiments, the CDRs are according to the Chothia definition (e.g., as set out in Table 9). In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to an amino acid sequence shown in Table 8, or encoded by a nucleotide sequence shown in Table 8. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 174, a VHCDR2 amino acid sequence of SEQ ID NO: 166, and a VHCDR3 amino acid sequence of SEQ ID NO: 168; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 175, a VLCDR2 amino acid sequence of SEQ ID NO: 176, and a VLCDR3 amino acid sequence of SEQ ID NO: 177, each disclosed in Table 9. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain variable region (VH) comprising a VHCDR1 amino acid sequence of SEQ ID NO: 174, a VHCDR2 amino acid sequence of SEQ ID NO: 185, and a VHCDR3 amino acid sequence of SEQ ID NO: 168; and a light chain variable region (VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 175, a VLCDR2 amino acid sequence of SEQ ID NO: 176, and a VLCDR3 amino acid sequence of SEQ ID NO: 177, each disclosed in Table 8. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 171, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 171. In one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 181. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 187, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 187. In one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 191, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 191. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 171 and a VL comprising the amino acid sequence of SEQ ID NO: 181. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 187 and a VL comprising the amino acid sequence of SEQ ID NO: 191. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 172, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 172. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 182, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 182. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 188, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 188. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 192, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 192. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 172 and a VL encoded by the nucleotide sequence of SEQ ID NO: 182. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 188 and a VL encoded by the nucleotide sequence of SEQ ID NO: 192. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 173, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 173. In one embodiment, the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 183, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 183. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 189, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 189. In one embodiment, the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 193. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 173 and a light chain comprising the amino acid sequence of SEQ ID NO: 183. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 189 and a light chain comprising the amino acid sequence of SEQ ID NO: 193. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 174, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 174. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 184, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 184. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 190, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 190. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 194, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 194. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 174 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 184. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 190 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 194. The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0218274, incorporated by reference in its entirety. Table 8. Amino acid and nucleotide sequences of exemplary anti-TIM-3 antibody molecules
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Other Exemplary TIM-3 Inhibitors In one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (AnaptysBio/Tesaro). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of TSR-022. In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of APE5137 or APE5121, e.g., as disclosed in Table 9. APE5137, APE5121, and other anti-TIM-3 antibodies are disclosed in WO 2016/161270, incorporated by reference in its entirety. In one embodiment, the anti-TIM-3 antibody molecule is the antibody clone F38-2E2. In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of F38-2E2. Further known anti-TIM-3 antibodies include those described, e.g., in WO 2016/111947, WO 2016/071448, WO 2016/144803, US 8,552,156, US 8,841,418, and US 9,163,087, incorporated by reference in their entirety. In one embodiment, the anti-TIM-3 antibody is an antibody that competes for binding with, and/or binds to the same epitope on TIM-3 as, one of the anti-TIM-3 antibodies described herein. Table 9. Amino acid sequences of other exemplary anti-TIM-3 antibody molecules
Figure imgf000111_0001
Cytokines In yet another embodiment, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more cytokines, including but not limited to, interferon, IL-2, IL-15, IL-7, or IL21. In certain embodiments, compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, are administered in combination with an IL-15/IL-15Ra complex. In some embodiments, the IL-15/IL-15Ra complex is selected from NIZ985 (Novartis), ATL-803 (Altor) or CYP0150 (Cytune). Exemplary IL-15/IL-15Ra complexes In one embodiment, the cytokine is IL-15 complexed with a soluble form of IL-15 receptor alpha (IL-15Ra). The IL-15/IL-15Ra complex may comprise IL-15 covalently or noncovalently bound to a soluble form of IL-15Ra. In a particular embodiment, the human IL-15 is noncovalently bonded to a soluble form of IL-15Ra. In a particular embodiment, the human IL-15 of the formulation comprises an amino acid sequence of SEQ ID NO: 199 in Table 10 or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 199, and the soluble form of human IL-15Ra comprises an amino acid sequence of SEQ ID NO: 200 in Table 10, or an amino acid sequence at least 85%, 90%, 95%, or 99% identical or higher to SEQ ID NO: 200, as described in WO 2014/066527, incorporated by reference in its entirety. The molecules described herein can be made by vectors, host cells, and methods described in WO 2007084342, incorporated by reference in its entirety. Table 10. Amino acid and nucleotide sequences of exemplary IL-15/IL-15Ra complexes
Figure imgf000112_0001
Other exemplary IL-15/IL-15Ra complexes In one embodiment, the IL-15/IL-15Ra complex is ALT-803, an IL-15/IL-15Ra Fc fusion protein (IL-15N72D:IL-15RaSu/Fc soluble complex). ALT-803 is described in WO 2008/143794, incorporated by reference in its entirety. In one embodiment, the IL-15/IL-15Ra Fc fusion protein comprises the sequences as disclosed in Table 11. In one embodiment, the IL-15/IL-15Ra complex comprises IL-15 fused to the sushi domain of IL- 15Ra (CYP0150, Cytune). The sushi domain of IL-15Ra refers to a domain beginning at the first cysteine residue after the signal peptide of IL-15Ra, and ending at the fourth cysteine residue after said signal peptide. The complex of IL-15 fused to the sushi domain of IL-15Ra is described in WO 2007/04606 and WO 2012/175222, incorporated by reference in their entirety. In one embodiment, the IL-15/IL-15Ra sushi domain fusion comprises the sequences as disclosed in Table 11. Table 11. Amino acid sequences of other exemplary IL-15/IL-15Ra complexes
Figure imgf000113_0001
In yet another embodiment, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more agonists of toll like receptors (TLRs, e.g., TLR7, TLR8, TLR9) to treat a disease, e.g., cancer. In some embodiments, a compound of the present disclosure can be used in combination with a TLR7 agonist or a TLR7 agonist conjugate. In some embodiments, the TLR7 agonist comprises a compound disclosed in International Application Publication No. WO2011/049677, which is hereby incorporated by reference in its entirety. In some embodiments, the TLR7 agonist comprises 3-(5-amino-2-(4-(2-(3,3-difluoro-3- phosphonopropoxy)ethoxy)-2-methylphenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic acid. In some embodiments, the TLR7 agonist comprises a compound of formula: In another embodiment, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more angiogenesis inhibitors to treat cancer, e.g., Bevacizumab (Avastin®), axitinib (Inlyta®); Brivanib alaninate (BMS-582664, (S)-((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1- f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); Sorafenib (Nexavar®); Pazopanib (Votrient®); Sunitinib malate (Sutent®); Cediranib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS 332012- 40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib (Gleevec®); Ponatinib (AP24534, CAS 943319-70-8); Tivozanib (AV951, CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037- 03-7); Vatalanib dihydrochloride (PTK787, CAS 212141-51-0); Brivanib (BMS-540215, CAS 649735-46-6); Vandetanib (Caprelsa® or AZD6474); Motesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3- pyridinecarboxamide, described in PCT Publication No. WO 02/066470); Dovitinib dilactic acid (TKI258, CAS 852433-84-2); Linfanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Lestaurtinib (CAS 111358-88-4); N-[5-[[[5-(1,1-Dimethylethyl)-2- oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5- yl)methyl)piperidin-3-ol (BMS690514); N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7- [[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]- 4-quinazolinamine (XL647, CAS 781613-23-8); 4-Methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4- yl]amino]-N-[3-(trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); or Aflibercept (Eylea®). In another embodiment, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more heat shock protein inhibitors to treat cancer, e.g., Tanespimycin (17-allylamino-17-demethoxygeldanamycin, also known as KOS-953 and 17-AAG, available from SIGMA, and described in US Patent No. 4,261,989); Retaspimycin (IPI504), Ganetespib (STA-9090); [6-Chloro-9-(4-methoxy-3,5- dimethylpyridin-2-ylmethyl)-9H-purin-2-yl]amine (BIIB021 or -CNF2024, CAS 848695-25-0); trans-4-[[2-(Aminocarbonyl)-5-[4,5,6,7-tetrahydro-6,6-dimethyl-4-oxo-3-(trifluoromethyl)-1H- indazol-1-yl]phenyl]amino]cyclohexyl glycine ester (SNX5422 or PF04929113, CAS 908115-27- 5); 5-[2,4-Dihydroxy-5-(1-methylethyl)phenyl]-N-ethyl-4-[4-(4-morpholinylmethyl)phenyl]- 3- Isoxazolecarboxamide (AUY922, CAS 747412-49-3); or 17-Dimethylaminoethylamino-17- demethoxygeldanamycin (17-DMAG). In yet another embodiment, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more HDAC inhibitors or other epigenetic modifiers. Exemplary HDAC inhibitors include, but not limited to, Voninostat (Zolinza®); Romidepsin (Istodax®); Treichostatin A (TSA); Oxamflatin; Vorinostat (Zolinza®, Suberoylanilide hydroxamic acid); Pyroxamide (syberoyl-3-aminopyridineamide hydroxamic acid); Trapoxin A (RF-1023A); Trapoxin B (RF-10238); Cyclo[(αS,2S)-α-amino-η-oxo-2- oxiraneoctanoyl-O-methyl-D-tyrosyl-L-isoleucyl-L-prolyl] (Cyl-1); Cyclo[(αS,2S)-α-amino-η-oxo-2- oxiraneoctanoyl-O-methyl-D-tyrosyl-L-isoleucyl-(2S)-2-piperidinecarbonyl] (Cyl-2); Cyclic[L- alanyl-D-alanyl-(2S)-η-oxo-L-α-aminooxiraneoctanoyl-D-prolyl] (HC-toxin); Cyclo[(αS,2S)-α- amino-η-oxo-2-oxiraneoctanoyl-D-phenylalanyl-L-leucyl-(2S)-2-piperidinecarbonyl] (WF-3161); Chlamydocin ((S)-Cyclic(2-methylalanyl-L-phenylalanyl-D-prolyl-η-oxo-L-α- aminooxiraneoctanoyl); Apicidin (Cyclo(8-oxo-L-2-aminodecanoyl-1-methoxy-L-tryptophyl-L- isoleucyl-D-2-piperidinecarbonyl); Romidepsin (Istodax®, FR-901228); 4-Phenylbutyrate; Spiruchostatin A; Mylproin (Valproic acid); Entinostat (MS-275, N-(2-Aminophenyl)-4-[N- (pyridine-3-yl-methoxycarbonyl)-amino-methyl]-benzamide); Depudecin (4,5:8,9-dianhydro- 1,2,6,7,11-pentadeoxy- D-threo-D-ido-Undeca-1,6-dienitol); 4-(Acetylamino)-N-(2-aminophenyl)- benzamide (also known as CI-994); N1-(2-Aminophenyl)-N8-phenyl-octanediamide (also known as BML-210); 4-(Dimethylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide (also known as M344); (E)-3-(4-(((2-(1H-indol-3-yl)ethyl)(2-hydroxyethyl)amino)-methyl)phenyl)-N- hydroxyacrylamide; Panobinostat(Farydak®); Mocetinostat, and Belinostat (also known as PXD101, Beleodaq®, or (2E)-N-Hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide), or chidamide (also known as CS055 or HBI-8000, (E)-N-(2-amino-5-fluorophenyl)-4-((3-(pyridin-3- yl)acrylamido)methyl)benzamide). Other epigenetic modifiers include but not limited to inhibitors of EZH2 (enhancer of zeste homolog 2), EED (embryonic ectoderm development), or LSD1 (lysine-specific histone demethylase 1A or KDM1A). In yet another embodiment, the compounds of Formula (I), or a pharmaceutically acceptable salt, thereof, of the present disclosure are used in combination with one or more inhibitors of indoleamine-pyrrole 2,3-dioxygenase (IDO), for example, Indoximod (also known as NLG-8189), α-Cyclohexyl-5H-imidazo[5,1-a]isoindole-5-ethanol (also known as NLG919), or (4E)-4-[(3- Chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine (also known as INCB024360), to treat cancer. Chimeric Antigen Receptors The present disclosure provides for the compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in combination with adoptive immunotherapy methods and reagents such as chimeric antigen receptor (CAR) immune effector cells, e.g., T cells and/or NK cells, or chimeric TCR-transduced immune effector cells, e.g., T cells. This section describes CAR technology generally that is useful in combination with the Compound of Formula (I), or a pharmaceutically acceptable salt thereof, and describes CAR reagents, e.g., cells and compositions, and methods. In general, aspects of the present disclosure pertain to or include an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor antigen as described herein, a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain described herein) (e.g., an intracellular signaling domain comprising a costimulatory domain (e.g., a costimulatory domain described herein) and/or a primary signaling domain (e.g., a primary signaling domain described herein). In other aspects, the present disclosure includes: host cells containing the above nucleic acids and isolated proteins encoded by such nucleic acid molecules. CAR nucleic acid constructs, encoded proteins, containing vectors, host cells, pharmaceutical compositions, methods of making, and methods of administration and treatment related to the present disclosure are disclosed in detail in International Patent Application Publication Nos., WO2020/047452, WO2019/241426, WO2016/164731, WO2021/108613, and WO2020/176397which are incorporated by reference in its entirety. In one aspect, the disclosure pertains to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain described herein) (e.g., an intracellular signaling domain comprising a costimulatory domain (e.g., a costimulatory domain described herein) and/or a primary signaling domain (e.g., a primary signaling domain described herein). In some embodiments, the tumor-supporting antigen is an antigen present on a stromal cell or a myeloid-derived suppressor cell (MDSC). In other aspects, the disclosure features polypeptides encoded by such nucleic acids and host cells containing such nucleic acids and/or polypeptides. Alternatively, aspects of the disclosure pertain to isolated nucleic acid encoding a chimeric T cell receptor (TCR) comprising a TCR alpha and/or TCR beta variable domain with specificity for a cancer antigen described herein. See for example, Dembic et al., Nature, 320, 232-238 (1986), Schumacher, Nat. Rev. Immunol., 2, 512-519 (2002), Kershaw et al., Nat. Rev. Immunol., 5, 928- 940 (2005), Xue et al., Clin. Exp. Immunol., 139, 167-172 (2005), Rossig et al., Mol. Ther., 10, 5- 18 (2004), and Murphy et al., Immunity, 22, 403-414 (2005); (Morgan et al. J. Immunol., 171, 3287-3295 (2003), Hughes et al., Hum. Gene Ther., 16, 1-16 (2005), Zhao et al., J. Immunol., 174, 4415-4423 (2005), Roszkowski et al., Cancer Res., 65, 1570-1576 (2005), and Engels et al., Hum. Gene Ther., 16, 799-810 (2005); US2009/03046557, the contents of which are hereby incorporated by reference in their entirety. Such chimeric TCRs may recognize, for example, cancer antigens such as MART-1, gp-100, p53, and NY-ESO-1, MAGE A3/A6, MAGEA3, SSX2, HPV-16 E6 or HPV-16 E7. In other aspects, the disclosure features polypeptides encoded by such nucleic acids and host cells containing such nucleic acids and/or polypeptides. Targets The present disclosure provides cells, e.g., immune effector cells (e.g., T cells, NK cells), that are engineered to contain one or more CARs that direct the immune effector cells to undesired cells (e.g., cancer cells). This is achieved through an antigen binding domain on the CAR that is specific for a cancer associated antigen. There are two classes of cancer associated antigens (tumor antigens) that can be targeted by the CARs of the instant disclosure: (1) cancer associated antigens that are expressed on the surface of cancer cells; and (2) cancer associated antigens that itself is intracellar, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by MHC (major histocompatibility complex). In some embodiments, the tumor antigen is chosen from one or more of: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1- 4)bDGlcp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase- like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type- A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight- melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta- specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP- 2); Cytochrome P4501B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY- TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE- 1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C- type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). A CAR described herein can comprise an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor- supporting antigen as described herein). In some embodiments, the tumor-supporting antigen is an antigen present on a stromal cell or a myeloid-derived suppressor cell (MDSC). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, the CAR-expressing cells destroy the tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival. In embodiments, the stromal cell antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin. In an embodiment, the FAP-specific antibody is, competes for binding with, or has the same CDRs as, sibrotuzumab. In embodiments, the MDSC antigen is chosen from one or more of: CD33, CD11b, C14, CD15, and CD66b. Accordingly, in some embodiments, the tumor-supporting antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CD11b, C14, CD15, and CD66b. Antigen Binding Domain Structures In some embodiments, the antigen binding domain of the encoded CAR molecule comprises an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab’)2, a single domain antibody (SDAB), a VH or VL domain, a camelid VHH domain or a bi-functional (e.g. bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol.17, 105 (1987)). In some instances, scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size see, e.g., Hollinger et al.1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. WO2006/020258 and WO2007/024715, is incorporated herein by reference. An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises amino acids glycine and serine. In another embodiment, the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1 ( SEQ ID NO: 205). In one embodiment, the linker can be (Gly4Ser)4 ( SEQ ID NO: 206) or (Gly4Ser)3( SEQ ID NO: 207). Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. In another aspect, the antigen binding domain is a T cell receptor (“TCR”), or a fragment thereof, for example, a single chain TCR (scTCR). Methods to make such TCRs are known in the art. See, e.g., Willemsen RA et al, Gene Therapy 7: 1369–1377 (2000); Zhang T et al, Cancer Gene Ther 11: 487–496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (references are incorporated herein by its entirety). For example, scTCR can be engineered that contains the Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is very useful to cancer associated target that itself is intracellar, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by MHC. In certain embodiments, the encoded antigen binding domain has a binding affinity KD of 10-4 M to 10-8 M. In one embodiment, the encoded CAR molecule comprises an antigen binding domain that has a binding affinity KD of 10-4 M to 10-8 M, e.g., 10-5 M to 10-7 M, e.g., 10-6 M or 10-7 M, for the target antigen. In one embodiment, the antigen binding domain has a binding affinity that is at least five- fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or 1,000-fold less than a reference antibody, e.g., an antibody described herein. In one embodiment, the encoded antigen binding domain has a binding affinity at least 5-fold less than a reference antibody (e.g., an antibody from which the antigen binding domain is derived). In one aspect such antibody fragments are functional in that they provide a biological response that can include, but is not limited to, activation of an immune response, inhibition of signal-transduction origination from its target antigen, inhibition of kinase activity, and the like, as will be understood by a skilled artisan. In one aspect, the antigen binding domain of the CAR is a scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived. In one aspect, the antigen binding domain of a CAR of the disclosure (e.g., a scFv) is encoded by a nucleic acid molecule whose sequence has been codon optimized for expression in a mammalian cell. In one aspect, entire CAR construct of the disclosure is encoded by a nucleic acid molecule whose entire sequence has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least US Patent Numbers 5,786,464 and 6,114,148. Antigen binding domains (and the targeted antigens) In one embodiment, an antigen binding domain against CD19 is an antigen binding portion, e.g., CDRs, of a CAR, antibody or antigen-binding fragment thereof described in, e.g., PCT publication WO2012/079000; PCT publication WO2014/153270; Kochenderfer, J.N. et al., J. Immunother.32 (7), 689-702 (2009); Kochenderfer, J.N., et al., Blood, 116 (20), 4099-4102 (2010); PCT publication WO2014/031687; Bejcek, Cancer Research, 55, 2346-2351, 1995; or U.S. Patent No. 7,446,190. In one embodiment, an antigen binding domain against mesothelin is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2015/090230. In one embodiment, an antigen binding domain against mesothelin is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in, e.g., PCT publication WO1997/025068, WO1999/028471, WO2005/014652, WO2006/099141, WO2009/045957, WO2009/068204, WO2013/142034, WO2013/040557, or WO2013/063419. In one embodiment, an antigen binding domain against mesothelin is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO/2015/090230. In one embodiment, an antigen binding domain against CD123 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2014/130635. In one embodiment, an antigen binding domain against CD123 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in, e.g., PCT publication WO2014/138805, WO2014/138819, WO2013/173820, WO2014/144622, WO2001/66139, WO2010/126066, WO2014/144622, or US2009/0252742. In one embodiment, an antigen binding domain against CD123 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO/2016/028896. In one embodiment, an antigen binding domain against EGFRvIII is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment or CAR described in, e.g., WO/2014/130657. In one embodiment, an antigen binding domain against CD22 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2016/164731; Haso et al., Blood, 121(7): 1165-1174 (2013); Wayne et al., Clin Cancer Res 16(6): 1894-1903 (2010); Kato et al., Leuk Res 37(1):83- 88 (2013); Creative BioMart (creativebiomart.net): MOM-18047-S(P). In one embodiment, an antigen binding domain against CS-1 is an antigen binding portion, e.g., CDRs, of Elotuzumab (BMS), see e.g., Tai et al., 2008, Blood 112(4):1329-37; Tai et al., 2007, Blood.110(5):1656-63. In one embodiment, an antigen binding domain against CLL-1 is an antigen binding portion, e.g., CDRs, of an antibody available from R&D, ebiosciences, Abcam, for example, PE-CLL1-hu Cat# 353604 (BioLegend); and PE-CLL1 (CLEC12A) Cat# 562566 (BD). In one embodiment, an antigen binding domain against CLL-1 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO/2016/014535. In one embodiment, an antigen binding domain against CD33 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Bross et al., Clin Cancer Res 7(6):1490-1496 (2001) (Gemtuzumab Ozogamicin, hP67.6),Caron et al., Cancer Res 52(24):6761-6767 (1992) (Lintuzumab, HuM195), Lapusan et al., Invest New Drugs 30(3):1121-1131 (2012) (AVE9633), Aigner et al., Leukemia 27(5): 1107-1115 (2013) (AMG330, CD33 BiTE), Dutour et al., Adv hematol 2012:683065 (2012), and Pizzitola et al., Leukemia doi:10.1038/Lue.2014.62 (2014). In one embodiment, an antigen binding domain against CD33 is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO/2016/014576. In one embodiment, an antigen binding domain against GD2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Mujoo et al., Cancer Res. 47(4):1098-1104 (1987); Cheung et al., Cancer Res 45(6):2642-2649 (1985), Cheung et al., J Clin Oncol 5(9):1430-1440 (1987), Cheung et al., J Clin Oncol 16(9):3053-3060 (1998), Handgretinger et al., Cancer Immunol Immunother 35(3):199-204 (1992). In some embodiments, an antigen binding domain against GD2 is an antigen binding portion of an antibody selected from mAb 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see e.g., WO2012033885, WO2013040371, WO2013192294, WO2013061273, WO2013123061, WO2013074916, and WO201385552. In some embodiments, an antigen binding domain against GD2 is an antigen binding portion of an antibody described in US Publication No.: 20100150910 or PCT Publication No.: WO 2011160119. In one embodiment, an antigen binding domain against BCMA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2012163805, WO200112812, WO2003062401, WO/2016/014565, and WO2019/241426. In one embodiment, an antigen binding domain against BCMA is an antigen binding portion, e.g., CDRs, of an antibody, antigen-binding fragment, or CAR described in WO2019/241426. In one embodiment, an antigen binding domain against Tn antigen is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US8,440,798, Brooks et al., PNAS 107(22):10056- 10061 (2010), and Stone et al., OncoImmunology 1(6):863-873(2012). In one embodiment, an antigen binding domain against PSMA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Parker et al., Protein Expr Purif 89(2):136-145 (2013), US 20110268656 (J591 ScFv); Frigerio et al, European J Cancer 49(9):2223-2232 (2013) (scFvD2B); WO 2006125481 (mAbs 3/A12, 3/E7 and 3/F11) and single chain antibody fragments (scFv A5 and D7). In one embodiment, an antigen binding domain against ROR1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hudecek et al., Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847; and US20130101607. In one embodiment, an antigen binding domain against FLT3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2011076922, US5777084, EP0754230, US20090297529, and several commercial catalog antibodies (R&D, ebiosciences, Abcam). In one embodiment, an antigen binding domain against TAG72 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hombach et al., Gastroenterology 113(4):1163-1170 (1997); and Abcam ab691. In one embodiment, an antigen binding domain against FAP is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Ostermann et al., Clinical Cancer Research 14:4584- 4592 (2008) (FAP5), US Pat. Publication No.2009/0304718; sibrotuzumab (see e.g., Hofheinz et al., Oncology Research and Treatment 26(1), 2003); and Tran et al., J Exp Med 210(6):1125- 1135 (2013). In one embodiment, an antigen binding domain against CD38 is an antigen binding portion, e.g., CDRs, of daratumumab (see, e.g., Groen et al., Blood 116(21):1261-1262 (2010); MOR202 (see, e.g., US8,263,746); or antibodies described in US8,362,211. In one embodiment, an antigen binding domain against CD44v6 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Casucci et al., Blood 122(20):3461-3472 (2013). In one embodiment, an antigen binding domain against CEA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Chmielewski et al., Gastoenterology 143(4):1095-1107 (2012). In one embodiment, an antigen binding domain against EPCAM is an antigen binding portion, e.g., CDRS, of an antibody selected from MT110, EpCAM-CD3 bispecific Ab (see, e.g., clinicaltrials.gov/ct2/show/NCT00635596); Edrecolomab; 3622W94; ING-1; and adecatumumab (MT201). In one embodiment, an antigen binding domain against PRSS21 is an antigen binding portion, e.g., CDRs, of an antibody described in US Patent No.: 8,080,650. In one embodiment, an antigen binding domain against B7H3 is an antigen binding portion, e.g., CDRs, of an antibody MGA271 (Macrogenics). In one embodiment, an antigen binding domain against KIT is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7915391, US 20120288506, and several commercial catalog antibodies. In one embodiment, an antigen binding domain against IL-13Ra2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2008/146911, WO2004087758, several commercial catalog antibodies, and WO2004087758. In one embodiment, an antigen binding domain against CD30 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7090843 B1, and EP0805871. In one embodiment, an antigen binding domain against GD3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7253263; US 8,207,308; US 20120276046; EP1013761; WO2005035577; and US6437098. In one embodiment, an antigen binding domain against CD171 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hong et al., J Immunother 37(2):93-104 (2014). In one embodiment, an antigen binding domain against IL-11Ra is an antigen binding portion, e.g., CDRs, of an antibody available from Abcam (cat# ab55262) or Novus Biologicals (cat# EPR5446). In another embodiment, an antigen binding domain again IL-11Ra is a peptide, see, e.g., Huang et al., Cancer Res 72(1):271-281 (2012). In one embodiment, an antigen binding domain against PSCA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Morgenroth et al., Prostate 67(10):1121-1131 (2007) (scFv 7F5); Nejatollahi et al., J of Oncology 2013(2013), article ID 839831 (scFv C5-II); and US Pat Publication No.20090311181. In one embodiment, an antigen binding domain against VEGFR2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Chinnasamy et al., J Clin Invest 120(11):3953-3968 (2010). In one embodiment, an antigen binding domain against LewisY is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Kelly et al., Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab (scFvs)); Dolezal et al., Protein Engineering 16(1):47-56 (2003) (NC10 scFv). In one embodiment, an antigen binding domain against CD24 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Maliar et al., Gastroenterology 143(5):1375-1384 (2012). In one embodiment, an antigen binding domain against PDGFR-beta is an antigen binding portion, e.g., CDRs, of an antibody Abcam ab32570. In one embodiment, an antigen binding domain against SSEA-4 is an antigen binding portion, e.g., CDRs, of antibody MC813 (Cell Signaling), or other commercially available antibodies. In one embodiment, an antigen binding domain against CD20 is an antigen binding portion, e.g., CDRs, of the antibody Rituximab, Ofatumumab, Ocrelizumab, Veltuzumab, or GA101; or antibodies described in WO2016/164731. In one embodiment, an antigen binding domain against Folate receptor alpha is an antigen binding portion, e.g., CDRs, of the antibody IMGN853, or an antibody described in US20120009181; US4851332, LK26: US5952484. In one embodiment, an antigen binding domain against ERBB2 (Her2/neu) is an antigen binding portion, e.g., CDRs, of the antibody trastuzumab, or pertuzumab. In one embodiment, an antigen binding domain against MUC1 is an antigen binding portion, e.g., CDRs, of the antibody SAR566658. In one embodiment, the antigen binding domain against EGFR is antigen binding portion, e.g., CDRs, of the antibody cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab. In one embodiment, an antigen binding domain against NCAM is an antigen binding portion, e.g., CDRs, of the antibody clone 2-2B: MAB5324 (EMD Millipore). In one embodiment, an antigen binding domain against Ephrin B2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Abengozar et al., Blood 119(19):4565-4576 (2012). In one embodiment, an antigen binding domain against IGF-I receptor is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US8344112 B2; EP2322550 A1; WO 2006/138315, or PCT/US2006/022995. In one embodiment, an antigen binding domain against CAIX is an antigen binding portion, e.g., CDRs, of the antibody clone 303123 (R&D Systems). In one embodiment, an antigen binding domain against LMP2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7,410,640, or US20050129701. In one embodiment, an antigen binding domain against gp100 is an antigen binding portion, e.g., CDRs, of the antibody HMB45, NKIbetaB, or an antibody described in WO2013165940, or US20130295007 In one embodiment, an antigen binding domain against tyrosinase is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US5843674; or US19950504048. In one embodiment, an antigen binding domain against EphA2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Yu et al., Mol Ther 22(1):102-111 (2014). In one embodiment, an antigen binding domain against GD3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7253263; US 8,207,308; US 20120276046; EP1013761 A3; 20120276046; WO2005035577; or US6437098. In one embodiment, an antigen binding domain against fucosyl GM1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US20100297138; or WO2007/067992. In one embodiment, an antigen binding domain against sLe is an antigen binding portion, e.g., CDRs, of the antibody G193 (for lewis Y), see Scott AM et al, Cancer Res 60: 3254-61 (2000), also as described in Neeson et al, J Immunol May 2013190 (Meeting Abstract Supplement) 177.10. In one embodiment, an antigen binding domain against GM3 is an antigen binding portion, e.g., CDRs, of the antibody CA 2523449 (mAb 14F7). In one embodiment, an antigen binding domain against HMWMAA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Kmiecik et al., Oncoimmunology 3(1):e27185 (2014) (PMID: 24575382) (mAb9.2.27); US6528481; WO2010033866; or US 20140004124. In one embodiment, an antigen binding domain against o-acetyl-GD2 is an antigen binding portion, e.g., CDRs, of the antibody 8B6. In one embodiment, an antigen binding domain against TEM1/CD248 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Marty et al., Cancer Lett 235(2):298-308 (2006); Zhao et al., J Immunol Methods 363(2):221-232 (2011). In one embodiment, an antigen binding domain against CLDN6 is an antigen binding portion, e.g., CDRs, of the antibody IMAB027 (Ganymed Pharmaceuticals), see e.g., clinicaltrial.gov/show/NCT02054351. In one embodiment, an antigen binding domain against TSHR is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US8,603,466; US8,501,415; or US8,309,693. In one embodiment, an antigen binding domain against GPRC5D is an antigen binding portion, e.g., CDRs, of the antibody FAB6300A (R&D Systems); or LS-A4180 (Lifespan Biosciences). In one embodiment, an antigen binding domain against CD97 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US6,846,911;de Groot et al., J Immunol 183(6):4127- 4134 (2009); or an antibody from R&D:MAB3734. In one embodiment, an antigen binding domain against ALK is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010). In one embodiment, an antigen binding domain against polysialic acid is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Nagae et al., J Biol Chem 288(47):33784- 33796 (2013). In one embodiment, an antigen binding domain against PLAC1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Ghods et al., Biotechnol Appl Biochem 2013 doi:10.1002/bab.1177. In one embodiment, an antigen binding domain against GloboH is an antigen binding portion of the antibody VK9; or an antibody described in, e.g., Kudryashov V et al, Glycoconj J.15(3):243-9 (1998), Lou et al., Proc Natl Acad Sci USA 111(7):2482-2487 (2014) ; MBr1: Bremer E-G et al. J Biol Chem 259:14773–14777 (1984). In one embodiment, an antigen binding domain against NY-BR-1 is an antigen binding portion, e.g., CDRs of an antibody described in, e.g., Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007). In one embodiment, an antigen binding domain against WT-1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Dao et al., Sci Transl Med 5(176):176ra33 (2013); or WO2012/135854. In one embodiment, an antigen binding domain against MAGE-A1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv). In one embodiment, an antigen binding domain against sperm protein 17 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Song et al., Target Oncol 2013 Aug 14 (PMID: 23943313); Song et al., Med Oncol 29(4):2923-2931 (2012). In one embodiment, an antigen binding domain against Tie 2 is an antigen binding portion, e.g., CDRs, of the antibody AB33 (Cell Signaling Technology). In one embodiment, an antigen binding domain against MAD-CT-2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., PMID: 2450952; US7635753. In one embodiment, an antigen binding domain against Fos-related antigen 1 is an antigen binding portion, e.g., CDRs, of the antibody 12F9 (Novus Biologicals). In one embodiment, an antigen binding domain against MelanA/MART1 is an antigen binding portion, e.g., CDRs, of an antibody described in, EP2514766 A2; or US 7,749,719. In one embodiment, an antigen binding domain against sarcoma translocation breakpoints is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Luo et al, EMBO Mol. Med. 4(6):453-461 (2012). In one embodiment, an antigen binding domain against TRP-2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Wang et al, J Exp Med.184(6):2207-16 (1996). In one embodiment, an antigen binding domain against CYP1B1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Maecker et al, Blood 102 (9): 3287-3294 (2003). In one embodiment, an antigen binding domain against RAGE-1 is an antigen binding portion, e.g., CDRs, of the antibody MAB5328 (EMD Millipore). In one embodiment, an antigen binding domain against human telomerase reverse transcriptase is an antigen binding portion, e.g., CDRs, of the antibody cat no: LS-B95-100 (Lifespan Biosciences) In one embodiment, an antigen binding domain against intestinal carboxyl esterase is an antigen binding portion, e.g., CDRs, of the antibody 4F12: cat no: LS-B6190-50 (Lifespan Biosciences). In one embodiment, an antigen binding domain against mut hsp70-2 is an antigen binding portion, e.g., CDRs, of the antibody Lifespan Biosciences: monoclonal: cat no: LS-C133261-100 (Lifespan Biosciences). In one embodiment, an antigen binding domain against CD79a is an antigen binding portion, e.g., CDRs, of the antibody Anti-CD79a antibody [HM47/A9] (ab3121), available from Abcam; antibody CD79A Antibody #3351 available from Cell Signalling Technology; or antibody HPA017748 - Anti- CD79A antibody produced in rabbit, available from Sigma Aldrich. In one embodiment, an antigen binding domain against CD79b is an antigen binding portion, e.g., CDRs, of the antibody polatuzumab vedotin, anti-CD79b described in Dornan et al., “Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma” Blood. 2009 Sep 24;114(13):2721-9. doi: 10.1182/blood-2009-02- 205500. Epub 2009 Jul 24, or the bispecific antibody Anti-CD79b/CD3 described in “4507 Pre- Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a Potential Therapy for B Cell Malignancies” Abstracts of 56th ASH Annual Meeting and Exposition, San Francisco, CA December 6-92014. In one embodiment, an antigen binding domain against CD72 is an antigen binding portion, e.g., CDRs, of the antibody J3-109 described in Myers, and Uckun, “An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia.” Leuk Lymphoma. 1995 Jun;18(1-2):119-22, or anti-CD72 (10D6.8.1, mIgG1) described in Polson et al., “Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection” Cancer Res March 15, 200969; 2358. In one embodiment, an antigen binding domain against LAIR1 is an antigen binding portion, e.g., CDRs, of the antibody ANT-301 LAIR1 antibody, available from ProSpec; or anti-human CD305 (LAIR1) Antibody, available from BioLegend. In one embodiment, an antigen binding domain against FCAR is an antigen binding portion, e.g., CDRs, of the antibody CD89/FCARAntibody (Catalog#10414-H08H), available from Sino Biological Inc. In one embodiment, an antigen binding domain against LILRA2 is an antigen binding portion, e.g., CDRs, of the antibody LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or Mouse Anti-LILRA2 antibody, Monoclonal (2D7), available from Lifespan Biosciences.. In one embodiment, an antigen binding domain against CD300LF is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CMRF35-like molecule 1 antibody, Monoclonal[UP-D2], available from BioLegend, or Rat Anti-CMRF35-like molecule 1 antibody, Monoclonal[234903], available from R&D Systems. In one embodiment, an antigen binding domain against CLEC12A is an antigen binding portion, e.g., CDRs, of the antibody Bispecific T cell Engager (BiTE) scFv-antibody and ADC described in Noordhuis et al., “Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug- Conjugates and Bispecific CLL-1xCD3 BiTE Antibody” 53rd ASH Annual Meeting and Exposition, December 10-13, 2011, and MCLA-117 (Merus). In one embodiment, an antigen binding domain against BST2 (also called CD317) is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CD317 antibody, Monoclonal[3H4], available from Antibodies-Online or Mouse Anti-CD317 antibody, Monoclonal [696739], available from R&D Systems. In one embodiment, an antigen binding domain against EMR2 (also called CD312) is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CD312 antibody, Monoclonal [LS-B8033] available from Lifespan Biosciences, or Mouse Anti-CD312 antibody, Monoclonal [494025] available from R&D Systems. In one embodiment, an antigen binding domain against LY75 is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-Lymphocyte antigen 75 antibody, Monoclonal [HD30] available from EMD Millipore or Mouse Anti-Lymphocyte antigen 75 antibody, Monoclonal[A15797] available from Life Technologies. In one embodiment, an antigen binding domain against GPC3 is an antigen binding portion, e.g., CDRs, of the antibody hGC33 described in Nakano K, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization. Anticancer Drugs.2010 Nov;21(10):907–916, or MDX-1414, HN3, or YP7, all three of which are described in Feng et al., “Glypican-3 antibodies: a new therapeutic target for liver cancer.” FEBS Lett.2014 Jan 21;588(2):377-82. In one embodiment, an antigen binding domain against FCRL5 is an antigen binding portion, e.g., CDRs, of the anti-FcRL5 antibody described in Elkins et al., “FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma” Mol Cancer Ther. 2012 Oct;11(10):2222-32. In one embodiment, an antigen binding domain against FCRL5 is an antigen binding portion, e.g., CDRs, of the anti-FcRL5 antibody described in, for example, WO2001/038490, WO/2005/117986, WO2006/039238, WO2006/076691, WO2010/114940, WO2010/120561, or WO2014/210064. In one embodiment, an antigen binding domain against IGLL1 is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-Immunoglobulin lambda-like polypeptide 1 antibody, Monoclonal [AT1G4] available from Lifespan Biosciences, Mouse Anti-Immunoglobulin lambda- like polypeptide 1 antibody, Monoclonal [HSL11] available from BioLegend. In one embodiment, the antigen binding domain comprises one, two three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, from an antibody listed above, and/or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above. In one embodiment, the antigen binding domain comprises a heavy chain variable region and/or a variable light chain region of an antibody listed above. In another aspect, the antigen binding domain comprises a humanized antibody or an antibody fragment. In some aspects, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. In one aspect, the antigen binding domain is humanized. In an embodiment, the antigen-binding domain of a CAR, e.g., a CAR expressed by a cell of the disclosure, binds to CD19. CD19 is found on B cells throughout differentiation of the lineage from the pro/pre-B cell stage through the terminally differentiated plasma cell stage. In an embodiment, the antigen binding domain is a murine scFv domain that binds to human CD19, e.g., the antigen binding domain of CTL019 (e.g., SEQ ID NO: 208). In an embodiment, the antigen binding domain is a humanized antibody or antibody fragment, e.g., scFv domain, derived from the murine CTL019 scFv. In an embodiment, the antigen binding domain is a human antibody or antibody fragment that binds to human CD19. Exemplary scFv domains (and their sequences, e.g., CDRs, VL and VH sequences) that bind to CD19 are provided in Table 12. The scFv domain sequences provided in Table 12 include a light chain variable region (VL) and a heavy chain variable region (VH). The VL and VH are attached by a linker comprising the sequence GGGGSGGGGSGGGGS ( SEQ ID NO: 5794), e.g., in the following orientation: VL-linker-VH. Table 12. Antigen Binding domains that bind CD19
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
The sequences of the CDR sequences of the scFv domains of the CD19 antigen binding domains provided in Table 12 are shown in Table 13 for the heavy chain variable domains and in Table 14 for the light chain variable domains. “ID” stands for the respective SEQ ID NO for each CDR. Table 13. Heavy Chain Variable Domain CDRs
Figure imgf000136_0001
In an embodiment, the antigen binding domain comprises an anti-CD19 antibody, or fragment thereof, e.g., an scFv. For example, the antigen binding domain comprises a variable heavy chain and a variable light chain listed in Table 15. The linker sequence joining the variable heavy and variable light chains can be any of the linker sequences described herein, or alternatively, can be GSTSGSGKPGSGEGSTKG ( SEQ ID NO: 229). The light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker- light chain variable region. Table 15. Additional Anti-CD19 antibody binding domains
Figure imgf000137_0001
In one embodiment, the CD19 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a CD19 binding domain described herein, e.g., provided in Table 12 or 13, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a CD19 binding domain described herein, e.g., provided in Table 12 or 14. In one embodiment, the CD19 binding domain comprises one, two, or all of LC CDR1, LC CDR2, and LC CDR3 of any amino acid sequences as provided in Table 14, incorporated herein by reference; and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of any amino acid sequences as provided in Table 13. Any known CD19 CAR, e.g., the CD19 antigen binding domain of any known CD19 CAR, in the art can be used in accordance with the instant disclosure to construct a CAR. For example, LG- 740; CD19 CAR described in the US Pat. No.8,399,645; US Pat. No.7,446,190; Xu et al., Leuk Lymphoma.201354(2):255-260(2012); Cruz et al., Blood 122(17):2965-2973 (2013); Brentjens et al., Blood, 118(18):4817-4828 (2011); Kochenderfer et al., Blood 116(20):4099-102 (2010); Kochenderfer et al., Blood 122 (25):4129-39(2013); and 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst 10. In one embodiment, an antigen binding domain against CD19 is an antigen binding portion, e.g., CDRs, of a CAR, antibody or antigen- binding fragment thereof described in, e.g., PCT publication WO2012/079000; PCT publication WO2014/153270; Kochenderfer, J.N. et al., J. Immunother.32 (7), 689-702 (2009); Kochenderfer, J.N., et al., Blood, 116 (20), 4099-4102 (2010); PCT publication WO2014/031687; Bejcek, Cancer Research, 55, 2346-2351, 1995; or U.S. Patent No.7,446,190. In an embodiment, the antigen-binding domain of CAR, e.g., a CAR expressed by a cell of the disclosure, binds to BCMA. BCMA is found preferentially expressed in mature B lymphocytes. In an embodiment, the antigen binding domain is a murine scFv domain that binds to human BCMA. In an embodiment, the antigen binding domain is a humanized antibody or antibody fragment, e.g., scFv domain, that binds human BCMA. In an embodiment, the antigen binding domain is a human antibody or antibody fragment that binds to human BCMA. In embodiments, exemplary BCMA CAR constructs are generated using the VH and VL sequences from PCT Publication WO2012/0163805 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the VH and VL sequences from PCT Publication WO2016/014565 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the VH and VL sequences from PCT Publication WO2014/122144 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the VH and VL sequences from PCT Publication WO2016/014789 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the VH and VL sequences from PCT Publication WO2014/089335 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the VH and VL sequences from PCT Publication WO2014/140248 (the contents of which are hereby incorporated by reference in its entirety). Any known BCMA CAR, e.g., the BMCA antigen binding domain of any known BCMA CAR, in the art can be used in accordance with the instant disclosure. For example, those described herein. Exemplary CAR Molecules In one aspect, a CAR, e.g., a CAR expressed by the cell of the disclosure, comprises a CAR molecule comprising an antigen binding domain that binds to a B cell antigen, e.g., as described herein, such as CD19 or BCMA. In one embodiment, the CAR comprises a CAR molecule comprising a CD19 antigen binding domain (e.g., a murine, human or humanized antibody or antibody fragment that specifically binds to CD19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain). Exemplary CAR molecules described herein are provided in Table 16. The CAR molecules in Table 16 comprise a CD19 antigen binding domain, e.g., an amino acid sequence of any CD19 antigen binding domain provided in Table 16. Table 16. Exemplary CD19 CAR molecules
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
In one aspect, a CAR, e.g., a CAR expressed by the cell of the disclosure, comprises a CAR molecule comprising an antigen binding domain that binds to BCMA, e.g., comprises a BCMA antigen binding domain (e.g., a murine, human or humanized antibody or antibody fragment that specifically binds to BCMA, e.g., human BCMA), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain). Exemplary CAR molecules of a CAR described herein are provided in Table 1 of WO2016/014565, which is incorporated by reference herein. Transmembrane domains With respect to the transmembrane domain, in various embodiments, a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR e.g., in one embodiment, the transmembrane domain may be from the same protein that the signaling domain, costimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell. The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. A transmembrane domain of particular use in this disclosure may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244,
Figure imgf000146_0001
Figure imgf000147_0001
In one aspect, the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one aspect a triplet of phenylalanine, tryptophan and valine can be found at each end of a recombinant transmembrane domain. Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 250). In some embodiments, the linker is encoded by a nucleotide sequence of
Figure imgf000147_0002
In one aspect, the hinge or spacer comprises a KIR2DS2 hinge. Signaling domains In embodiments of the disclosure having an intracellular signaling domain, such a domain can contain, e.g., one or more of a primary signaling domain and/or a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a sequence encoding a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain. The intracellular signaling sequences within the cytoplasmic portion of the CAR of the disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker. In one aspect, the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, e.g., a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue. Primary Signaling domains A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary intracellular signaling domains that are of particular use in the disclosure include those of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR of the disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta. Costimulatory Signaling Domains In some embodiments, the encoded intracellular signaling domain comprises a costimulatory signaling domain. For example, the intracellular signaling domain can comprise a primary signaling domain and a costimulatory signaling domain. In some embodiments, the encoded costimulatory signaling domain comprises a functional signaling domain of a protein chosen from one or more of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, or NKG2D. In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD27. In one aspect, the signaling domain of CD27 comprises an amino acid sequence of
Figure imgf000149_0001
Vectors In another aspect, the disclosure pertains to a vector comprising a nucleic acid sequence encoding a CAR described herein. In one embodiment, the vector is chosen from a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In one embodiment, the vector is a lentivirus vector. These vectors or portions thereof may, among other things, be used to create template nucleic acids, as described herein for use with the CRISPR systems as described herein. Alternatively, the vectors may be used to deliver nucleic acid directly to the cell, e.g., the immune effector cell, e.g., the T cell, e.g., the allogeneic T cell, independent of the CRISPR system. The present disclosure also provides vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco- retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. A retroviral vector may also be, e.g., a gammaretroviral vector. A gammaretroviral vector may include, e.g., a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and a transgene of interest, e.g., a gene encoding a CAR. A gammaretroviral vector may lack viral structural gens such as gag, pol, and env. Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, e.g., in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses.2011 Jun; 3(6): 677–713. In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the disclosure is an adenoviral vector (A5/35). In another embodiment, the expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al.2009Nature Reviews Immunology 9.10: 704-716, is incorporated herein by reference. The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Disclosed herein are methods for producing an in vitro transcribed RNA CAR. The present disclosure also includes a CAR encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequence (“UTR”), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the CAR. Non-viral delivery methods In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject. In some embodiments, the non-viral method includes the use of a transposon (also called a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome. For example, a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition. In some embodiments, cells, e.g., T or NK cells, are generated that express a CAR described herein by using a combination of gene insertion using the SBTS and genetic editing using a nuclease (e.g., Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system, or engineered meganuclease re-engineered homing endonucleases). In some embodiments, cells of the disclosure, e.g., T or NK cells, e.g., allogeneic T cells, e.g., described herein, (e.g., that express a CAR described herein) are generated by contacting the cells with (a) a composition comprising one or more gRNA molecules, e.g., as described herein, and one or more Cas molecules, e.g., a Cas9 molecule, e.g., as described herein, and (b) nucleic acid comprising sequence encoding a CAR, e.g., described herein (such as a template nucleic acid molecule as described herein). Without being bound by theory, said composition of (a), above, will induce a break at or near the genomic DNA targeted by the targeting domain of the gRNA molecule(s), and the nucleic acid of (b) will incorporate, e.g., partially or wholly, into the genome at or near said break, such that upon integration, the encoded CAR molecule is expressed. In embodiments, expression of the CAR will be controlled by promoters or other regulatory elements endogenous to the genome (e.g., the promoter controlling expression from the gene in which the nucleic acid of (b) was inserted). In other embodiments, the nucleic acid of (b) further comprises a promoter and/or other regulatory elements, e.g., as described herein, e.g., an EF1-alpha promoter, operably linked to the sequence encoding the CAR, such that upon integration, expression of the CAR is controlled by that promoter and/or other regulatory elements. Additional features of the disclosure relating to use of CRISPR/Cas9 systems, e.g., as described herein, to direct incorporation of nucleic acid sequence encoding a CAR, e.g., as described herein, are described elsewhere in this application, e.g., in the section relating to gene insertion and homologous recombination. In embodiments, the composition of a) above is a composition comprising RNPs comprising the one or more gRNA molecules. In embodiments, RNPs comprising gRNAs targeting unique target sequences are introduced into the cell simultaneously, e.g., as a mixture of RNPs comprising the one or more gRNAs. In embodiments, RNPs comprising gRNAs targeting unique target sequences are introduced into the cell sequentially. In some embodiments, use of a non-viral method of delivery permits reprogramming of cells, e.g., T or NK cells, and direct infusion of the cells into a subject. Advantages of non-viral vectors include but are not limited to the ease and relatively low cost of producing sufficient amounts required to meet a patient population, stability during storage, and lack of immunogenicity. Promoters In one embodiment, the vector further comprises a promoter. In some embodiments, the promoter is chosen from an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, an ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter. In one embodiment, the promoter is an EF-1 promoter. Host cells for CAR expression As noted above, in some aspects the disclosure pertains to a cell, e.g., an immune effector cell, (e.g., a population of cells, e.g., a population of immune effector cells) comprising a nucleic acid molecule, a CAR polypeptide molecule, or a vector as described herein. In certain aspects of the present disclosure, immune effector cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and, optionally, to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. It is recognized that the methods of the application can utilize culture media conditions comprising 5% or less, for example 2%, human AB serum, and employ known culture media conditions and compositions, for example those described in Smith et al., “Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement” Clinical & Translational Immunology (2015) 4, e31; doi:10.1038/cti.2014.31. In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation. The methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells, that are a T regulatory cell-depleted population, CD25+ depleted cells, using, e.g., a negative selection technique, e.g., described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells. In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein. In one embodiment, the T regulatory cells, e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from MiltenyiTM. In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7 cells to 20 uL, or 1e7 cells to15 uL, or 1e7 cells to 10 uL, or 1e7 cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In one embodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greater than 500 million cells/ml is used. In a further aspect, a concentration of cells of 600, 700, 800, or 900 million cells/ml is used. In one embodiment, the population of immune effector cells to be depleted includes about 6 x 109 CD25+ T cells. In other aspects, the population of immune effector cells to be depleted include about 1 x 109 to 1x 1010 CD25+ T cell, and any integer value in between. In one embodiment, the resulting population T regulatory depleted cells has 2 x 109 T regulatory cells, e.g., CD25+ cells, or less (e.g., 1 x 109, 5 x 108, 1 x 108, 5 x 107, 1 x 107, or less CD25+ cells). In one embodiment, the T regulatory cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system with a depletion tubing set, such as, e.g., tubing 162-01. In one embodiment, the CliniMAC system is run on a depletion setting such as, e.g., DEPLETION2.1. Without wishing to be bound by a particular theory, decreasing the level of negative regulators of immune cells (e.g., decreasing the number of unwanted immune cells, e.g., TREG cells), in a subject prior to apheresis or during manufacturing of a CAR-expressing cell product can reduce the risk of subject relapse. For example, methods of depleting TREG cells are known in the art. Methods of decreasing TREG cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25-depletion, and combinations thereof. In some embodiments, the manufacturing methods comprise reducing the number of (e.g., depleting) TREG cells prior to manufacturing of the CAR-expressing cell. For example, manufacturing methods comprise contacting the sample, e.g., the apheresis sample, with an anti- GITR antibody and/or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand), e.g., to deplete TREG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product. In an embodiment, a subject is pre-treated with one or more therapies that reduce TREG cells prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment. In an embodiment, methods of decreasing TREG cells include, but are not limited to, administration to the subject of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof, can occur before, during or after an infusion of the CAR-expressing cell product. In an embodiment, a subject is pre-treated with cyclophosphamide prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR- expressing cell treatment. In an embodiment, a subject is pre-treated with an anti-GITR antibody prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment. In one embodiment, the population of cells to be removed are neither the regulatory T cells or tumor cells, but cells that otherwise negatively affect the expansion and/or function of CART cells, e.g., cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potentially immune suppressive cells. In one embodiment, such cells are envisioned to be removed concurrently with regulatory T cells and/or tumor cells, or following said depletion, or in another order. The methods described herein can include more than one selection step, e.g., more than one depletion step. Enrichment of a T cell population by negative selection can be accomplished, e.g., with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail can include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. The methods described herein can further include removing cells from the population which express a tumor antigen, e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 or CD11b, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted, and tumor antigen depleted cells that are suitable for expression of a CAR, e.g., a CAR described herein. In one embodiment, tumor antigen expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-tumor antigen antibody, or fragment thereof, can be attached to the same substrate, e.g., bead, which can be used to remove the cells or an anti-CD25 antibody, or fragment thereof, or the anti-tumor antigen antibody, or fragment thereof, can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the tumor antigen expressing cells is sequential, and can occur, e.g., in either order. Also provided are methods that include removing cells from the population which express a check point inhibitor, e.g., a check point inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted cells, and check point inhibitor depleted cells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary check point inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA and LAIR1. In one embodiment, check point inhibitor expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-check point inhibitor antibody, or fragment thereof, can be attached to the same bead which can be used to remove the cells, or an anti-CD25 antibody, or fragment thereof, and the anti-check point inhibitor antibody, or fragment there, can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the check point inhibitor expressing cells is sequential, and can occur, e.g., in either order. Methods described herein can include a positive selection step. For example, T cells can isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours, e.g., 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. In one embodiment, a T cell population can be selected that expresses one or more of IFN-^, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO 2013/126712. For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml, or 5 billion/ml is used. In one aspect, a concentration of 1 billion cells/ml is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further aspects, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one aspect, the concentration of cells used is 5 x 106/ml. In other aspects, the concentration used can be from about 1 x 105/ml to 1 x 106/ml, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10oC or at room temperature. T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen. In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure. Also contemplated in the context of the disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in immune effector cell therapy for any number of diseases or conditions that would benefit from immune effector cell therapy, such as those described herein. In one aspect a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. In a further aspect of the present disclosure, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain aspects, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system. In one embodiment, the immune effector cells expressing a CAR molecule, e.g., a CAR molecule described herein, are obtained from a subject that has received a low, immune enhancing dose of an mTOR inhibitor. In an embodiment, the population of immune effector cells, e.g., T cells, to be engineered to express a CAR, are harvested after a sufficient time, or after sufficient dosing of the low, immune enhancing, dose of an mTOR inhibitor, such that the level of PD1 negative immune effector cells, e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g., T cells/ PD1 positive immune effector cells, e.g., T cells, in the subject or harvested from the subject has been, at least transiently, increased. In other embodiments, population of immune effector cells, e.g., T cells, which have, or will be engineered to express a CAR, can be treated ex vivo by contact with an amount of an mTOR inhibitor that increases the number of PD1 negative immune effector cells, e.g., T cells or increases the ratio of PD1 negative immune effector cells, e.g., T cells/ PD1 positive immune effector cells, e.g., T cells. In one embodiment, a T cell population is diaglycerol kinase (DGK)-deficient. DGK-deficient cells include cells that do not express DGK RNA or protein or have reduced or inhibited DGK activity. DGK-deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent DGK expression. Alternatively, DGK- deficient cells can be generated by treatment with DGK inhibitors described herein. In one embodiment, a T cell population is Ikaros-deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity, Ikaros-deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, e.g., lenalidomide. In embodiments, a T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros- deficient cells can be generated by any of the methods described herein. In an embodiment, the NK cells are obtained from the subject. In another embodiment, the NK cells are an NK cell line, e.g., NK-92 cell line (Conkwest). In some aspects, the cells of the disclosure (e.g., the immune effector cells of the disclosure, e.g., the CAR-expressing cells of the disclosure) are induced pluripotent stem cells (“iPSCs”) or embryonic stem cells (ESCs), or are T cells generated from (e.g., differentiated from) said iPSC and/or ESC. iPSCs can be generated, for example, by methods known in the art, from peripheral blood T lymphocytes, e.g., peripheral blood T lymphocytes isolated from a healthy volunteer. As well, such cells may be differentiated into T cells by methods known in the art. See e.g., Themeli M. et al., Nat. Biotechnol., 31, pp.928-933 (2013); doi:10.1038/nbt.2678; WO2014/165707, the contents of each of which are incorporated herein by reference in their entirety. Methods of Manufacture CARTs disclosed herein can be manufactured ex vivo by any known methods in the art. For example, methods described in WO2012/079000, or WO2020/047452 (both incorporated herein by reference). CARTs disclosed herein can also be manufactured in vivo by any known methods in the art. For example, methods described in WO2020/176397 (incorporated herein by reference). An immune effector cell (e.g., T cell or NK cell) may express one CAR, or two or more CARs. In some embodiments, the methods disclosed herein may manufacture immune effector cells engineered to express one or more CARs in less than 24 hours. Without wishing to be bound by theory, the methods provided herein preserve the undifferentiated phenotype of T cells, such as naive T cells, during the manufacturing process. These CAR-expressing cells with an undifferentiated phenotype may persist longer and/or expand better in vivo after infusion. In some embodiments, CART cells produced by the manufacturing methods provided herein comprise a higher percentage of stem cell memory T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein comprise a higher percentage of effector T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein better preserve the sternness of T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein show a lower level of hypoxia, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein show a lower level of autophagy, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. In some embodiments, the immune effector cells are engineered to comprise a nucleic acid molecule encoding one or more CARs disclosed herein. In some embodiments, the methods disclosed herein do not involve using a bead, such as Dynabeads® (for example, CD3/CD28 Dynabeads®), and do not involve a de-beading step. In some embodiments, the CART cells manufactured by the methods disclosed herein may be administered to a subject with minimal ex vivo expansion, for example, less than 1 day, less than 12 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or no ex vivo expansion. Accordingly, the methods described herein provide a fast manufacturing process of making improved CAR-expressing cell products for use in treating a disease in a subject. In some embodiments, the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) comprising: (i) contacting a population of cells (for example, T cells, for example, T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells; (ii) contacting the population of cells (for example, T cells) with a nucleic acid molecule(s) (for example, a DNA or RNA molecule) encoding the CAR(s), thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and (iii) harvesting the population of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration, wherein: (a) step (ii) is performed together with step (i) or no later than 20 hours after the beginning of step (i), for example, no later than 12, 13, 14, 15, 16, 17, or 18 hours after the beginning of step (i), for example, no later than 18 hours after the beginning of step (i), and step (iii) is performed no later than 26 hours after the beginning of step (i), for example, no later than 22, 23, or 24 hours after the beginning of step (i), for example, no later than 24 hours after the beginning of step (i); (b) step (ii) is performed together with step (i) or no later than 20 hours after the beginning of step (i), for example, no later than 12, 13, 14, 15, 16, 17, or 18 hours after the beginning of step (i), for example, no later than 18 hours after the beginning of step (i), and step (iii) is performed no later than 30 hours after the beginning of step (ii), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the beginning of step (ii); or (c) the population of cells from step (iii) are not expanded, or expanded by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, for example, no more than 10%, for example, as assessed by the number of living cells, compared to the population of cells at the beginning of step (i). In some embodiments, the nucleic acid molecule in step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is on a viral vector, for example, a viral vector chosen from a lentivirus vector, an adenoviral vector, or a retrovirus vector. In some embodiments, the nucleic acid molecule in step (ii) is on a non- viral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not on any vector. In some embodiments, step (ii) comprises transducing the population of cells (for example, T cells) a viral vector(s) comprising a nucleic acid molecule encoding the CAR(s). In some embodiments, the population of cells (for example, T cells) is collected from an apheresis sample (for example, a leukapheresis sample) from a subject. In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject and shipped as a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility. Then the frozen apheresis sample is thawed, and T cells (for example, CD4+ T cells and/or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and/or CD8+ T cells) are then seeded for CART manufacturing using the activation process described herein. In some embodiments, the selected T cells (for example, CD4+ T cells and/or CD8+ T cells) undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing. In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject and shipped as a fresh product (for example, a product that is not frozen) to a cell manufacturing facility. T cells (for example, CD4+ T cells and/or CD 8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and/or CD8+ T cells) are then seeded for CART manufacturing using the activation process described herein. In some embodiments, the selected T cells (for example, CD4+ T cells and/or CD8+ T cells) undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing. In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject. T cells (for example, CD4+ T cells and/or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and/or CD8+ T cells) are then shipped as a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility. The selected T cells (for example, CD4+ T cells and/or CD8+ T cells) are later thawed and seeded for CART manufacturing using the activation process described herein. In some embodiments, cells (for example, T cells) are contacted with anti-CD3 and anti-CD28 antibodies for, for example, 12 hours, followed by transduction with a vector (for example, a lentiviral vector) (e.g. one or more vectors) encoding a CAR (e.g. one or more CARs).24 hours after culture initiation, the cells are washed and formulated for storage or administration. Without wishing to be bound by theory, brief CD3 and CD28 stimulation may promote efficient transduction of self-renewing T cells. Compared to traditional CART manufacturing approaches, the activation process provided herein does not involve prolonged ex vivo expansion. Similar to the cytokine process, the activation process provided herein also preserves undifferentiated T cells during CART manufacturing. In some embodiments, the population of cells is contacted with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells. In some embodiments, the agent that stimulates a CD3/TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28. In some embodiments, the agent that stimulates a CD3/TCR complex is chosen from an antibody (for example, a single-domain antibody (for example, a heavy chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (for example, a naturally-existing, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates a CD3/TCR complex is an antibody. In some embodiments, the agent that stimulates a CD3/TCR complex is an anti-CD3 antibody. In some embodiments, the agent that stimulates a costimulatory molecule is chosen from an antibody (for example, a single-domain antibody (for example, a heavy chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (for example, a naturally-existing, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates a costimulatory molecule is an antibody. In some embodiments, the agent that stimulates a costimulatory molecule is an anti-CD28 antibody. In some embodiments, the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead. In some embodiments, the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™. In some embodiments, the matrix comprises or consists of a polymeric, for example, biodegradable or biocompatible inert material, for example, which is non-toxic to cells. In some embodiments, the matrix is composed of hydrophilic polymer chains, which obtain maximal mobility in aqueous solution due to hydration of the chains. In some embodiments, the mobile matrix may be of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. A polysaccharide may include for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectins, xanthan, guar gum or alginate. Other polymers may include polyesters, polyethers, poly acrylates, polyacrylamides, polyamines, polyethylene imines, polyquaternium polymers, polyphosphazenes, polyvinylalcohols, poly vinylacetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. In some embodiments, the mobile matrix is a polymer of dextran. In some embodiments, the population of cells is contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, the population of cells is transduced with a DNA molecule (e.g., one or more DNA molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, in the case of a co-transduction of two nucleic acid molecules (e.g., lentiviral vectors), each of which encodes a CAR disclosed herein, each of the vectors containing nucleic acid molecules encoding the CAR can be added to the reaction mixture (e.g., containing a cell population) at a different multiplicity of infection (MOI). Without wishing to be bound by theory, it is believed that, in some embodiments, using different MOIs for the vectors containing nucleic acid molecules which encode distinct CAR molecules may affect the final composition of the cellular population. For example, in the case of a co transduction of a lentiviral vector encoding one CAR and a lentiviral vector encoding another CAR targeting a different target, different MOIs can be used to maximize the percent of preferred mono CART cells and dual CART cells, while resulting in fewer undesired mono CART cells and untransduced cells. The precise MOI used for each vector can be adjusted or determined based on a number of factors, including, but not limited to, properties of the batch of viral vector, characteristics of the cells to be transduced, and transduction efficiency. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs simultaneously with contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 20 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 19 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 18 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 17 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 16 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 15 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 14 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 14 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 13 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 12 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 11 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 10 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 9 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 8 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 7 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 6 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 5 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 4 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 3 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 2 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 1 hour after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 30 minutes after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration. In some embodiments, the population of cells is harvested for storage or administration no later than 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 26 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 25 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 24 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 23 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 22 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is not expanded ex vivo. In some embodiments, the population of cells is expanded by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 5%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 10%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 15%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 20%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 25%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 30%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 35%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 40%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above. In some embodiments, the activation process is conducted in serum free cell media. In some embodiments, the activation process is conducted in cell media comprising one or more cytokines chosen from: IL-2, IL-15 (for example, hetIL-15 (IL15/sIL-15Ra)), or IL-6 (for example, IL-6/sIL- 6Ra). In some embodiments, hetIL-15 comprises the amino acid sequence of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSITCPPPMSVEHADIWVK SYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVT TAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTP SQT TAKNWELTASASHQPPGVYPQG (SEQ ID NO: 254). In some embodiments, the activation process is conducted in cell media comprising a LSD1 inhibitor. In some embodiments, the activation process is conducted in cell media comprising a MALT1 inhibitor. In some embodiments, the serum free cell media comprises a serum replacement. In some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR). In some embodiments, the level of ICSR can be, for example, up to 5%, for example, about 1%, 2%, 3%, 4%, or 5%. Without wishing to be bound by theory, using cell media, for example, Rapid Media shown in Table 21 or Table 25, comprising ICSR, for example, 2% ICSR, may improve cell viability during a manufacture process described herein. In some embodiments, the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) comprising: (a) providing an apheresis sample (for example, a fresh or cryopreserved leukapheresis sample) collected from a subject; (b) selecting T cells from the apheresis sample (for example, using negative selection, positive selection, or selection without beads); (c) seeding isolated T cells at, for example, 1 x 106 to 1 x 107 cells/mL; (d) contacting T cells with an agent that stimulates T cells, for example, an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells (for example, contacting T cells with anti-CD3 and/or anti- CD28 antibody, for example, contacting T cells with TransAct); (e) contacting T cells with a nucleic acid molecule(s) (for example, a DNA or RNA molecule) encoding the CAR(s) (for example, contacting T cells with a virus comprising a nucleic acid molecule(s) encoding the CAR(s)) for, for example, 6-48 hours, for example, 20-28 hours; and (f) washing and harvesting T cells for storage (for example, reformulating T cells in cryopreservation media) or administration. In some embodiments, step (f) is performed no later than 30 hours after the beginning of step (d) or (e), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the beginning of step (d) or (e). In some embodiments, provided herein is a population of cells (for example, immune effector cells, for example, T cells or NK cells) made by any of the manufacturing processes described herein. In some embodiments, the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) (1) is the same as, (2) differs, for example, by no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, from, or (3) is increased, for example, by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, as compared to, the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) shows a higher percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% higher), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days). In some embodiments, the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) is not less than 20, 25, 30, 35, 40, 45, 50, 55, or 60%. In some embodiments, the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) (1) is the same as, (2) differs, for example, by no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% from, or (3) is decreased, for example, by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, as compared to, the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) shows a lower percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% lower), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days). In some embodiments, the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) is no more than 40, 45, 50, 55, 60, 65, 70, 75, or 80%. In some embodiments, the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) after being administered in vivo, persists longer or expands at a higher level (for example, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days). In some embodiments, the population of cells has been enriched for IL6R-expressing cells (for example, cells that are positive for IL6Ra and/or I L6 Kb) prior to the beginning of the manufacturing process (for example, prior to the beginning of the cytokine process or the activation process described herein). In some embodiments, the population of cells comprises, for example, no less than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% of IL6R-expressing cells (for example, cells that are positive for IL6Ra and/or I L6 Kb) at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein).
EXAMPLES PREPARATION OF COMPOUNDS AND EXAMPLES The disclosure is further illustrated by the following examples and synthesis schemes, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims. Compounds of the present disclosure may be prepared by methods known in the art of organic synthesis. In all of the methods it is understood that protecting groups for sensitive or reactive groups may be employed where necessary in accordance with general principles of chemistry. Protecting groups are manipulated according to standard methods of organic synthesis (T.W. Green and P.G.M. Wuts (1999) Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons). These groups are removed at a convenient stage of compound synthesis using methods that are readily apparent to those skilled in the art. Temperatures are given in degrees Celsius. As used herein, unless specified otherwise, the term “room temperature” or “ambient temperature” means a temperature of from 15°C to 30°C, such as of from 20°C to 30°C, such as of from 20°C to 25°C. If not mentioned otherwise, all evaporations are performed under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (= 20 - 133 mbar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR. Abbreviations used are those conventional in the art. Methods Employed in the Purification of the Examples Purification of intermediates and final products was carried out via either normal, reverse phase chromatography or supercritical fluid chromatography (SFC). Normal phase chromatography was carried out using prepacked SiO2 cartridges (e.g., RediSep® Rf columns from Teledyne Isco, Inc.) eluting with gradients of appropriate solvent systems (e.g., heptane and ethyl acetate; DCM and MeOH; or unless otherwise indicated). Reverse phase preparative HPLC was carried out using the methods described below or unless otherwise indicated in the experimental section: (1) Basic method: XBridge 5μm column, 5 mM NH4OH in acetonitrile and Water. (2) TFA method: Sunfire 5μm column, 0.1% TFA in acetonitrile and Water. (3) Formic acid method: XBridge 5μm column; 0.1% formic acid in acetonitrile and Water. Analytical Methods, Materials, and Instrumentation Unless otherwise noted, reagents and solvents were used as received from commercial suppliers. Proton nuclear magnetic resonance (NMR) spectra were obtained on either Bruker Avance spectrometer or Varian Oxford 400 MHz spectrometer unless otherwise noted. Spectra are given in ppm (δ) and coupling constants, J, are reported in Hertz. Tetramethylsilane (TMS) was used as an internal standard. Chemical shifts are reported in ppm relative to dimethyl sulfoxide (δ 2.50), methanol (δ 3.31), chloroform (δ 7.26) or other solvent as indicated in NMR spectral data. A small amount of the dry sample (2-5 mg) is dissolved in an appropriate deuterated solvent (1 mL). The chemical names were generated using ChemBioDraw Ultra v12 from CambridgeSoft. Mass spectra (ESI-MS) were collected using a Waters System (Acquity UPLC and a Micromass ZQ mass spectrometer) or Agilent-1260 Infinity (6120 Quadrupole); all masses reported are the m/z of the protonated parent ions unless recorded otherwise. The sample was dissolved in a suitable solvent such as MeCN, DMSO, or MeOH and was injected directly into the column using an automated sample handler. The analysis is performed on Waters Acquity UPLC system (Column: Waters Acquity UPLC BEH C181.7µm, 2.1 x 30mm; Flow rate: 1 mL/min; 55°C (column temperature); Solvent A: 0.05% formic acid in water, Solvent B: 0.04% formic acid in MeOH; gradient 95% Solvent A from 0 to 0.10 min; 95% Solvent A to 20% Solvent A from 0.10 to 0.50 min; 20% Solvent A to 5% Solvent A from 0.50 to 0.60 min; hold at 5% Solvent A from 0.6 min to 0.8 min; 5% Solvent A to 95% Solvent A from 0.80 to 0.90 min; and hold 95% Solvent A from 0.90 to 1.15 min. Analytical HPLC Method Information The analytical HPLC of final compounds is carried out using columns and specific conditions as described below. The analytical equipment (Waters, Agilent, Shimadzu) is also equipped with a mass detector in each case.
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
METHOD OF SYNTHESIZING THE COMPOUNDS OF THE INVENTION The compounds of the present invention may be prepared in accordance with the definition of compound of formula (I), (II) and (III), by the routes described in the following Schemes or Examples. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as”) provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. In the following general methods, R1, R2, R3, R4, R5, R6, are defined as above, or limited to designations in the Schemes. Unless otherwise stated, starting materials are either commercially available or are prepared by known methods. Compounds in formula (1-4a – 1-4b) according to the invention can be prepared stepwise starting with the synthesis depicted in scheme 1. Key intermediate 1-2a-b can be prepared via cyclization (step 1.a) of the corresponding 5-amino pyrazole bearing either a 4-cyano (1-2a) or 4- ethyl ester (1-2b) functionality and (E)-3-(dimethylamino)acrylonitrile. Bromination of amino- pyrazolopyrimidines (1-2a-b) with N-bromosuccinimide in dichloromethane (step 1.b) yields intermediates 1-3a-b and subsequent –Boc protection (step 1.c) provide the final compounds of formula 1-4a and 1-4b.
Compounds in formula (2-3a-b) according to the invention can be prepared stepwise starting with the synthesis depicted in scheme 2. Key intermediate 2-2a-b can be prepared via cyclization of amino-pyrazole 2-1 and 2-bromomalonaldehyde (step 2.a). Coupling of the resulting pyrazolopyrimidines 2-2a-b and 4,4,4,4,5,5,5,5-Octamethyl-2,2-bi-(1,3,2-dioxaborolane using a palladium catalyst and potassium acetate (step 2.b) to provide boronic acids of formula 2-3a – 2- 3b. Compounds in formula (3-3) according to the invention can be prepared stepwise starting with the synthesis depicted in scheme 3. Key intermediate 3-2 can be prepared via bromination of 3-1 with N-bromosuccinimide in acetonitrile (step 3.a). Coupling of the resulting naphthyl compounds 3-2 and 4,4,4,4,5,5,5,5-Octamethyl-2,2-bi-(1,3,2-dioxaborolane) using a palladium catalyst and potassium acetate (step 3.b) to provide boronic acids of formula 3-3.
Compounds in formula (4-6) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 4. Key intermediate 4-2 can be prepared via treatment of fluorobenzenes 4-1 with LDA (step 4.a) followed by addition of appropriate acylating reagent (i.e. DMF). Intermediates 4-2 were heated with ethyl 2-mercaptoacetate and potassium carbonate (step 4.b) to give benzothiophenes 4-3. Hydrolysis of 4-3 (step 4.d) with NaOH and subsequent decarboxylation of 4-4 (step 4.d) in the presence of DBU under microwave irradiation affords bromo benzothiophenes 4-5. Coupling of the resulting bromobenzothiophenes 4-5 with and 4,4,4,4,5,5,5,5-Octamethyl-2,2-bi-(1,3,2-dioxaborolane) using a palladium catalyst (step 4.e) to provide boronic esters of formula 4-6. Compounds in formula (5-6) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 5. Key intermediate 5-2 can be prepared via treatment of fluorobenzenes 5-1 with LDA (step 5.a) followed by addition of appropriate acylating reagent (i.e. DMF). Intermediates 5-2 were heated with ethyl 2-mercaptoacetate and potassium carbonate (step 5.b) to give benzothiophenes, 5-3. Hydrolysis of 5.3 (step 5.c) with NaOH and subsequent decarboxylation of 5-4 (step 5.d) in the presence of DBU under microwave irradiation affords bromo benzothiophenes 5-5. Coupling of the resulting bromobenzothiophenes 5-5 with and 4,4,4,4,5,5,5,5-Octamethyl-2,2-bi-(1,3,2-dioxaborolane) using a palladium catalyst (step 5.e) to provide boronic esters of formula 5-6. General synthesis of compounds of general formula (I) of the present invention Compounds in formula (I) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 6. Key intermediate 6-1 can be prepared via Suzuki reaction (step 6.a) of the corresponding pyrazolopyrimidine bromide core bearing either a 7-Boc protected amine (1-4a), a 2,7-hydrogen (2-2a) or an 2-amine functionality (2-2b) and appropriate biaryl boronate, which are either commercially available or can be synthesized as described in scheme 3-5. The cyano pyazolopyrimidines (6-1 to 6-3) subjected with sodium azide in step 6.b to provide the final compounds formula (I)
Compounds in formula (I) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 7. Key intermediates 7-1 and 7-2 can be prepared via Suzuki reaction (step 7.a) of the corresponding pyrazolopyrimidine boronic acid core bearing either a 2,7- hydrogen (2-3a) or an 2-amine functionality (2-3b) and appropriate biaryl bromide, which are either commercially available or synthesized as described in schemes 3-5. The cyano pyazolopyrimidines (7-1 to 7-2) subjected with sodium azide (step 7.b) to provide the final compounds of formula (I).
Compounds in formula (I) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 8. Key intermediate 8-1 can be prepared via a tandem Suzuki cross-coupling/isoxazole ring opening (step 9.a) of the corresponding biaryl bromide 3-2 and 4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole in surfactant using a palladium catalyst. Cyclization of the biaryl enols 8-1 and commercially available 5-amino-1H-pyrazole-4-carbonitrile (step 9.c), in toluene with pTSA affords biaryl pyrazolopyrimidines 8-2. Subsequent treatment of 8.2 with sodium azide provides the final compounds of formula (I). General synthesis of compounds of general formula (II) of the present invention Compounds in formula (II) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 9. Key intermediates 9-2 and 9-3 can be prepared by heating pyrazolopyrimidines 8-2 or 6-1 (preparations described in schemes 6 and 8) in the presence of hydroxylamine hydrochloride and sodium carbonate (step 9a) Cyclization of N-hydroxyamidines 9-2 and 9-3 with 2-ethylhexyl carbonochloridate in the presence of pyridine provide the final compounds of formula (II). General synthesis of compounds of general formula (III) of the present invention
Compounds in formula (III) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 10. Key intermediate 10-1 can be prepared Suzuki reaction (step 10.a) of the corresponding pyrazolopyrimidine bromide 1-4b and appropriate biaryl boronic acid (3-3), which are either commercially available or synthesized as described in schemes 3. The resulting biaryl pyrazolopyrimidines 10-1 were treated with hydrochloric acid to give esters 10-2 that upon hydrolysis with NaOH provide the final compounds of formula III. Compounds in formula (III) according to the invention can be prepared stepwise starting with a synthesis depicted in scheme 11. Key intermediate 11-1 can be prepared via cyclization (step 11.a) of 5-amino-1H-pyrazole-4-carbonitrile and the corresponding enol intermediate 8-1, which are synthesized as described in scheme 8, in toluene with pTSA. The resulting esters (11- 1) were hydrolyzed (step 11.b) to provide the final compounds of formula (III). Synthesis of Intermediates Intermediate 1-4a. tert-butyl (6-bromo-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)(tert butoxycarbonyl) carbamate Step 1.a.7-aminopyrazolo[1,5-a]pyrimidine-3-carbonitrile (1-2a) To a stirred solution of (E)-3-(dimethylamino) acrylonitrile (10 g,104 mmol) in acetic acid (60 mL) and ethanol (60 mL) was added 5-Amino -1H-pyrazole-4-carbonitrile (1-1) (11.23 g,104 mmol) and stirred the reaction mixture at 90°C for 16h. The progress of the reaction was monitored by TLC, using mobile phase:50% EtOAc in hexane. Reaction mixture was cooled at room temperature and quenched with saturated NaHCO3 solution (100 mL), the solid precipitated, filtered, washed with n-pentane and diethyl ether, dried under vacuum to afford 7- aminopyrazolo[1,5-a]pyrimidine-3-carbonitrile (1-2a) (10.2g, 64.1 mmol, 62.5%) as yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.63 (s, 1H), 8.32 (bs, 1H), 8.23-8.22 (d, J=5.6Hz, 1H), 6.33- 6.32 (d, J=6Hz, 1H). LC-MS (ESI): m/z = 160.1 [M+H]+ Step 1.b. Preparation of 7-amino-6-bromopyrazolo[1,5-a]pyrimidine-3-carbonitrile (1-3a) To a stirred solution of 7-aminopyrazolo[1,5-a]pyrimidine-3-carbonitrile (1-2a) (5.0 g, 31.41 mmol) in acetonitrile (120 mL) and dichloromethane (120 mL) was added N- bromosuccinimide (5.59 g, 31.41 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2h. The progress of the reaction was monitored by TLC, using mobile phase:50 % EtOAc in hexane. The reaction mixture was diluted with water (30mL), basified with saturated NaHCO3 solution (500 mL), the solid precipitated, filtered and dried under vacuum to get crude. The crude was triturated with diethyl ether to afford 7-amino-6-bromopyrazolo[1,5- a]pyrimidine-3-carbonitrile (1-3a) (7.1g, 29.82mmol,95.94%) as yellow solid. 1H NMR: 1H NMR (300 MHz, DMSO-d6) δ 8.66 (s, 1H), 8.64 (bs, 2H), 8.44 (s, 1H). LC-MS (ESI): m/z = 240.0 [M+H]+ Step 1.c. tert-butyl (6-bromo-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)(tert butoxycarbonyl) carbamate (1-4a) To a stirred solution of 7-amino-6-bromopyrazolo[1,5-a]pyrimidine-3-carbonitrile (1-3a) (7.0 g, 29.41 mmol) in dichloromethane (120 mL) at room temperature was added N, N dimethylpyridine-4-amine (0.360 g, 2.94 mmol) and triethylamine (6.47g, 64.11 mmol). Di-tert- butyl-dicarbonate (16g, 73.50 mmol) was added portion-wise to this mixture and was stirred at 38°C for 3h. The progress of the reaction was monitored by TLC, using mobile phase:50 % EtOAc in hexane. The reaction mixture was diluted with water (50 mL) and extracted with DCM (100 mL). The organic layer was washed with brine solution and dried over anhydrous Na2SO4 and concentrated under vacuum. The crude was purified by Combi flash, eluting the product at 9.8% EtOAc in hexane to afford tert-butyl (6-bromo-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)(tert- butoxycarbonyl)carbamate (1-4a) (5.8 g, 13.23 mmol, 44.99%) as off-white solid.1H NMR (300 MHz, CDCl3): δ 8.81 (s, 1H), 8.38 (s, 1H), 1.37 (s, 18H). LC-MS (ESI): m/z = 338.0 [M+H]+ Intermediate 1-4b. ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-bromopyrazolo[1,5- a]pyrimidine-3-carboxylate Step 1.a. ethyl 7-aminopyrazolo[1,5-a]pyrimidine-3-carboxylate (1-2b) Mixture of ethyl 5-amino-1H-pyrazole-4-carboxylate (1-1) (10.0g, 65.41 mmol) and (E)-3- (dimethylamino)acrylonitrile (9.2g, 96.77 mmol) taken in acetic acid (70.0 mL) and HCl in Ethanol (70.0 mL) at room temperature. The reaction mixture irradiated under microwave at 110°C for 8h. The completion of the reaction was monitored by TLC, using mobile phase 80% EtOAc in hexane. Cooled the reaction mixture to room temperature and evaporated to dryness under vacuum. Diluted with saturated NaHCO3 solution and extracted the product with ethyl acetate (40 mL x2). The combined organic layer was washed with brine solution and dried (Na2SO4) and concentrated under reduced pressure. The crude was purified by flash chromatography eluting the product at 5% MeOH in DCM to afford ethyl 7-aminopyrazolo[1,5-a]pyrimidine-3-carboxylate (1-2b) (10.0 g, 48.54 mmol, 75.2%) as yellow solid.1H NMR (400 MHz, DMSO-d6): δ 8.48 (s, 1H), 8.25-8.24 (d, J=5.2 Hz, 1H), 8.12 (bs, 2H), 6.31-6.29 (d, J=5.2 Hz ,1H), 4.28-4.22 (q, 2H), 1.31-1.27(t, 3H). LC- MS (ESI): m/z = 207.2 [M+H]+ Step 1.b. ethyl 7-amino-6-bromopyrazolo[1,5-a]pyrimidine-3-carboxylate (1-3b) To a stirred solution of ethyl 7-aminopyrazolo[1,5-a]pyrimidine-3-carboxylate (1-2b) (3.1 g, 15.04 mmol) in acetonitrile (80 mL) and dichloromethane (80 mL) was added N- bromosuccinimide (2.67 g, 15.04 mmol) at 0°C. The reaction mixture was stirred at room temperature for 2h. The progress of the reaction was monitored by TLC, using mobile phase:50 % EtOAc in hexane. The reaction mixture was diluted with water (30mL), basified with saturated NaHCO3 solution (500 mL), precipitated solid filtered and dried under vacuum. The crude was triturated with diethyl ether to afford ethyl 7-amino-6-bromopyrazolo[1,5-a]pyrimidine-3- carboxylate (1-3b) (3.72g, 1304 mmol, 86.91%) as yellow solid.1H NMR (300 MHz, DMSO-d6): δ 8.47 (s, 1H), 8.45 (s, 1H), 8.42 (bs, 2H), 4.27-4.20 (q, 2H), 1.29-1.24 (t, 3H). LC-MS (ESI): m/z = 286.9 [M+H]+ Step 1.c. ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-bromopyrazolo[1,5-a]pyrimidine-3- carboxylate (1-4b) To a stirred solution of ethyl 7-amino-6-bromopyrazolo[1,5-a]pyrimidine-3-carboxylate (1- 3b) (3.70 g, 12.97 mmol) in DCM (111.0 mL), was added DMAP (0.158 gm,1.297 mmol), followed by the addition of triethylamine (3.93 mL, 28.27 mmol) and Boc anhydride (7.08 gm, 32.44 mmol) and allowed to stir the reaction mixture at 38oC for 3h. The progress of the reaction was monitored by TLC, using mobile phase 50% EtOAc in hexane. The reaction mixture was diluted with water (25 mL) and extracted the crude product with DCM. The combined organic layer was washed with brine solution (10mL x 3) and dried with anhy. Na2SO4 and concentrated the organic layer under reduced pressure to afford the crude. The crude was purified by using combi flash (24 g Silicycle column cartridge) chromatography using 10% EtOAc in hexane as eluent and concentrated the eluent under reduced pressure to afford ethyl 7-(bis(tert-butoxycarbonyl)amino)-6- bromopyrazolo[1,5-a]pyrimidine-3-carboxylate (1-4b) (3.10 g, 6.38 mmol, 49.24 %) as white solid. 1H NMR (400 MHz, DMSO-d6): δ 9.14 (s, 1H), 8.75 (s, 1H), 4.35-4.30 (q, 2H), 1.34-1.30 (t, 3H),1.29 (s, 12H). LC-MS (ESI): m/z = 485.05 [M+H]+ Intermediate 2-3a. (3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)boronic acid Step 2.a.6-bromopyrazolo[1,5-a]pyrimidine-3-carbonitrile (2-2a) To a solution of 5-Amino -1H-pyrazole-4-carbonitrile (2-1) (3.90 g, 36.07 mmol) in ethanol (30 mL) at room temperature was added 2-bromomalonaldehyde (7.50 g, 50.51 mmol). The reaction mixture was irradiated under microwave for 0.5h at 110°C temperature. (Reaction was performed in 3 batches of 1.30 g). The completion of the reaction was monitored by TLC, using mobile phase 30% EtOAc in hexane. Cooled the reaction mixture to room temperature and evaporated to dryness. The residue was diluted with saturated sodium bicarbonate solution, precipitated solid filtered and dried under vacuum to afford 6-bromopyrazolo[1,5-a]pyrimidine-3- carbonitrile (2-2a) (7.0g, 30.820 mmol, 85.47%) as off-white solid. 1H NMR: (300MHz, DMSO- d6): 9.89 (s, 1H), 8.96 (s, 1H), 8.82 (s, 1H). Step 2.b. (3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)boronic acid (2-3a)
Figure imgf000192_0001
The solution of 6-bromopyrazolo(1,5-a)pyrimidine-3-carbonitrile (2-2a) (5.0 g, 22.42 mmol) and 4,4,4,4,5,5,5,5-Octamethyl-2,2-bi-(1,3,2-dioxaborolane (11.38 g, 44.84 mmol) in Dioxane (50 mL) was added potassium acetate (6.60 g, 67.26 mmol) and purged with nitrogen for 30 minutes. Then added [1,1′-Bis (diphenylphosphino)ferrocene]dichloropalladium(II)dichloromethane(3.6 g,4.48 mmol) at room temperature and heated 100°C for 3h. The completion of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. To the reaction mixture saturated NaHCO3 solution (50 mL) and water(100mL) was added and extracted with EtOAc (100 mL) and separated the layer. The aqueous layer was acidified with 1M HCl (100 mL) and extracted with EtOAc (100 mL). The organic layer was washed with water, brine solution, dried over anhy. sodium sulphate and concentrated to afford (3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)boronic acid (2-3a) (2.5 g, 13.3 mmol, 59%) as an off-white solid. LC-MS (ESI): m/z = 189.1 [M+H]+ Intermediate 2-3b. (2-amino-3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)boronic acid Step 2.a.2-amino-6-bromopyrazolo[1,5-a]pyrimidine-3-carbonitrile (2-2b) To a stirred solution of 3,5-diamino-1H-pyrazole-4-carbonitrile (2-1) (8.6 g, 69.91 mmol) in EtOH (80 mL) at 0°C was added 2-bromomalonaldehyde (12.6 g, 83.90 mmol) was dissolved in EtOH (50 mL). The reaction mixture was stirred at room temperature for 1h. The color of the reaction mixture changes from yellow to brown. The progress of the reaction checked by TLC with mobile phase 30% EtOAc in hexane. The reaction mass was poured into cold ice water (400 mL) and added saturated NaHCO3 solution (100 mL) to this aqueous portion and extracted the product with EtOAc (800 mL) and washed with 0.5N HCl (100 mL) and organic layer dried over anhy. Na2SO4 and concentrated under vacuum to get the crude. The crude was triturated with n-pentane (20 mL) and diethyl ether (40 mL) to afford 2-amino-6-bromopyrazolo[1,5-a]pyrimidine-3- carbonitrile (2-2b) (8.6g, 36.13mmol, 51.6 %) as a pale brown solid.1H NMR: 1H NMR (300 MHz, DMSO-d6) δ 9.35 (s, 1H), 8.56 (s, 1H), 7.00-6.6 (bs, 2H). Step 2.b. (2-amino-3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)boronic acid (2-3b) To a stirred solution of 2-amino-6-bromopyrazolo[1,5-a]pyrimidine-3-carbonitrile (2-2b) (3.3 g, 13.86 mmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (5.3 g, 20.79 mmol) in Dioxane (33.0 mL) was added Potassium Acetate (4.0 g, 41.56 mmol) at room temperature and degassed with argon gas for 20 minutes. Then added PdCl2(dppf).CH2Cl2 (1.13 g, 1.386 mmol) and purged for another 10 minutes. The reaction mixture was heated at 100°C for 6h. The color changes from brown to black. The progress of the reaction checked by TLC with mobile phase 30% EtOAc in hexane. Reaction was cooled to rt and added brine solution (500 ml), extracted the product with EtOAc (1000 mL). Added sat. NaHCO3 solution (300 mL) to organic layer and stirred for 15 minutes and extracted the product with EtOAc (800 mL). The aqueous layer was acidified with 1N HCl (pH~ 4.0), the solid precipitated was filtered off under vacuum to afford (2-amino-3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)boronic acid (2-3b) (1.0 g, 4.9 mmol, 35.5%) as off-white solid. LC-MS (ESI): m/z = 203.6 [M+H]+ Intermediate 3-2a.1-bromo-6-fluoro-2-methylnaphthalene Step 1. 2-fluoro-6-methylnaphthalene To a stirred solution of 2-bromo-6-fluoronaphthalene (3.0 g, 13.32 mmol) in DME: H2O (90 ml) were added methyl boronic acid (2.39 g, 39.98 mmol) and Na2CO3 (7.0 g, 66.6 mmol) and purged the reaction mixture with argon for 10 minutes. Then added Pd(PPh3)4 (0.307 g, 0.266 mmol) and the reaction mixture was further purged with argon for 30 minutes. Sealed the reaction mixture and stirred at 90°C for 12 hours. The completion of the reaction was monitored by TLC, using mobile phase 100% hexane. Cooled the reaction mixture to room temperature and diluted with water (25 mL) and extracted the product with ethyl acetate (40 mL x 3). The organic layer was washed with brine solution (10 mL x 3) and dried with anhydrous Na2SO4 then concentrated under reduced pressure to afford the crude product. The crude was purified by flash Buchi (silica column 24 g), eluting the product at 0-2% EtOAc in hexane to afford 2-fluoro-6-methylnaphthalene (1.20g, 7.49mmol, 56.20%) as off-white solid.1H NMR: (300 MHz, CDCl3): δ 7.76-7.67 (m, 2H), 7.60 (s, 1H), 7.43-7.32 (m, 2H),7.23-7.19 (m, 1H), 2.50 (s, 3H). Step 2. 1-bromo-6-fluoro-2-methylnaphthalene (3-2a) To a stirred solution of 2-fluoro-6-methylnaphthalene (From step 1, 0.500 g, 3.121 mmol) in acetonitrile (21.0 ml) was added NBS (0.628 g, 3.532 mmol) at 0°C and the reaction mixture was stirred at room temperature for 16h. The completion of the reaction was monitored by TLC, using mobile phase: 100% Hexane. To the reaction mixture was added saturated NaHCO3 solution (50 mL) and water(100m) and extracted with EtOAc (100 mL). The organic layer was washed with water, brine solution, dried over anhy. sodium sulphate and concentrated to afford 1-bromo-6-fluoro-2-methylnaphthalene (3-2a) (0.310 g, 1.25mmol, 40.20%) as pale-yellow gummy solid.1H NMR (300 MHz, CDCl3): δ 8.32-8.27(m, 1H),7.72-7.60(m, 1H),7.43-7.29(m, 3H), 2.60(s,3H); 19FNMR (300 MHz, CDCl3): δ -116.12 δ ppm confirmed the formation of desired product. Intermediate 3-2b. (1-bromonaphthalen-2-yl)methanol Step 1. 1-bromo-2-(bromomethyl)naphthalene To a stirred solution of 1-bromo-2-methylnaphthalene (2.0 g, 9.049 mmol) in ACN (20 mL) at room temperature, N-bromosuccinimide (1.70 g, 9.095 mmol) and benzoic peroxyanhydride (0.021 g, 0.090 mmol) were added and reaction mixture was heated at 85°C for 6h. The completion of the reaction was monitored by TLC using mobile phase: 5% EtOAc in hexane. Reaction mixture was diluted with water and extracted with diethyl ether, washed the organic layer with brine solution, dried over anhy. Na2SO4 and concentrated under vacuum to get the crude. The crude was triturated with n-hexane to afford 1-bromo-2-(bromomethyl) naphthalene (1.46 g, 4.866 mmol, 53.7 %) as off-white solid. LCMS: Not ionization. The obtained product was taken to next step. Step 2. (1-bromonaphthalen-2-yl)methanol (3-2b) To a stirred solution of 1-bromo-2-(bromomethyl)naphthalene (From step 1, 0.500 g, 1.666 mmol) in Dioxane: water (1:1, 10 mL), was added calcium carbonate (0.083 g, 8.333 min) at room temperature and reaction mixture was heated at 100°C 10h duration. The completion of the reaction was monitored by TLC, using mobile phase: 30 % EtOAc in hexane. Cooled the reaction mixture to room temperature. Reaction mixture was diluted with water and 1N HCl solution and extracted with EtOAc. The organic layer was washed with brine solution, dried over anhy. Na2SO4 and concentrated under vacuum to get the crude. The crude was triturated with diethyl ether to afford (1-bromonaphthalen-2-yl)methanol (3-2b) (0.265 g, 1.118 mmol, 67.1 %) as off-white solid. HPLC:95.3%; LCMS: Not ionised. The obtained product was taken to next step based on TLC. Intermediate 3-2c.1-bromo-2-methoxy-7-methylnaphthalene Step 1. 2-bromo-7-methoxynaphthalene To a stirred solution of 7-bromonaphthalen-2-ol (5.0 g, 22.41 mmol) in DMF (150.0 mL) under nitrogen atmosphere, were added methyl iodide (10.5 mL, 168.10 mmol) and K2CO3(9.2 g, 67.24 mmol) and the reaction mixture was stirred at room temperature for 3h. The progress of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. The reaction mixture was quenched with saturated ammonium chloride and extracted the product with ethyl acetate (3*100 mL) and organic layer washed with water. Concentrated the ethyl acetate layer to afford 2-bromo-7-methoxynaphthalene (4.31 g, 18.17 mmol, 81%) as off-white solid. The formation of the product was confirmed by 1H NMR (400 MHz, CDCl3): δ 7.89 (s, 1H), 7.70-7.68 (d, J=8.8 Hz, 1H), 7.63-7.61 (d, J=8.4 Hz, 1H), 7.41-7.38 (m, 1H), 7.15-7.12 (m, 1H), 7.30 (s, 1H), 3.91 (s, 3H). Step 2. 2-methoxy-7-methylnaphthalene To a stirred suspension of 2-bromo-7-methoxynaphthalene (From step 1, 3.0 g, 12.65 mmol) in DME (60.0 mL) and water (30.0 mL) were added methyl boronic acid (4.54 g, 75.91 mmol), Na2CO3 (6.70 g, 63.26 mmol) and purged the reaction mixture with argon for 30 min. After 30 minutes, added Pd(PPh3)4 (0.73 g, 0.63 mmol) and purged again for another 10 minutes. The reaction mixture was heated to 90°C for 8h. The progress of the reaction was monitored by TLC, using mobile phase 100% hexane. Cooled the reaction mixture to room temperature. Diluted the reaction mass with water and extracted with ethyl acetate (3x50mL) and organic layer was washed with water, brine solution and dried over anhy. sodium sulphate. Concentrated the organic layer under vacuum to get the crude. The crude was purified by using combi flash 24 g cartridges, eluting the product at neat hexane to afford 2-methoxy-7-methylnaphthalene (1.37 g, 7.954 mmol, 63%) as off-white solid. The formation of the product was confirmed by 1H NMR (400 MHz, CDCl3): δ 7.70-7.68 (t, J=9.2Hz, 2H), 7.52 (s, 1H), 7.19-7.17 (m, 1H), 7.09-7.07 (m, 2H), 3.92 (s, 3H), 2.50 (s, 3H). Step 3. 1-bromo-2-methoxy-7-methylnaphthalene (3-2c) To a stirred suspension of 2-methoxy-7-methylnaphthalene (From step 2, 1.3 g, 7.54 mmol) in MeCN (13.0 mL, 10.0 V), NBS (1.47 g, 8.30 mmol) was added and stirred the reaction mixture at room temperature for 3h. The progress of the reaction was monitored by TLC, using mobile phase 100% hexane. Quenched the reaction mixture with saturated sodium thiosulphate solution and extracted with ethyl acetate (3*100 mL) and organic layer washed with water. Concentrated the organic layer to get crude. The crude was purified by combi flash using 24 g cartridge, eluting the product at neat hexane to afford 1-bromo-2-methoxy-7-methylnaphthalene (3-2c) (0.91 g, 2.36 mmol, 48%) as white colored solid. The formation of product was confirmed by 1H NMR (400 MHz, CDCl3): δ 7.99 (s, 1H), 7.77-7.75 (d, J=9.2Hz,1H), 7.69-7.67 (J=8.0 Hz, 1H), 7.23-7.19 (m, 2H), 4.02 (s, 3H), 2.55 (s, 3H). Intermediate 3-2d.1-bromo-2-(difluoromethyl)naphthalene (3-2d) To a stirred solution of 1-bromo-2-naphthaldehyde (1.00 g,4.27 mmol) in DCM (10.0 ml) was added DAST (1.13 ml, 8.55 mmol) at 0oC and the reaction mixture was stirred at room temperature for 12h. The progress of the reaction was monitored by TLC, using mobile phase:100% hexane. The reaction mixture was quenched with saturated sodium bicarbonate solution (10 mL) and extracted to ethyl acetate (2*10 mL). The organic layer was separated, dried over anhy. sodium sulfate, filtered and concentrated under reduced pressure to get the crude. The crude product was purified by combi-flash using a 4g cartridge and the product eluted with hexane to afford 1-bromo-2-(difluoromethyl)naphthalene (3-2d) (0.830g, 3.24mmol, 76.15%) as white solid. 1H NMR (400 MHz, CDCl3): δ 8.38-8.36 (d, J=8.4Hz, 1H), 7.92-7.87 (m, 2H), 7.72- 7.64 (m, 3H), 7.38-7.10 (t, J= 55.2Hz, 1H); 19F NMR: -117.54 δ ppm. LCMS: No ionization. Intermediate 3-2e.1-(1-bromonaphthalen-2-yl)-N,N-dimethylmethanamine To a stirred solution of 1-bromo-2-(bromomethyl)naphthalene (1.0 g, 3.333 mmol) in EtOH(10 mL), was added dimethylamine (33% in EtOH; 3.0 mL) at room temperature and reaction mixture was heated at 80°C for 5h. The completion of the reaction was monitored by TLC, using mobile phase: 10 % EtOAc in hexane. Cooled the reaction mixture to room temperature. Reaction mixture was extracted with EtOAc twice. The organic layer was washed with brine solution, dried over anhy. Na2SO4 and concentrated under vacuum to get the crude. The crude was purified by Buchi, (12 g Silcycle column cartridge) using 5-10% EtOAc in hexane to afford 1-(1- bromonaphthalen-2-yl)-N,N-dimethylmethanamine (3-2e) (0.50 g, 1.89 mmol, 56.78 %) as yellow gum.1H NMR (300 MHz, DMSO-d6): δ 8.24-8.21 (d, J=8.4 Hz, 1H), 7.98-7.93 (t, J=7.8 Hz, 2H), 7.69-7.59 (m, 3H), 3.71 (s, 2H), 2.23 (s, 6H). LC-MS (ESI): m/z = 263.9 [M+H]+ Intermediate 3-2f.1-bromo-2,7-dimethylnaphthalene To a solution of 2,7-dimethylnaphthalene (3.0 g ,19.2 mmol) in DCM (60.0 mL) in two necked round bottom flask at 0°C was added Bromine (0.493 mL,19.2 mmol) in DCM (60 mL) over 30min. The color changes to brown and reaction mixture was stirred at room temperature for 3h. The progress of the reaction checked by TLC using mobile phase: 100% hexane. Solvents are removed under vacuum. Reaction mass was quenched with saturated Na2S2O7 solution and extracted with DCM (50 mL x 2). The organic layer was washed with Na2CO3 solution and brine solution, dried over anhy. Na2SO4 and then concentrated under vacuum. The crude product was purified by combi-flash using 24 g cartridge eluted with 100% Hexane to afford 1-bromo-2,7- dimethylnaphthalene (3-2f) (3.4 g) as clear oil.1H NMR (400MHz, CDCl3): δ 8.06 (s, 1H), 7.70- 7.64 (m, 1H), 7.30-7.26 (m, 1H), 2.59 (s, 3H), 2.56 (s, 3H). Intermediate 3-2g. 4,4,5,5-tetramethyl-2-(7-methylnaphthalen-1-yl)-1,3,2-dioxaborolane (3- 2g) Step 1. (7-methyl-3,4-dihydronaphthalen-1(2H)-ylidene)hydrazine To a stirred solution of 7-methyl-3,4-dihydronaphthalen-1-(2H)-one (5.0 g, 31.21 mmol) in ethanol (50 mL) was added hydrazine monohydrate (20 mL, 624.2 mmol) dropwise and then stirred reaction mixture at 70°C for 16h.The progress of reaction was monitored by TLC, using mobile phase 20% EtOAc in hexane. The reaction mass was cooled to room temperature and the volatiles were removed on high vacuum. To the solid residue obtained was added water and extracted with EtOAc washed with brine and dried over anhy. Na2SO4 and concentrated in vacuum to afford 7-methyl-3,4-dihydronaphthalen-1(2H)-ylidene) hydrazine (5.20 g, crude) as pale-yellow solid. 1H NMR: (400 MHz, DMSO-d6): δ 7.63 (s, 1H), 6.96-6.92 (dd, 2H),6.24 (s, 2H),2.60-2.59 (t, 2H),2.39-2.36 (t, 2H), 2.23 (s, 3H), 1.75-1.70 (m, 2H). LC-MS (ESI): m/z = 174.7 [M+H]+ Step 2. 4-bromo-6-methyl-1,2-dihydronaphthalene To a stirred solution of 7-methyl-3,4-dihydronaphthalen-1(2H)-ylidene) hydrazine (From step 1, 2.70 g, 15.49 mmol) in THF (27 mL), was added KOtBu (1.75 g, 15.49 mmol) and reaction mixture was stirred for 20 min then added copper bromide (7.0 g, 30.98 mmol) in portion wise at 0°C. The resulting reaction mixture was stirred at room temperature for 2h. The completion of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. The reaction mixture was filtered on a celite bed, and the filtrate was extracted with ethyl acetate. The organic layer was washed with brine solution and dried with Na2SO4 and concentrated under reduced pressure to get the crude. The crude was purified by combi-flash using 40g column, eluted the product with 0-5% EtOAc in hexane to afford 4-bromo-6-methyl-1,2-dihydronaphthalene (0.760 g, 3.406 mmol, 21.98%) as pale brown solid.1H NMR: (400 MHz, CDCl3): δ 7.36 (s, 1H), 6.99 (s, 2H),6.44-6.41 (t, J=9.6 Hz, 1H), 2.81-2.77 (t, 2H), 2.35 (s, 3H), 2.36-2.30 (m, 2H). Step 3. 1-bromo-7-methylnaphthalene To a stirred solution of 4-bromo-6-methyl-1,2-dihydronaphthalene (From step 2, 1.50 g, 6.72 mmol) in toluene (15 mL) at 0°C was added DDQ (3.05 g, 13.44 mmol) and stirred reaction mixture at room temperature for 16h. The completion of the reaction was monitored by TLC using mobile phase:100% pentane. Cooled the reaction to room temperature and the volatiles were removed on high vacuum from reaction mixture and solid residue was quenched by sat. NaHCO3 solution and extracted with EtOAc, separated the layer. The organic layer was washed with water, brine solution, dried over anhy. Na2SO4 and concentrated to get the crude. The crude was purified by using Combi flash using 4 g cartridge, 0-100% EtOAc in hexane as eluent and concentrated the fractions under reduced pressure to afford 1-bromo-7-methylnaphthalene (0.660 g, 2.985 mmol, 44.59%) as pale-yellow solid. LC-MS (ESI): m/z = 220.2 [M+H]+ Step 4. 4,4,5,5-tetramethyl-2-(7-methylnaphthalen-1-yl)-1,3,2-dioxaborolane (3-2g) To a stirred solution of 1-bromo-7-methylnaphthalene (From step 3, 0.260 g, 1.17 mmol) in dioxane (2.50 mL), were added 4,4,4’,4’,5,5,5’,5’-octamethyl-2,2-bi(1,3,2-dioxaborolane (0.447 g, 1.76 mmol) and Potassium Acetate (0.173 g, 1.76 mmol) and the mixture was purged with Argon for 30 min. Then added [1,1'Bis (diphenylphosphino) ferrocene] palladium(II)dichloridedichloromethane complex (0.480 g, 5.87 mmol) to the reaction mixture and further purged with Argon for 5 min. Then the reaction mixture was stirred at 100°C for 12h.The progress of the reaction was monitored by TLC, using mobile phase:10 % EtOAc in hexane. Reaction mixture was cooled to room temperature and concentrated under reduced pressure to get the crude. The crude was purified by combi flash using 4 g cartridge, eluted the product with 0-10% EtOAc in hexane to afford 4,4,5,5-tetramethyl-2-(7-methylnaphthalen-1-yl)-1,3,2- dioxaborolane (3-2g) (0.320 g, 1.193 mmol, 100.0%) as yellow solid. LC-MS (ESI): m/z = 267.0 [M+H]+ Intermediate 3-2h.1-bromo-6-methoxynaphthalene Step 1. (Z)-(6-methoxy-3,4-dihydronaphthalen-1(2H)-ylidene)hydrazine To a stirred solution 6-methoxy-3,4-dihydronaphthalen-1(2H)-one (10.0 g.56.75 mmol) in ethanol (100 mL) under nitrogen atmosphere were added hydrazine hydrate (36.37 mL,1135.07 mmol) at room temperature and refluxed for 70°C for 16h. The completion of the reaction was monitored by TLC using mobile phase: 20% hexane. Cooled the reaction mass to room temperature. To this reaction mixture 300 mL water was added and extracted with ethyl acetate (100 mL x 3). The organic layer was washed with brine solution (50 mL) and dried with anhy. Na2SO4 and concentrated under reduced under vacuum to get the crude product (Z)-(6-methoxy- 3,4-dihydronaphthalen-1(2H)-ylidene)hydrazine (7.30 g, 38.37 mmol, 67.65 %) as yellow solid. 1H NMR:, (400 MHz, DMSO-d6): δ 7.75-7.73 (d, J=8.8 Hz, 1H), 6.73-6.70 (m, 1H), 6.67-6.66 (m, 1H), 6.60 (bs, 2H), 3.72 (s, 3H), 2.65-2.62 (t, J=5.6 Hz, 2H), 2.39-2.36 (t, J=6.8 Hz, 2H), 1.80- 1.75 (m, 2H). Step 2. 4-bromo-7-methoxy-1,2-dihydronaphthalene To a stirred solution of tertiary butoxide (1.76 g,15.77 mmol) in THF (20 mL) was added copper bromide (7.045 g ,31.54 mmol) at 0°C and stirred at same temperature for 15 minutes. After stirring for 15 min, (Z)-(6-methoxy-3,4-dihydronaphthalen-1(2H)-ylidene)hydrazine (From step 1, 3.0 g 15.77 mmol) in THF (10 mL) was added dropwise using dropping funnel and allowed to stir the reaction mixture for 2h at room temperature.The completion of the reaction was monitored by TLC using mobile phase: 5% EtOAc in hexane. The reaction mixture was partitioned between water (200 mL) and the aqueous layer was extracted with EtOAc (300 mL x 2). Filtered the solvents on celite bed. Combined organic phases were washed with saturated brine solution, dried over Na2SO4 and the solvent was removed under reduced pressure to afford 4-bromo-7- methoxy-1,2-dihydronaphthalene (1.1 g,4.60 mmol, 29.17%) as brown liquid. 1H NMR: the formation of desired product.1H NMR: (400 MHz, CDCl3): δ 7.47-7.45 (d, J=8.8 Hz, 1H), 6.75- 6.73 (dd, 1H), 6.67- 6.66 (s, 1H), 6.30-6.28 (t, J=4.8 Hz, 1H), 3.81 (s, 3H), 2.83-2.79 (t, J=8.0 Hz, 2H), 2.36-2.31 (m, 2H). Step 3. 1-bromo-6-methoxynaphthalene (3-2h) To a stirred solution of 4-bromo-7-methoxy-1,2-dihydronaphthalene (From step 2, 1.8 g 7.5 mmol) in toluene (18 mL) was added DDQ (1.87 g,8.25 mmol) at room temperature and refluxed the reaction mass at 110°C for 12h. The completion of the reaction was monitored by TLC using mobile phase: 10 % EtOAc in hexane. The reaction mixture was quenched with dil. 10% H2SO4, water (50 mL) and extracted with dichloromethane (50 mL x 3). The combined organic layer was washed with brine solution (100 mL) and dried with Na2SO4 and concentrated under vacuum to get crude. The crude was purified using 12g column, eluting the product at 100% hexane to afford 1-bromo-6-methoxynaphthalene (3-2h) (0.220 g, 0.927 mmol, 12.94 %) as yellow semiliquid.1H NMR: (400 MHz, CDCl3): δ 8.14-8.12 (d, J=9.2 Hz, 1H), 7.70-7.68 (d, J=8.4 Hz 1H), 7.62-7.60 (dd, 1H), 7.28-7.22 (m, 2H), 7.12 (s, 1H), 3.93 (s, 3H). Intermediate 3-3i. 2-(2,6-dimethoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane Step 1. 1-bromo-2,6-dimethoxynaphthalene(3-2i) To a stirred suspension of 2,6-dimethoxynaphthalene (3.00 g, 15.94 mmol) in ACN (30.0 mL), NBS (2.85 g, 15.94 mmol) was added at 0oC and stirred the reaction mixture at room temperature for 1h. The progress of the reaction was monitored by TLC, using mobile phase 100% hexane. The reaction mixture was quenched with water (10.0 mL) and extracted with ethyl acetate (2*20 mL). The organic layer was washed with brine solution, dried over anhy. Na2SO4 and then concentrated under vacuum to get the crude. The crude was purified by combi flash using (4g Silicycle cartridge), eluting the product at neat hexane to afford 1-bromo-2,6- dimethoxynaphthalene (3-2i) (3.10 g, 11.61 mmol, 72.94%) as white colored solid. The formation of product was confirmed by 1H NMR (400 MHz, DMSO-d6): δ 7.99 -7.97 (d, J = 9.2 Hz,1H), 7.90- 7.88 (d, J = 8.4 Hz,1H), 7.49-7.47 (d, J = 9.2 Hz,1H), 7.38-7.37 (s, 1H), 7.30-7.25 (m, 1H), 3.95 (s, 3H), 3.87 (s, 3H). Step 2. 2-(2,6-dimethoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3i) To a stirred solution of 1-bromo-2,6-dimethoxynaphthalene (3-2i) (1.50 g, 5.61 mmol) in dioxane (40mL), was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (2.85 g, 11.23 mmol) followed by addition of potassium acetate (1.65 g, 16.84 mmol) and purged the reaction mixture with argon for 30 min. To this mixture Pd(dppf)Cl2.DCM (0.458 g, 0.561 mmol) was added. The reaction mixture was further purged with argon for 5 minutes, sealed the reaction mixture and stirred at 90°C for 16h. The completion of the reaction was monitored by TLC, using mobile phase 20% EtOAc in hexane. Cooled the reaction mixture to room temperature and diluted the reaction mixture with water (10.0 mL) and extracted with ethyl acetate (2 x 20 mL). The organic layer was washed with brine solution, dried over anhy. Na2SO4 and then concentrated under vacuum to get crude. The crude product was purified by combi-flash (24g Silicycle cartridge), eluted with 15% EtOAc: Hexane to afford 2-(2,6-dimethoxynaphthalen-1-yl)-4,4,5,5-tetramethyl- 1,3,2-dioxaborolane (3-3i) (2.10 g, 6.68 mmol) as a green gummy solid. LC-MS (ESI): m/z = 315.1 [M+H]+ Intermediate 3-3j.2-(3-methoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane Step 1.2,4-dibromonaphthalen-1-amine To a stirred solution of Napthalene -1-amine (10.0g, 69.83mmol) in acetic acid (70mL) at 0°C was added bromine (7.87mL,152.24mmol) dropwise at same temperature and the reaction mixture was allowed to stir at 70°C for 12h. The color changes from brown to black. The progress of the reaction was monitored by TLC, using mobile phase:10% EtOAc in hexane. The reaction mixture was cooled at room temperature and the solid precipitated filtered the solid. The solid was washed with water and dried under vacuum to get the crude. The crude was purified by chromatography, eluted (40 g Silicycle cartridge) with 5-10% EtOAc: hexane to afford 2,4- dibromonaphthalen-1-amine (7.0g, 23.26 mmol, 33.30%) as brown solid. 1H NMR (300 MHz, DMSO-d6): δ 8.30-8.28(d, J= 7.8Hz, 1H),8.00-7.98(d, J= 7.8Hz, 1H),7.79 (s, 1H),7.67-7.64(t, J= 8.4Hz, 1H),7.58-7.52(m, 1H),6.12 (bs,2H). LC-MS (ESI): m/z = 301.9 [M+H]+ Step 2.4-bromo-1-(diazen-1-ium-2-yl)naphthalen-2-olate To a stirred solution of 2,4-dibromonaphthalen-1-amine (From step 1, 5.0g, 16.61mmol) in acetic acid(80mL) at 0° C were added NaNO2 (1.14, 16.61mmol) and propionic acid (15mL) in portion wise at same temperature and the reaction mixture was allowed to stir at 0°C for 30 mins. The progress of the reaction was monitored by TLC, using mobile phase:10% EtOAc in hexane. The reaction mixture was poured to ice cold water, the solid precipitated. Filtered the solid, washed with water, dried under vacuum for overnight to afford 4-bromo-1-(diazen-1-ium-2- yl)naphthalen-2-olate (2.50g, 10.44 mmol, 62.84%) as brown solid.1H NMR (300 MHz, DMSO- d6): δ 7.99-7.96 (d, J= 7.8Hz, 1H),7.64(bs, 2H),7.44-7.40 (m, 1H),7.19 (s, 1H). Step 3.4-bromonaphthalen-2-ol To a stirred solution of 4-bromo-1-(diazen-1-ium-2-yl)naphthalen-2-olate (From step 2, 2.48g, 11.21 mmol) in EtOH (24.8mL) at 0° C, was added sodium borohydride (0.445g, 11.17 mmol) in portion wise at same temperature and the reaction mixture was allowed to stir at 0°C for 30 min. The progress of the reaction was monitored by TLC, using mobile phase: 15 % EtOAc in hexane. The reaction mixture was partitioned between ice cold water and extracted the product with ethyl acetate. The organic layer was washed with brine solution and dried with anhy. Na2SO4 and concentrated under reduced pressure to get the crude. The crude was purified by using (24 g Silicycle cartridge) Buchi, 5-15% EtOAc in hexane as eluent to afford 4-bromonaphthalen-2-ol (5) (1.10g, 4.98mmol, 49.97%) as grey solid.1H NMR: (300 MHz, DMSO-d6): δ 10.12(s, 1H),7.98- 7.95(d, J= 8.7 Hz, 1H),7.77-7.74(d, J= 8.1 Hz, 1H),7.50-7.38 (m, 3H), 7.20 (s, 1H). Step 4.1-bromo-3-methoxynaphthalene (3.2j) To a stirred solution of 4-bromonaphthalen-2-ol (From step 3, 0.60g, 2.68mmol) in THF (12mL) was added MeI (0.334mL, 5.378mmol). Then added sodium hydride (0.083g, 3.496mmol) in portion wise at 0°C and the reaction mixture was stirred at room temperature for 6h. The progress of the reaction was monitored by TLC, using mobile phase: 20 % EtOAc in hexane. The reaction mixture was portioned between ice cold water and extracted the product with ethyl acetate. The organic layer was washed with brine solution and dried with anhy. Na2SO4 and concentrated under reduced pressure to get the crude. The crude was purified by using Buchi, (24 g Silicycle cartridge) ,5-15% EtOAc in hexane as eluent to afford 1-bromo-3- methoxynaphthalene (3.2j) (0.63g, 2.66mmol,98.79%) as yellow liquid. 1H NMR (300 MHz, CDCl3): δ8.15-8.12(d, J= 8.1 Hz, 1H),7.73-7.70(d, J= 9.3 Hz, 1H),7.50-7.43(m, 3H),7.12 (s, 1H),3.91(s, 3H). Step 5.2-(3-methoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3j) To a stirred solution of 1-bromo-3-methoxynaphthalene (3-2j) (0.62g, 2.95mmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (0.895g, 5.904 mmol) in dioxane (6.20 mL), was added potassium acetate(0.434g, 4.428mml) and purged the reaction mixture with argon for 30 minutes .Then added PdCl2(dppf)DCM(0.482g, 0.590mmol) to reaction mixture and purged again with argon for 5 minutes. The reaction mixture was stirred at 100°C for 12h. The progress of the reaction was monitored by TLC, using mobile phase: 10 % EtOAc in hexane. Cooled the reaction mixture to room temperature and was concentrated under reduced pressure to afford the crude. The crude was purified by using (4 g Silicycle cartridge) Buchi, 8-12% EtOAc in hexane as eluent to afford 2-(3-methoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (3-3j) (0.56g, 1.97mmol, 75.04%) as yellow liquid. 1H NMR (300 MHz, CDCl3): δ8.66-8.64(d, J= 7.8 Hz, 1H),7.75-7.7(m, 1H), 7.45-7.36 (m, 3H), 7.25-7.22 (m, 1H), 3.92 (s, 3H),1.41 (s, 12H). Intermediate 3-2k.1-bromo-2-methoxy-6-methylnaphthalene Step 1.2-methoxy-6-methylnaphthalene To a stirred suspension of 2-bromo-6-methoxynaphthalene (7 g ,29.5 mmol) in DME (70.0 mL) methyl boronic acid (6.9 g, 118.1 mmol), 2M K2CO3 (70 mL, 147.5 mmol) was added and purged the reaction mixture with argon for 20 min. After 30 minutes, added Pd(PPh3)4 (0.601 g, 0.735 mmol) and purged again for another 10 minutes. The reaction mixture was heated to 90°C for 18h. The progress of the reaction was monitored by TLC, using mobile phase 100% hexane. Cooled the reaction mixture to room temperature. Diluted the reaction with ethyl acetate and filtered on celite bed, and the organic layer was washed with water, brine solution and dried over anhy. sodium sulphate. Concentrated the organic layer under vacuum to get the crude. The crude was purified by combi flash, eluting the product at neat hexane and further purified by reverse phase HPLC using Column ZORBAX ( C18 , 21.2mm X 250mm), Flow: 18 mL/min, Mobile Phase: A= 0.1% TFA IN WATER,B= MeCN, Gradient Program:(Time, %B):(0,60),(2,65),(10, 70), followed by the evaporation of purified sample to afford 2-methoxy-6-methylnaphthalene (1.20 g, 6.92 mmol, 23.4%) as white solid. The formation of the product was confirmed by 1H NMR: (300 MHz, CDCl3): δ 7.67-7.66 (m, 2H), 7.54 (s, 1H), 7.30-7.25 (m, 1H), 7.14-7.10 (m, 2H), 3.91 (s, 3H), 2.48 (s, 3H). Step 2.1-bromo-2-methoxy-6-methylnaphthalene (3-2k) To a stirred suspension of 2-methoxy-6-methylnaphthalene (From step 1, 1.00 g, 5.81 mmol) in MeCN (30 mL), was added NBS (1.14 g, 6.39 mmol) and stirred the reaction mixture at room temperature for 2h. The progress of the reaction was monitored by TLC, using mobile phase 100% hexane. Quenched the reaction mixture with saturated sodium thiosulphate solution (10mL), extracted with ethyl acetate (2 x 30 mL) and washed organic layer with water. Concentrated the organic layer to get the crude. The crude was purified by combi flash using 4g cartridge, eluting the product at neat hexane to afford 1-bromo-2-methoxy-6-methylnaphthalene (3-2k) (1.10 g, 4.44 mmol, 75.86%) as white solid.1H NMR (300 MHz, CDCl3): δ 8.12 - 8.09 (d, J = 9.3 Hz,1H), 7.74-7.71 (d, J = 9.0 Hz,1H), 7.55 (s, 1H), 7.41-7.38 (m, 1H), 7.25-7.22 (d, J = 9.3 Hz,1H), 4.01 (s, 3H), 2.49 (s, 3H). Intermediate 3-3l.2-(3,4-dihydronaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane Step 1.3,4-dihydronaphthalen-1-yl trifluoromethanesulfonate To a stirred solution of 3,4-dihydronaphthalen-1(2H)-one (1.0 g ,6.83 mmol) in DCM (26.0 mL) at 0°C, were added triethylamine (1.43 mL,10.25 mmol) and Trifluromethanesulfonic anhydride (1.68 mL,10.25 mmol) and the reaction mixture was heated to 40°C for 12h. The completion of the reaction was monitored by TLC using mobile phase: 5% EtOAc in hexane. The reaction mixture was diluted with water and the product was extracted with DCM (3x50 mL). The organic layer was washed with brine solution (100.0 mL) and dried over anhydrous Na2SO4 and concentrated under reduced pressure to get the crude. The crude was purified by Buchi, (12 g, Silicycle column) using 5-10% ethyl acetate: hexane as eluent to afford 3,4-dihydronaphthalen-1- yl trifluoromethanesulfonate (1.06 g, 3.809 mmol, 55.78 %) as yellow liquid. HPLC: 97.33% at RT = 7.155 min. Step 2.2-(3,4-dihydronaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3l) To a stirred suspension 3,4-dihydronaphthalen-1-yl trifluoromethanesulfonate (From step 1, 0.500 g ,1.79 mmol) in Dioxane (5.0 mL) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2- dioxaborolane (0.68 g, 2.69 mmol), potassium acetate (0.266 g, 2.69 mmol) and the reaction mixture was purged with argon for 15 min. Then added dppf (0.099 g ,0.179 mmol) and repeated the purging for another 5 minutes and added Pd(dppf)Cl2.DCM (0.293 g ,0.359 mmol) and the reaction mixture was heated to 90oC for 16h. The completion of the reaction was monitored by TLC using mobile phase: 5 % EtOAc in hexane. The reaction mixture was partitioned between water (100 mL) and EtOAc (3 x 50 mL), Combined organic phases were washed with saturated brine solution, dried over anhy. Na2SO4 and the solvent was removed under reduced pressure to get the crude. The crude was purified by combi flash (12 g, Silicycle column) using 10% EtOAc in hexane as eluent to afford 2-(3,4-dihydronaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (3-3l) (0.07 g, 0.273 mmol, 15.21 %) as off-white solid.1H NMR;(400MHz, CDCl3) δ 7.70 (d, 1H), 7.30 (m, 1H), 7.20 (t, 1H), 7.09 (m, 1H), 6.90 (m, 1H), 2.73-2.70 (t, 2H), 2.31-2.29 (m, 2H),1.33 (s, 12H). Intermediate 3-3m. (3-methylnaphthalen-1-yl)boronic acid Step 1. N-(2-methylnaphthalen-1-yl)acetamide To a stirred solution of 2-methylnaphthalen-1-amine (10.0 g, 63.69 mmol) in DCM (200.00 mL) was added acetic anhydride (11.8 mL, 127.3 mmol) dropwise at room temperature and the reaction mixture was allowed to stir at room temperature for 3h. The completion of the reaction was monitored by TLC using mobile phase:40% EtOAc in hexane. The reaction mixture was evaporated under vacuum to dryness and the solid was triturated with diethyl ether to afford N- (2-methylnaphthalen-1-yl) acetamide (10.85 g, 54.52 mmol, 85.6 %) as off-white solid.1H NMR (300 MHz, DMSO-d6): (300 MHz, DMSO-d6) δ 9.69 (s, 1H), 7.90-7.87 (t, J=7.2Hz, 2H), 7.78-7.75 (d, J=8.7Hz, 1H), 7.53-7.39 (m, 3H), 2.30 (s, 3H), 2.17 (s, 3H) LC-MS (ESI): m/z = 200.1 [M+H]+ Step 2. N-(4-bromo-2-methylnaphthalen-1-yl)acetamide To a stirred solution of N-(2-methylnaphthalen-1-yl)acetamide (From step 1, 10.8g, 54.27 mmol) in acetic acid (162.0 mL) was added bromine (3.35 mL, 65.12 mmol) at room temperature dropwise with 4h duration. The reaction mixture heated at 55°C for 6h. The completion of the reaction was monitored by TLC, using mobile phase: 40% EtOAc in hexane. Reaction mixture was poured into ice water, the solid precipitated. Filtered the solid under vacuum and washed with diethyl ether, dried under vacuum to afford N-(4-bromo-2-methylnaphthalen-1-yl)acetamide (15.05 g, 54.13 mmol, 99.7 %) as off-white solid.1H NMR (300 MHz, DMSO-d6) δ 9.79 (s, 1H), 8.10-8.07 (m, 1H), 7.95-7.93 (m, 1H), 7.85 (s, 1H), 7.66-7.59 (m, 2H), 2.30 (s, 3H), 2.17 (s, 3H). LC-MS (ESI): m/z = 275.7 [M+H]+ Step 3.4-bromo-2-methylnaphthalen-1-amine To a stirred solution of N-(4-bromo-2-methylnaphthalen-1-yl)acetamide (From step 2, 14.9g, 53.95 mmol) in EtOH (300 mL) was added concentrated HCl (75.0 mL) at room temperature . The reaction mixture was stirred at 85°C for 36h. The completion of the reaction was monitored by TLC using mobile phase: 40% EtOAc in hexane. Cooled the reaction mass to room temperature. The solid precipitated, filtered the solid and dried under vacuum to afford 4- bromo-2-methylnaphthalen-1-amine (9.3 g, 39.57 mmol, 73.3 %) as light brown liquid.1H NMR (300 MHz, DMSO-d6) δ 8.23-8.20 (d, J=7.8Hz, 1H), 8.00-7.97 (d, J=9.3Hz, 1H), 7.61-7.49 (m, 3H), 4.79 (bs, 2H), 2.32 (s, 3H). Step 4.1-bromo-3-methylnaphthalene To a stirred solution of 4-bromo-2-methylnaphthalen-1-amine (From step 3, 9.2g,38.98mmol) in concentrated HCl (10mL) and acetic acid (73mL) was added sodium nitrate solution (3.22g, 46.77 mmol) in water (25.0mL) dropwise at 5°C. The reaction mixture was stirred at 5°C for 0.5h. H3PO2(73.0 mL) was added to the reaction mixture at 5°C. The reaction mixture stirred at room temperature for 12h, followed by 100°C for 1h. The completion of the reaction was monitored by TLC using mobile phase: 10% EtOAc in hexane. Reaction mixture was diluted with water and extracted with diethyl ether, washed the organic layer with brine solution, dried over anhy. Na2SO4 and concentrated under vacuum to get the crude product. The crude was purified by Combi flash, eluting the product at 1% EtOAc in hexane to afford 1-bromo-3- methylnaphthalene (5.55g, 25.11 mmol, 64.4 %) as light brown liquid.1H NMR (300 MHz, DMSO- d6) δ 8.07-8.04 (d, J=7.2Hz, 1H), 7.90-7.87 (m, 1H), 7.76 (s, 2H), 7.63-7.55 (m, 2H), 2.46 (s, 3H). Step 5. (3-methylnaphthalen-1-yl)boronic acid (3-3m) To a solution of 1-bromo-3-methylnaphthalene (From step 4, 0.5g, 2.262 mmol) in diethyl ether (200 mL) was added n-BuLi (2.0 M in cyclohexane, 1.69 mL, 3.396 mmol) at -78°C. The reaction mixture was stirred at room temperature for 2h, and added triisopropylborate (0.77 g, 3.396 mmol) to this reaction mixture at –78 oC. The reaction mixture was stirred for 12 hr at room temperature. The progress of the reaction was monitored by TLC (30% EtOAc in hexane). The reaction mixture was diluted with water and 2N HCl solution and stirred for 15 min. Extracted the product with ether, washed the organic layer with brine solution and dried over anhydrous Na2SO4 and concentrated under vacuum. The crude was triturated with hexane to afford (3- methylnapthalen-1yl)boronic acid (3-3m) (0.24 g, 1.451 mmol, 64.1%) as off-white solid.1H NMR (400 MHz, DMSO-d6) ^ 9.13-9.11 (d, J = 8.4 Hz, 1H), 8.12 (s, 1H), 7.85-7.83 (d, J = 7.2 Hz, 1H), 7.74 (s, 1H), 7.51-7.45 (m, 2H), 2.52 (s, 3H) complies. Intermediate 3-3n. 2-(7-methoxy-6-methylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane Step 1.1-bromo-7-methoxy-6-methylnaphthalene (3-2n) To a solution of 2-methoxy-3-methylnaphthalene (1 g, 5.81 mmol) in dry DCM (40 mL) at -78 °C was treated dropwise with Bromine (0.299 mL, 5.81 mmol) and the resulting reddish solution was allowed to stir at the same temperature for 3 hr. 20% aq. sodium thiosulfate (10 mL) was added and stirred for 10 min. Org. layer was separated, washed with saturated aq. NaHCO3, brine and dried over Na2SO4. The Organic phase was filtered and evaporated to dryness to provide the crude compound. It was purified via SiO2 column (40 g, 0-10% EtOAc/Hept gradient) to provide the desired compound 1-bromo-7-methoxy-6-methylnaphthalene (3-2n) (1150 mg, 4.58 mmol, 79 % yield). LC-MS (ESI): m/z = 249.1 [M+H]+ Step 2. 2-(7-methoxy-6-methylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3- 3n) 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (607 mg, 2.389 mmol), 3-2n (300 mg, 1.195 mmol), potassium acetate (703 mg, 7.17 mmol) were dissolved in Dioxane (4 mL). The mixture was degassed thoroughly for 15 min then PdCl2(dppf).CH2Cl2 adduct (98 mg, 0.119 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen for 15 min and then heated to 85°C. overnight. The reaction mixture was allowed to come to ambient temperature and diluted with EtOAc (50 mL), filtered through celite to remove Pd residues then washed with EtOAc, combined filtrate and concentrated. It was purified via silica gel column (120 gram, 0-20% EtOAc/Hept gradient) to provide the desired product 2-(7-methoxy-6-methylnaphthalen-1-yl)- 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3n) (254 mg, 0.852 mmol, 71.3 % yield). LC-MS (ESI): m/z = 299.0 [M+H]+ Intermediate 3-3o.2-(2-ethoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (607 mg, 2.389 mmol), 1-bromo- 2-ethoxynaphthalene (300 mg, 1.195 mmol), potassium acetate (703 mg, 7.17 mmol) were dissolved in Dioxane (4 mL). The mixture was degassed thoroughly for 15 min then PdCl2(dppf).CH2Cl2 adduct (98 mg, 0.119 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen for 15 min and then heated to 85°C. overnight. The reaction mixture was allowed to come to ambient temperature and diluted with EtOAc (50 mL), filtered through celite to remove Pd residues then washed with EtOAc and concentrated. It was purified via silica gel column (120 gram, 0-20% EtOAc/Heptane gradient) to provide the desired product 2-(2-ethoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3o) (quantitative yield). LC-MS (ESI): m/z = 299.4 [M+H]+ Intermediate 3-2p.1-bromo-7-isopropylnaphthalene To a solution of 2-isopropylnaphthalene (5.27 g, 31.0 mmol) in MeCN (155 ml) at 0 °C, NBS (8.26 g, 46.4 mmol) was added. The resulting mixture was stirred at 0 °C for 20 min and at rt for 12 h at which time TLC showed the disappearance of the starting material. The reaction mixture was concentrated, and the crude was purified by silica gel plug using 100% heptane as eluant to afford the desired 1-bromo-7-isopropylnaphthalene (3-2p) (7.1 g, 28.5 mmol, 92 % yield, mixture). 1H NMR (400 MHz, DMSO-d6) δ 8.25 (dd, J = 8.7, 1.1 Hz, 1H), 7.97 (ddd, J = 7.6, 4.7, 3.4 Hz, 2H), 7.68 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.63 - 7.55 (m, 2H), 3.67 (p, J = 6.9 Hz, 1H), 1.39 - 1.04 (m, 6H). Intermediate 3-3q. 2-(2,6-dimethylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane, (2,6-dimethylnaphthalen-1-yl)boronic acid BISPIN (864 mg, 3.40 mmol), 1-Bromo-2,6-dimethylnapthalene (400 mg, 1.70 mmol), Potassium Acetate (501 mg, 5.10 mmol) were dissolved in DMF (10 mL) and the mixture was degassed thoroughly refilling with nitrogen. PdCl2(dppf).CH2Cl2 adduct (139 mg, 0.17 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was then heated to 85°C. over 16hrs. Filtered through celite washing with Ethyl Acetate to remove Palladium residues then diluted with water (200 mL). The aqueous was extracted with Ethyl Acetate (x2) and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on the ISCO 40g cartridge, eluting with 0-10% Ethyl Acetate in heptane to give as a clear oil 2-(2,6-dimethylnaphthalen-1-yl)-4,4,5,5-tetramethyl- 1,3,2-dioxaborolane (3-3q) (241 mg, 0.854 mmol, 50.2 % yield). LC-MS (ESI): m/z = not ionized Intermediate 3-3r.2-(6,7-dimethylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane BISPIN (864 mg, 3.40 mmol), 1-bromo-6, 7, dimethylnapthalene (400 mg, 1.701 mmol) and Potassium Acetate (501 mg, 5.10 mmol) were dissolved in DMF (10 mL) and the mixture was degassed thoroughly refilling with nitrogen. PdCl2(dppf).CH2Cl2 adduct (69.5 mg, 0.085 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was then heated to 85°C. Overnight. Filtered through celite washing with Ethyl Acetate, to remove Pd residues then diluted with water (200 mL). The aqueous was washed with Ethyl Acetate and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on the ISCO 40g cartridge, eluting with 0-10% Ethyl Acetate in heptane to give as a clear oil 2-(6,7-dimethylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3r) (263 mg, 0.932 mmol, 54.8 % yield). LC-MS (ESI): m/z = 283.2 [M+H]+ Intermediate 3-2s.2-(4-bromo-3-methylnaphthalen-2-yl)-1,3,4-oxadiazole Step 1.4-bromo-3-methyl-2-naphthoic acid 3-methyl-2-naphthoic acid (140 mg, 0.752 mmol) in DMF (1 mL) was treated with NBS (141 mg, 0.789 mmol) overnight. Added water and filtered off the precipitate. Collected precipitate in Ethyl Acetate. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness. Residue was recrystallized from DCM to give as a white solid. 4-bromo-3-methyl-2- naphthoic acid (82 mg, 0.309 mmol, 41.1 % yield). LC-MS (ESI): m/z = 263.2 [M+H]+ Step 2.4-bromo-N'-formyl-3-methyl-2-naphthohydrazide 4-bromo-3-methyl-2-naphthoic acid (From step 1, 78.7 mg, 0.297 mmol) in DMF (1 mL) was treated with HATU (135 mg, 0.356 mmol) and formohydrazide (26.7 mg, 0.445 mmol) at r.t. then DIPEA (0.156 mL, 0.891 mmol) was added dropwise. After stirring for 2hrs, added water, aqueous was then extracted with Ethyl Acetate (x 2) the combined organic layers were dried (MgSO4), filtered and evaporated to dryness to give 4-bromo-N'-formyl-3-methyl-2- naphthohydrazide. LC-MS (ESI): m/z = 307.0 [M+H]+ Step 3.2-(4-bromo-3-methylnaphthalen-2-yl)-1,3,4-oxadiazole (3-2s) 4-bromo-N'-formyl-3-methyl-2-naphthohydrazide (From step 2, 213 mg, 0.693 mmol) in dry DCM (10 mL) was cooled to -20 °C then treated with pyridine (0.561 mL, 6.93 mmol) and dropwise with 1M Triflic Anhydride in DCM (0.693 mL, 0.693 mmol) allowing to warm to room temperature overnight. 1M Triflic Anhydride (3 mL, 3.00 mmol) in DCM, was added and the mixture was stirred for an extra 3 hours, then added saturated aq. NaHCO3, stirred for 10 mins before the layers were separated extracting the aqueous with DCM. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on the ISCO 24g cartridge, eluting with 0-100% Ethyl Acetate in heptane. 2-(4-bromo-3- methylnaphthalen-2-yl)-1,3,4-oxadiazole (3-2s) (50 mg, 0.173 mmol, 24.94 % yield) as a white solid. LC-MS (ESI): m/z = 287.0 [M+H]+ Intermediate 3-2t.1-((4-bromo-3-methylnaphthalen-2-yl)methyl)-1H-pyrazole (4-bromo-3-methylnaphthalen-2-yl)methanol (472 mg, 1.880 mmol) in dry Tetrahydrofuran (10 mL) was treated with Triphenylphosphine (739 mg, 2.82 mmol) and pyrazole (640 mg, 9.40 mmol) then dropwise with DIAD (0.548 mL, 2.82 mmol) after stirring for 16hrs the mixture was evaporated to dryness. The residue was chromatographed on the ISCO 40g cartridge, eluting with 0-20% Ethyl Acetate in heptane to give 1-((4-bromo-3-methylnaphthalen-2-yl)methyl)-1H- pyrazole (3-2t) (500 mg, 1 mmol, 53.0 % yield). LC-MS (ESI): m/z = 303.9 [M+H]+ Intermediate 3-3u.2-(3-(tert-butyl)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane BISPIN (1135 mg, 4.47 mmol), 3-tbutyl-1-bromonapthalene (294 mg, 1.117 mmol), and Potassium Acetate (1316 mg, 13.41 mmol) were dissolved in DMF (20 mL) and the mixture was degassed thoroughly refilling with nitrogen. PdCl2(dppf).CH2Cl2 adduct (91 mg, 0.112 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was then heated to 85°C. Overnight. Filtered through celite to remove Pd residues then diluted with water (200 mL) and Water and Ethyl Acetate were added and the mixture was separated. The aqueous was washed with Ethyl Acetate and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on the ISCO 40g cartridge, eluting with 0-10% Ethyl Acetate in heptane to give as a clear oil 2-(3-(tert-butyl)naphthalen-1-yl)-4,4,5,5- tetramethyl-1,3,2-dioxaborolane (3-3u) (140 mg, 0.451 mmol, 40.4 % yield) LC-MS (ESI): m/z = not ionized Intermediate 3-3v.2-(7-(tert-butyl)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane BISPIN (1135 mg, 4.47 mmol), 7-tbutyl-1-bromonapthalene (294 mg, 1.117 mmol), Potassium Acetate (1.31 g, 13.41 mmol) were dissolved in DMF (20 mL) and the mixture was degassed thoroughly refilling with nitrogen. PdCl2(dppf).CH2Cl2 adduct (91 mg, 0.112 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was then heated to 85°C. overnight. Filtered through celite to remove Pd residues then diluted with water (200 mL) and Water and Ethyl Acetate were added and the mixture was separated. The aqueous was washed with Ethyl Acetate and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on the ISCO 40g cartridge, eluting with 0-10% Ethyl Acetate in heptane to give as a clear oil 2-(7-(tert-butyl)naphthalen-1-yl)-4,4,5,5- tetramethyl-1,3,2-dioxaborolane (3-3v) (140 mg, 0.451 mmol, 40.4 % yield) LC-MS (ESI): m/z = not ionized Intermediate 3-3w.2-(6-(tert-butyl)naphthalen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane BISPIN (1135 mg, 4.47 mmol), 6-tbutyl-2-bromonapthalene (294 mg, 1.117 mmol), Potassium Acetate (1.31 g, 13.41 mmol) were dissolved in DMF (20 mL) and the mixture was degassed thoroughly refilling with nitrogen. PdCl2(dppf).CH2Cl2 adduct (91 mg, 0.112 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was then heated to 85°C. overnight. Filtered through celite to remove Pd residues then diluted with water (200 mL) and Water and Ethyl Acetate were added and the mixture was separated. The aqueous was washed with Ethyl Acetate and the organic layers were each individually washed with 0.5M aqueous Lithium Chloride solution. The combined organic layers were dried (MgSO4), filtered and evaporated to dryness. The residue was chromatographed on the ISCO 40g cartridge, eluting with 0-10% Ethyl Acetate in heptane to give as a clear oil 2-(6-(tert-butyl)naphthalen-2-yl)-4,4,5,5- tetramethyl-1,3,2-dioxaborolane (3-3w) (70 mg, 0.23 mmol, 20 % yield) LC-MS (ESI): m/z = not ionized Intermediate 3-2x.1-bromo-6-isopropyl-2-methoxynaphthalene Step 1.2-isopropyl-6-methoxynaphthalene 2-bromo-6-methoxynaphthalene (570 mg, 2.404 mmol) and PdCl2(dppf) (196 mg, 0.240 mmol, 0.1eqv) were placed under an atmosphere of nitrogen and taken up in THF (6 mL). The solution was cooled to 0ºC and isopropyl magnesium bromide (3.21 mL, 2.404 mmol, 1eqv) was added. Once added, the reaction was heated to 60 ºC for 3 h. At this time, the reaction was quenched with sat. NH4Cl(aq) and the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with saturated NH4Cl (aq), passed through a phase separator, and concentrated under reduced pressure to give 2-isopropyl-6- methoxynaphthalene as an orange solid (637 mg, 3.18 mmol, 132 % yield).1H NMR (400 MHz, Methylene Chloride-d2) ^ 7.69 (s, 1H), 7.67 (s, 1H), 7.58 (d, J = 1.3 Hz, 1H), 7.36 (dd, J = 8.4, 1.9 Hz, 1H), 7.12 (d, J = 2.6 Hz, 1H), 7.10 (dd, J = 8.8, 2.6 Hz, 1H), 3.90 (s, 3H), 3.04 (hept, J = 6.9 Hz, 1H), 1.33 (s, 6H). Step 2.1-bromo-6-isopropyl-2-methoxynaphthalene (3-2x) NBS (471 mg, 2.64 mmol) was added to a solution of 2-isopropyl-6-methoxynaphthalene (From step 1, 481 mg, 2.404 mmol) in Acetonitrile (Volume: 8 mL) and the reaction was stirred at rt. After 1 h, the reaction was concentrated under reduced pressure. The residue was taken up in EtOAc, washed with sat Na2S2O3(aq) and the layers were separated. The organic layer was washed with brine, passed through a phase separator, and concentrated under reduced pressure to give 1-bromo-6-isopropyl-2-methoxynaphthalene (3-2x) as a brown solid (782 mg, 2.80 mmol, 117 % yield). 1H NMR (400 MHz, Methylene Chloride-d2) ^ 8.15 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.9 Hz, 1H), 7.65 (d, J = 1.2 Hz, 1H), 7.53 (dd, J = 8.8, 1.9 Hz, 1H), 7.32 (d, J = 9.0 Hz, 1H), 4.04 (s, 3H), 3.11 (hept, J = 6.8 Hz, 1H), 1.37 (d, J = 6.9 Hz, 6H). Intermediate 3-3y.2-(2-isopropylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (5.50 g, 21.67 mmol, mixture), 1- bromo-3-isopropylnaphthalene (1.35 g, 5.42 mmol, mixture), potassium acetate (6.38 g, 65.0 mmol) were dissolved in Dioxane (30 mL). The mixture was degassed thoroughly for 15 min then PdCl2(dppf).CH2Cl2 adduct (0.442 g, 0.542 mmol) was added and the mixture was again degassed thoroughly refilling with nitrogen for 15 min. The resulting suspension mixture was then heated to 85°C. overnight. The reaction mixture was allowed to come to ambient temperature. It was diluted with EtOAc (50 mL), filtered through celite to remove Pd residues then wahsed with EtOAc, concentrated. It was purified via silica gel column (80 gram, 0-100% EtOAc/Hept., 30 min). Desired fractions were combined, concentrated and further dired under high vac to provide 2-(3- isopropylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3y) (1.278 g, 4.31 mmol, 80 %, major required isomer and contains minor regio isomer).). It was taken as is for further steps and isolated the desired product in the final step. LC-MS (ESI): m/z = 298.2 [M+H]+ Intermediate 3-3z.2-(2-ethylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (6.8 g, 26.8 mmol, mixture), 1- bromo-2-ethylnaphthalene (3.15 g, 13.4 mmol, mixture), potassium acetate (7.69 g, 80.0 mmol) were dissolved in Dioxane (60 mL). The mixture was degassed thoroughly for 15 min then PdCl2(dppf).CH2Cl2 adduct (1.09 g, 1.34 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen for 15 min. The resulting suspension mixture was then heated to 85°C. overnight. The reaction mixture was allowed to come to ambient temperature. It was diluted with EtOAc (50 mL), filtered through celite to remove Pd residues then washed with EtOAc, concentrated. It was purified via silica gel column (120 gram, 0-20% EtOAc/Hept., 30 min). Desired fractions were combined and concentrated under high vac to provide 2-(2- ethylnaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (3-3z) (3.78 g, 10.49 mmol, 78 %, major required isomer and contains minor regio isomer). It was taken as it is for further steps and isolated the desired product in the final step. LC-MS (ESI): m/z = 283.3 [M+H]+ Intermediate 3-2aa.1-((1-bromonaphthalen-2-yl)methyl)pyrrolidin-2-one To a stirred solution of 1-bromo-2-(bromomethyl)naphthalene (1.00 g,3.35 mmol), and pyrrolidin-2-one (0.397 g, 4.667 mmol) in DMF (10.0 ml) was added KH (10.147 g, 3.66 mmol) at 0oC and the reaction mixture was stirred at room temperature for 12h. The reaction mixture was quenched with saturated NH4Cl solution (10 mL) and extracted to ethyl acetate (2*10 mL). The organic layer was separated, dried over anhy. sodium sulfate filtered and concentrated under reduced pressure to get the crude. The crude product was purified by combi-flash using 4g cartridge and product eluted with eluted with 0-20% ethyl acetate in heptane to afford 1-((1- bromonaphthalen-2-yl)methyl)pyrrolidin-2-one (3-2aa) (1.01 g, 3.33 mmol, 99%) as a viscous oil. LC-MS (ESI): m/z = 306.0 [M+2H]+ Intermediate 4-5a.7-bromo-5-ethylbenzo[b]thiophene Step 1.3-bromo-5-ethyl-2-fluorobenzaldehyde To a solution of 2-bromo-4-ethyl-1-fluorobenzene (3.00 g, 14.85 mmol) in THF (30.00 mL) was added LDA (2M in THF) (11.13 mL, 22.27 mmol) dropwise at –78oC (color of the reaction mixture changed from white to yellowish) and reaction mixture was allowed to stir at -78 oC for 30 minutes. After 30 minutes, added N, N-dimethyl formamide (3.45 mL, 44.55 mmol) dropwise and the reaction continued at same temperature for 3h. The progress of the reaction was monitored by TLC, using mobile phase 100% hexane. Quenched the reaction mixture with saturated ammonium chloride solution (50.0 mL) and extracted with ethyl acetate (2 x 25 mL). The combined organic layer was washed with brine solution (20.0 mL), dried over anhy. Na2SO4 and concentrated the organic layer to afford 3-bromo-5-ethyl-2-fluorobenzaldehyde (4.40 g) as brown gummy liquid. The formation of the product was confirmed by LC-MS (ESI): m/z = 226.5 [M-2H]+ Step 2. ethyl 7-bromo-5-ethylbenzo[b]thiophene-2-carboxylate To a solution of 3-bromo-5-ethyl-2-fluorobenzaldehyde (From step 1, 3.80 g, 16.52 mmol) in DMF (38.0 mL) potassium carbonate (3.42 g, 24.78 mmol) were added and ethyl 2- mercaptoacetate (1.98 g, 16.52 mmol) and the reaction mixture was heated to 50oC for 1h and continued to 70oC for 4h. The progress of the reaction was monitored by TLC, using mobile phase 100% hexane. Cooled the reaction mixture to room temperature. Diluted the reaction mixture with water (10mL) and extracted with ethyl acetate (2 x 10mL) and the organic layer was washed with brine solution and dried over anhy. sodium sulphate. Concentrated the organic layer under vacuum to get crude. The crude was purified by combi flash using 4g cartridge, eluting the product at neat hexane to afford ethyl 7-bromo-5-ethylbenzo[b]thiophene-2-carboxylate (2.90 g, 9.29 mmol, 56.31%) as pale-yellow solid. The formation of the product was confirmed by TLC, LC-MS (ESI): m/z = no ionization. The crude product was taken to the next step. Step 3.7-bromo-5-ethylbenzo[b]thiophene-2-carboxylic acid To a solution ethyl 7-bromo-5-ethylbenzo[b]thiophene-2-carboxylate (From step 2, 2.90 g, 9.29 mmol) in THF (29.0 mL) was added 4N NaOH (34.80 mL) and the reaction mixture was heated to 70oC for 16h.The progress of the reaction was monitored by TLC, using mobile phase 50% hexane. The reaction mixture was concentrated to dryness to get residue. Diluted the residue with water and aqueous layer was acidified to pH = 6 using acetic acid at 0 oC. The solid formed was filtered, washed with pentane, and dried under vacuum to afford 7-bromo-5- ethylbenzo[b]thiophene-2-carboxylic acid (1.40 g, 4.93 mmol, 53.2 %) as white colored solid. LC- MS (ESI): m/z = 284.5 [M-H]+ Step 4.7-bromo-5-ethylbenzo[b]thiophene (4-5a) To a stirred solution of 7-bromo-5-ethylbenzo[b]thiophene-2-carboxylic acid (From step 3, 1.00 g, 3.51 mmol) in DMA (10.0 mL) was added DBU (1.71 g, 11.22 mmol) and heated to 200oC for 70 minutes under microwave radiation. The progress of the reaction was monitored by TLC, using mobile phase 30% EtOAc in hexane. Cooled the reaction mass to room temperature. Diluted the reaction mixture with water (10mL) and extracted with ethyl acetate (2 x 20mL). The organic layer was washed with brine solution and dried over anhy. sodium sulphate. Concentrated the organic layer under vacuum to get crude. The crude product was purified by combi-flash using 4g cartridge and eluted with neat hexane to afford 7-bromo-5-ethylbenzo[b]thiophene (4-5a) (0.740 g, 3.08 mmol, 88.09%) as colorless oil. The required product formed confirmed by 1H NMR (300 MHz, CDCl3): δ 7.58(s,1H),7.48-7.46(d, J=5.4 Hz,1H),7.37-7.36 (m, 2H),2.79-2.71 (q,2H), 1.31-1.26 (t, 3H). LC-MS (ESI): m/z = no ionization. Intermediate 4-5b. 4,4,5,5-tetramethyl-2-(5-methylbenzo[b]thiophen-7-yl)-1,3,2- dioxaborolane Step 1. ethyl 7-bromo-5-methylbenzo[b]thiophene-2-carboxylate To a stirred solution of 3-bromo-2-fluoro-5-methylbenzaldehyde (3.0 g, 13.82 mmol) in DMF (30 mL, 10 vol) under nitrogen atmosphere were added ethyl 2-mercaptoacetate (1.52 mL, 13.82 mmol) and potassium carbonate (1.91 g,13.82 mmol) the reaction mixture was heated at 70oC for 2h. The progress of reaction was monitored by TLC, using mobile phase 10% EtOAc in hexane. The reaction mass was cooled to room temperature and poured to ice cold water, solid was precipitated. Filtered the solid and dried under vacuum to afford ethyl 7-bromo-5- methylbenzo[b]thiophene-2-carboxylate (6.3 g, 21.72 mmol, 92.64 %) as white solid. 1H NMR (300 MHz, CDCl3): δ 8.04(s, 1H),7.60 (s, 1H),7.44 (s, 1H), 4.44-4.37 (q, 2H), 2.46 (s, 3H), 1.44- 1.39 (t, 3H). LC-MS (ESI): m/z = no ionization Step 2.7-bromo-5-methylbenzo[b]thiophene-2-carboxylic acid To a solution of ethyl 7-bromo-5-methylbenzo[b]thiophene-2-carboxylate (From step 1, 2.0 g, 6.68 mmol) in THF (20 mL), was added 4N NaOH (20.0 mL) and the reaction mixture was heated to 100oC for 6h. The progress of the reaction was monitored by TLC, using mobile phase 50% EtOAc in hexane. Evaporated the reaction mixture to dryness to get the residue. Diluted the residue with water, acidified the aqueous layer with conc. HCl, the solid precipitated. Filtered the solid and dried under vacuum to afford 7-bromo-5-methylbenzo[b]thiophene-2-carboxylic acid (1.7 g, 6.27 mmol, 93.0%) as white solid.1H NMR (300 MHz, DMSO-d6): δ 8.15 (s,1H),7.82(s, 1H),7.62(s, 1H),2.43 (s,3H). LC-MS (ESI): m/z = 270.9 [M-H]+ Step 3.7-bromo-5-methylbenzo[b]thiophene (4-5b) To a solution of 7-bromo-5-methylbenzo[b]thiophene-2-carboxylic acid (From step 2, 0.9 g, 3.31 mmol) in Quinoline (113.5 mL, 15 vol) under nitrogen atmosphere, was added copper powder (0.42 g, 6.63 mmol) and the reaction mixture was heated to 120oC for 12h. The progress of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. The reaction mixture was quenched with 10% HCl (9.0 mL) and extracted to ethyl acetate (3 x 9 mL). The organic layer was separated, washed with water and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford 7-bromo-5-methylbenzo[b]thiophene (4-5b) (1.0 g,4.40 mmol,99 %) as yellow oil.1H NMR (400 MHz, CDCl3): δ 7.56(s, 1H),7.47-7.46(d, J=5.2 Hz, 1H),7.35-7.34 (m, 2H),2.46 (s, 3H). Step 4.4,4,5,5-tetramethyl-2-(5-methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (4-6b) To a stirred compound 7-bromo-5-methylbenzo[b]thiophene (4-5b) (0.3 g, 1.32 mmol) in dioxane (3.0 mL), were added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane (0.67 g, 2.64 mmol), followed by potassium acetate (0.38 g, 3.96 mmol). The reaction mixture was purged with argon for 25 minutes. After 25 minutes, added Pd(dppf)Cl2.DCM (0.10 g, 0.13 mmol) and purged again for 10 minutes. Sealed the reaction mixture and heated the reaction mixture 90°C for 3h. The progress of the reaction was monitored by TLC, using mobile phase 10% EtOAc in hexane. Cooled the reaction mixture to room temperature. Diluted the reaction mixture with brine solution and extracted the product with EtOAc. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated under vacuum to get the crude. The crude was purified using 5% EtOAc in hexane as eluent to afford 4,4,5,5-tetramethyl-2-(3- methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (4-6b) (0.26 g, 0.94 mmol, 72%) as pale- yellow solid. LC-MS (ESI): m/z = 274.6 [M+H]+ Intermediate 4-6c. 4,4,5,5-tetramethyl-2-(6-methylbenzo[b]thiophen-7-yl)-1,3,2- dioxaborolane Step 1.3-bromo-2-fluoro-4-methylbenzaldehyde To a solution of 2-bromo-1-fluoro-3-methylbenzene (3.00 g, 15.87 mmol) in THF (30.00 mL) at -78oC added LDA (2M in THF, 11.90 mL, 23.81 mmol) dropwise, (colour of the reaction mixture changed from white to yellowish) and reaction mixture was allowed to stir at -78 o C for 30minutes. After 30 minutes, N, N-dimethyl formamide (3.68 mL, 47.61 mmol) was added dropwise, and the reaction was continued at same temperature for 2h. The completion of the reaction was monitored by TLC using mobile phase: 5% EtOAc in hexane. The reaction mixture was quenched with saturated ammonium chloride solution (10.0 mL) and extracted the product with ethyl acetate (2x20 mL). The organic layer was washed with brine solution (1x10.0 mL) and dried over anhydrous sodium sulphate and concentrated under reduced pressure to get crude. The crude was purified by combi flash using 12 g cartridge using 10% ethyl acetate: hexane as eluent to afford 3-bromo-2-fluoro-4-methylbenzaldehyde (1.60 g, 7.37 mmol, 46.51 %) as white solid.1H NMR (300MHz, DMSO-d6): δ10.14 (s, 1H),7.77-7.72(t, J=7.2 Hz,1H),7.42-7.39(d, J=7.8 Hz,1H),2.47 (s, 3H). Step 2. ethyl 7-bromo-6-methylbenzo[b]thiophene-2-carboxylate To a solution of 3-bromo-2-fluoro-4-methylbenzaldehyde (From step 1, 1.5 g, 6.91 mmol) in DMF (15.0 mL) were added potassium carbonate (1.43 g, 10.36 mmol) and ethyl 2- mercaptoacetate (1.25 g, 10.36 mmol), reaction mixture was heated to 70oC for 4h. The completion of the reaction was monitored by TLC using mobile phase:100 % hexane. The reaction mixture was partitioned between water (50 mL) and EtOAc (25 mL), and the aqueous layer was extracted with EtOAc (20 mL x 2). Combined organic phases were washed with saturated brine solution, dried over Na2SO4 and the solvent was removed under reduced pressure to furnish the desired compound in its crude form. The crude was purified by combi flash using 12 g cartridge using 100% hexane as eluent to afford ethyl 7-bromo-6-methylbenzo[b]thiophene-2-carboxylate (1.40 g, 4.67 mmol, 67.96 %) as pale-yellow solid.1H NMR (300 MHz, DMSO-d6): δ 8.30 (s, 1H), 7.97-7.94 (d, J=7.2 Hz ,1H), 7.49-7.46 (d, J=7.2 Hz ,1H), 4.39- 4.32 (q, 2H), 1.36-1.31 (t, 3H). Step 3.7-bromo-6-methylbenzo[b]thiophene-2-carboxylic acid To a solution of ethyl 7-bromo-6-methylbenzo[b]thiophene-2-carboxylate (From step 2, 1.4 g, 4.67 mmol) in THF (14.0 mL) 4N NaOH (16.8 mL) was added, and reaction mixture was heated to 70 oC for 5h. The completion of the reaction was monitored by TLC using mobile phase: 100% hexane. The reaction mixture was concentrated to remove the volatiles and acidified to pH=3 using conc. HCl at 0o C. The solid formed was filtered, washed with n-pentane and dried under vacuum to afford 7-bromo-6-methylbenzo[b]thiophene-2-carboxylic acid (1.20 g, 4.79 mmol, 95.23 %) as white solid.1H NMR (300 MHz, DMSO-d6): δ 7.75-7.72(d, J=8.0 Hz ,1H), 7.6 (s,1H), 7.39-7.29(d, J=8.0 Hz ,1H),2.47(s, 3H). LC-MS (ESI): m/z = 270.9 [M-H]+ Step 4.7-bromo-6-methylbenzo[b]thiophene (4-5c) To a solution of 7-bromo-6-methylbenzo[b]thiophene-2-carboxylic acid (From step 3, 1.20 g, 4.42 mmol) in quinoline (18.0 mL), copper powder (0.562 g, 8.85 mmol) was added and reaction mixture was heated to 150oC for 4h. The completion of the reaction was monitored by TLC using 20% EtOAc in hexane. The reaction mixture was diluted with water (10 mL) and extracted to ethyl acetate (2x15 mL). The organic layer was separated, washed with 2N HCl wash (2x10 mL), dried over sodium sulfate filtered, and concentrated under reduced pressure. The crude was purified by combi flash using 12 g cartridge using hexane as eluent to afford 7-bromo-6- methylbenzo[b]thiophene (4-5c) (0.850 g, 3.74, 84.83 %) as colorless oil. 1H NMR (300 MHz, DMSO-d6): δ 7.82-7.75 (m, 2H), 7.57-7.55 (d, J=5.1 Hz ,1H), 7.38-7.35 (d, J=8.1 Hz ,1H), Methyl peak merges with d6-DMSO. LC-MS (ESI): m/z = not ionized. Step 5.4,4,5,5-tetramethyl-2-(6-methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (4-6c) To a stirred solution of 7-bromo-6-methylbenzo[b]thiophene (4.5c) (0.500 g, 2.20 mmol), was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane (1.12 g, 4.40 mmol) and potassium acetate (0.648 g, 6.60 mmol) in dioxane (10.0 mL) at room temperature. Rection mixture was degassed with argon gas for 20 min. Pd(dppf)Cl2. DCM (0.179 g, 0.220 mmol) was added to reaction mixture at room temperature. The reaction mixture was heated at 80°C for 16h. The completion of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. Reaction mass was cooled and added brine solution (10 mL), then extracted with EtOAc (2x10mL) and dried over Na2SO4 and concentrated under vacuum. The crude product was purified by combi-flash eluted with 3.0% EtOAc/Hexane 4 g Cartridge to afford 4,4,5,5-tetramethyl-2-(6- methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (4-6c) (0.400 g, 1.45 mmol, 66.33 %) as a white solid.1H NMR (300 MHz, DMSO-d6): δ 7.84-7.81 (m, 1H), 7.62-7.60 (d, J=5.7 Hz ,1H), 7.36- 7.34 (d, J=5.1 Hz ,1H), 7.22-7.19 (d, J=7.8 Hz ,1H), 2.57 (s, 3H), 1.36 (s, 12H). LC-MS (ESI): m/z = not ionized. Intermediate 4-6d. 7-bromo-N,N-dimethylbenzo[b]thiophen-3-amine Step 1. ethyl 3-amino-7-bromobenzo[b]thiophene-2-carboxylate To a stirred solution of 3-bromo-2-fluorobenzonitrile (7.40 g, 36.99 mmol) in DMF (74.0 ml), K2CO3(15.31 g, 110.97 mmol) and Ethyl 2-mercaptoacetate (4.8 mL, 44.39 mmol) were added. The reaction mixture was stirred at 100°C for 16 h. The completion of the reaction was monitored by TLC, 30% EtOAc in hexane. Cooled the reaction mixture to room temperature and poured into ice water. The resulting precipitate filtered off and washed with hexane, dried under reduced pressure to afford ethyl 3-amino-7-bromobenzo[b]thiophene-2-carboxylate (10.10 g, 33.65 mmol, 90.94 %) as off-white solid.1H NMR (300 MHz, CDCL3) δ 7.63-7.59 (m, 2H), 7.28- 7.23 (m, 1H), 5.86 (bs, 2H), 4.40-4.33 (m, 2H), 1.42-1.37 (t, J=7.2Hz, 3H). LC-MS (ESI): m/z = 301.1 [M+H]+ Step 2. ethyl 7-bromo-3-(dimethylamino)benzo[b]thiophene-2-carboxylate To a stirred solution of ethyl 3-amino-7-bromobenzo[b]thiophene-2-carboxylate (From step 1, 2.0 g, 6.662 mmol) in DMF (20.0 ml), were added K2CO3 (4.603 g, 33.31 mmol) and MeI (4.147 ml, 66.62 mmol) at 0°C. Then the reaction mixture was stirred at 60° C for 12h. The progress of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. Cooled the reaction mixture to room temperature. The reaction mixture was extracted with water and Ethyl acetate (50 mL x 3) twice. The organic layer was washed with brine solution (30 mL x 3) and dried with Na2SO4 and concentrated under reduced pressure to get the crude. The crude was purified by Chromatography using 40g silicycle column, eluting the product at neat hexane to afford ethyl 7-bromo-3-(dimethylamino)benzo[b]thiophene-2-carboxylate (2.10 g, 6.40 mmol, 96.03%) as yellow liquid. The formation of the product was confirmed by 1H NMR (300 MHz, CDCL3) δ 7.90-7.88 (d, J=8.1Hz, 1H), 7.58-7.55 (d, J=7.5Hz, 1H), 7.25-7.20 (t, J=8.1Hz, 1H), 4.39-4.32 (m, 2H), 3.11 (s, 6H), 1.42-1.38 (t, J=7.2Hz, 3H). Step 3.7-bromo-3-(dimethylamino)benzo[b]thiophene-2-carboxylic acid To a solution ethyl 7-bromo-3-(dimethylamino)benzo[b]thiophene-2-carboxylate (From step 2, 2.0 g, 6.093 mml) in THF (20 mL) NaOH (8N, (24 mL) was added and the reaction mixture was heated to 75oC for 5h. The progress of the reaction was monitored by TLC, using mobile phase 90% EtOAc in hexane. The reaction mixture was concentrated to remove the volatiles to dryness to get residue. The residue was acidified with HCl (pH=7). The solid formed was filtered, washed with n-pentane and dried under vacuum to afford 7-bromo-3- (dimethylamino)benzo[b]thiophene-2-carboxylic acid (1.81 g, 6.00 mmol, 98.41 %) as off-white colored solid. LC-MS (ESI): m/z = 302.1 [M+H]]+ Step 4.7-bromo-N,N-dimethylbenzo[b]thiophen-3-amine (4-5d) To a stirred solution of 7-bromo-3-(dimethylamino)benzo[b]thiophene-2-carboxylic acid ( From step 3, 1.80 g, 5.996 mmol) in DMA (14.4 mL) was added DBU (2.86 mL, 19.18 mmol) in microwave vial and heated to 200oC for 70 minutes under microwave radiation. The progress of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. Cooled the reaction mixture to room temperature. Concentrated the reaction mixture under vacuum to get crude. The crude product dissolved in DCM, adsorbed in silica and was purified by combi-flash using 24g column and eluted with 2-3% EtOAc in hexane to afford 7-bromo-N,N-dimethylbenzo[b]thiophen- 3-amine (4-5d) (1.41 g, 5.47 mmol, 91.41%) as colorless oil. The required product formed confirmed by 1H NMR: δ 7.80-7.78 (d, J=7.2Hz, 1H), 7.51-7.48 (d, J=7.5Hz,1H), 7.28-7.23 (t, J=8.1Hz, 1H), 6.60 (s, 1H), 2.87 (s, 6H). Intermediate 4-6e. 4,4,5,5-tetramethyl-2-(3-methylbenzo[b]thiophen-7-yl)-1,3,2- dioxaborolane Step 1.3-bromo-2-fluoro-N-methoxy-N-methylbenzamide To a solution of 3-bromo-2-fluorobenzoic acid (5.0 g, 22.83 mmol) in DMF (50.0 mL) in two necked round bottom flask, were added diisopropylethylamine (11.80 mL,68.49 mmol), followed by addition of EDCI (6.56 g,34.24 mmol), HOBt (4.62 g,34.24 mmol) at 0°C and stirred for 15 minutes. After 15 minutes, added N,O-dimethylhydroxylamine ( 2.44 g ,25.11 mmol) and stirred the reaction mixture at room temperature for 16h. The progress of the reaction was monitored by TLC, using mobile phase 30% EtOAc in hexane. The reaction mixture was diluted with ice water (200 mL) and extracted the product with ethyl acetate (100mL x 3). The combined organic layer was washed with brine solution, dried over anhydrous sodium sulphate and concentrated the organic layer. The crude was purified by Combi flash using 24g cartridge, eluting the product at 50% EtOAc in hexane to afford 3-bromo-2-fluoro-N-methoxy-N-methylbenzamide (4.4 g, 16.78 mmol, 73.54 %).1H NMR (300MHz, DMSO-d6): δ 7.83-7.78 (t, 1H),7.52-7.47 (t, 1H), 7.27-7.22 (t, 1H), 3.48 (m, 3H), 3.27 (m, 3H). Step 2.1-(3-bromo-2-fluorophenyl)ethan-1-one To a solution of 3-bromo-2-fluoro-N-methoxy-N-methylbenzamide (From step 1, 4.4 g ,16.78 mmol) in dry THF (45.0 mL) in two necked round bottom flask under nitrogen atmosphere was added Methyl Magnesium bromide (3M in THF, 16.86 mL,50.36 mmol) dropwise at -78°C and stirred the reaction mixture for 6h. The progress of the reaction was monitored by TLC, using mobile phase 10% EtOAc in hexane. Quenched the reaction mixture with saturated ammonium chloride (200 mL) and extracted the product with ethyl acetate (100mL x 3). The combined organic layer was washed with brine solution, dried over anhydrous sodium sulphate and concentrated the organic layer under vacuum. The crude was purified by 12g cartridge, using 14% EtOAc in hexane as eluent to afford 1-(3-bromo-2-fluorophenyl) ethan-1-one (3.3 g, 15.20 mmol, 91.66 %) as pale-yellow solid. 1H NMR (300MHz, CDCl3): δ 7.81-7.77 (m, 1H),7.75-7.71 (m, 1H), 7.13-7.09 (t, 1H), 2.66 (s, 3H). Step 3. ethyl 7-bromo-3-methylbenzo[b]thiophene-2-carboxylate To a stirred solution of sodium hydride (497 mg,20.73 mmol) in two necked round bottom flask, to this DMSO (30 mL) was added at 0°C and stirred for 15 minutes. To this mixture ethyl 2- mercaptoacetate in DMSO dissolved solution (1.51 mL,13.82 mmol) was added dropwise, followed by the addition of 1-(3-bromo-2-fluorophenyl)ethan-1-one (From step 2, 3.0 g,13.82 mmol) the reaction mixture was stirred for 2h at room temperature. The progress of the reaction was monitored by TLC, using mobile phase 10% EtOAc in hexane. Quenched the reaction mixture with saturated ammonium chloride (120 mL) and extracted the product with ethyl acetate (150 mL). The organic layer was washed with brine solution, dried over anhydrous sodium sulphate and concentrated the organic layer under vacuum to get crude. The crude was purified by 12g cartridge, using 5% EtOAc in hexane as eluent to afford ethyl 7-bromo-3- methylbenzo[b]thiophene-2-carboxylate (1.70 g, 5.862 mmol, 41.16 %) as yellow solid.1H NMR: (300MHz, DMSO-d6): δ 8.04-8.01 (d, J=8.7 Hz, 1H),7.82-7.79 (d, J= 7.8 Hz, 1H), 7.48-7.43 (t, 1H), 4.38-4.31 (q, 2H), 2.71 (s, 3H), 1.36-1.31 (t, 3H). Step 4.7-bromo-3-methylbenzo[b]thiophene-2-carboxylic acid To a solution of ethyl 7-bromo-3-methylbenzo[b]thiophene-2-carboxylate (From step 3, 1.6 g, 0.5.34 mmol) in THF (16 mL), was added 4N NaOH (19.2 mL) and the reaction mixture was heated to 70oC for 12h. The progress of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Evaporated the reaction mixture to dryness to residue. Diluted the residue with water, acidified the aqueous layer with conc. HCl to pH=3, the solid precipitated. Filtered the solid and dried under vacuum to afford 7-bromo-3-methylbenzo[b]thiophene-2-carboxylic acid (1.2 g, 4.425 mmol, 83.33 %) as white solid.1H NMR: (300MHz, DMSO-d6): δ 13.6 (bs, 1H), 8.02- 7.99 (d, J= 8.1 Hz, 1H), 7.80-7.77 (d, J= 7.2 Hz, 1H), 7.48-7.42 (t, 1H), 2.70 (s, 3H). Step 5.7-bromo-3-methylbenzo[b]thiophene (4-5e) To a solution of 7-bromo-3-methylbenzo[b]thiophene-2-carboxylic acid (From step 4, 1.2 g,4.42 mmol) in Quinoline (14.0 mL), was added copper powder (421.90 mg ,6.63 mmol) and the reaction mixture was heated to 130oC for 12h. The progress of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. The reaction mixture was diluted with water (100 mL) and extracted to ethyl acetate (2 x 100 mL). The organic layer was separated, washed with 2N HCl (2 x 20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to get the crude. The crude was purified by 24g column, using neat hexane as eluent to afford 7-bromo-3-methylbenzo [b]thiophene (4-5e) (1.0 g,4.40 mmol,99 %) as yellow solid. LC- MS (ESI): m/z = 225.6 [M+H]+ Step 6. 4,4,5,5-tetramethyl-2-(3-methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (4-6e) To a stirred compound 7-bromo-3-methylbenzo[b]thiophene (4-5e) (0.200g, 0.880 mmol) in dioxane (2.0 mL), were added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane (0.447 g 1.761 mmol), followed by potassium acetate (0.216 g,2.20 mmol). The reaction mixture was purged with argon for 25 minutes. After 25 minutes, added Pd(dppf)Cl2.DCM (0.114 g ,0.176 mmol) and purged again for 10 minutes. Sealed the reaction mixture and heated the reaction mixture 90°C for 16h. The progress of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. Cooled the reaction mixture to rt. Diluted the reaction mixture with EtOAc and filtered the solvents on a celite bed, washed the celite bed with EtOAc. The solvents were evaporated under vacuum. The crude was purified by 12g column, using 5% EtOAc in hexane as eluent to afford 4,4,5,5-tetramethyl-2-(3-methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (4-6e) (0.130 g, 0.378 mmol, 43.03%) as pale-yellow solid.1H NMR: (400MHz, DMSO-d6): δ 7.90-7.88 (d, 1H), 7.72-7.70 (d, 1H), 7.44-7.38 (m, 2H), 2.50 (s, 3H), 1.34 (s, 12H). Intermediate 4-6f. 2-(3-ethylbenzo[b]thiophen-7-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane Step 1.1-(3-bromo-2-fluorophenyl)propan-1-one To a stirred solution of 1-bromo-2-fluorobenzene (5.0 g, 28.571 mmol) in THF (50.0 mL) was added LDA (1M in THF, 21.0 mL, 42.856 mmol) dropwise at -78oC over 30 min. After 30 minutes, N-methoxy-N-methylpropionamide (6.16 mL, 42.856 mmol) was added and the reaction was allowed to stir at same temperature for 4h. The completion of the reaction was monitored by TLC, using mobile phase: 100% hexane. The reaction mixture was quenched with saturated ammonium chloride solution (100 mL) and extracted the product with ethyl acetate (3 x 100.0mL). The combined organic layer was washed with brine solution (3 x 50.0 mL), dried the organic layer over anhy. sodium sulphate and concentrated under reduced pressure to get the crude. The crude was purified by chromatography using 40g column using 0-5% ethyl acetate: hexane as eluent to afford 1-(3-bromo-2-fluorophenyl)propan-1-one (3.21g, 13.89 mmol, 48.62 %) as pale yellow solid.1H NMR: 1H NMR (300 MHz, CDCl3) δ 7.80-7.69 (m, 2H), 7.71-7.08 (m, 1H), 3.04-2.96 (m, 2H),1.23-1.18 (t, 3H). Step 2. ethyl 7-bromo-3-ethylbenzo[b]thiophene-2-carboxylate To a stirred solution of 1-(3-bromo-2-fluorophenyl)propan-1-one (From step 1, 3.1 g, 12.98 mmol) in DMF (31.0 mL) K2CO3(5.37 g, 38.94 mmol) and ethyl 2-mercaptoacetate(1.7 mL, 15.58 mmol) were added . Then the reaction mixture was stirred at 100°C for 16h. The completion of the reaction was monitored by TLC, using mobile phase: 100% hexane. Cooled the reaction mixture to room temperature and poured the reaction mixture to ice cold water, the solid precipitated. Filtered the solid, dried under vacuum to get the crude. The crude was purified by chromatography using 40 g column using 0-5% ethyl acetate: hexane as eluent to afford ethyl 7- bromo-3-ethylbenzo[b]thiophene-2-carboxylate (3.1 g, 9.90 mmol, 73.77%).1H NMR: (400 MHz, CDCl3): δ 7.82-7.80 (d, J=8.4 Hz, 1H), 7.62-7.60 (d, J=7.6 Hz, 1H), 7.33-7.29 (t, J=8.0 Hz, 1H), 4.43-4.38 (q, 2H), 3.30-3.24 (m, 2H), 1.44-1.40 (t, 3H), 1.32-1.26 (t, 3H). Step 3.7-bromo-3-ethylbenzo[b]thiophene-2-carboxylic acid To a stirred solution of ethyl 7-bromo-3-ethylbenzo[b]thiophene-2-carboxylate (From step 2, 3.0 g,9.57 mmol) in THF (30 mL) was added NaOH (4N, 36 mL) and the reaction mixture was stirred at 75°C for 2h. The completion of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Cooled the reaction mixture to room temperature and concentrated under vacuum to remove the volatiles. Diluted the evaporated residue with water and acidified to using conc. HCl at 0o C to pH = 7. The solid precipitated, filtered, washed with pentane and dried under vacuum to afford 7-bromo-3-ethylbenzo[b]thiophene-2-carboxylic acid (2.5 g, 8.77 mmol, 91.53%) as off-white solid.1H NMR: (400 MHz, DMSO-d6): δ 8.04-8.02 (d, J=8.0 Hz, 1H), 7.79-7.77 (d, J=7.6 Hz, 1H), 7.47-7.43 (t, J=7.2 Hz, 1H), 4.40 (s, 2H), 3.27-3.21 (q, 3H), 1.21-1.17 (t, 3H). Step 4.7-bromo-3-ethylbenzo[b]thiophene (4-5f) To a stirred solution of 7-bromo-3-(dimethylamino)benzo[b]thiophene-2-carboxylic acid (From step 3, 1.0 g, 3.506 mmol) in dimethylacetamide (8.0 mL) was added DBU (1.67 mL, 11.22 mmol) in microwave vial and the reaction mixture was irradiated in microwave at 200°C at 12 bar pressure for 70 min. The completion of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Cooled the reaction mixture to room temperature and evaporated under vacuum to get the crude. The crude was purified by combi flash using 24g cartridge and 2-5% EtOAc in hexane as eluent to afford 7-bromo-3-ethylbenzo[b]thiophene (4-5f) (0.710 g, 2.94 mmol, 83.96%) as off-white solid.1H NMR: 1H NMR (400 MHz, DMSO-d6) δ 7.84-7.81 (d, J=8.0 Hz, 1H), 7.62-7.60 (d, J=7.2 Hz, 1H), 7.51 (s, 1H), 7.38-7.34 (t, J=8.0 Hz, 1H), 2.84-2.78 (q, 3H), 1.34-1.26 (t, 3H). Step 5.2-(3-ethylbenzo[b]thiophen-7-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (4-6f) To a stirred solution of 7-bromo-3-ethylbenzo[b]thiophene (4-5f) (0.70g, 2.902 mmol) in 1,4-dioxane (7.0 mL) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane (0.696 g, 4.354 mmol) and potassium acetate (0.427 g, 4.354 mmol) and purged with argon for 15 min. After 15 mins, added Pd(dppf)Cl2DCM (0.473 g, 0.580 mmol) and purged again for 10 minutes. Then sealed the reaction mixture and heated at 100°C for 3h. The completion of the reaction was monitored by TLC, using mobile phase: 5% EtOAc in hexane. Cooled the reaction mixture to room temperature. The reaction mixture was evaporated under vacuum to dryness to get crude. The crude product was purified by Buchi using 24g column and eluted with 2-5% EtOAc in hexane. to afford 2-(3-ethylbenzo[b]thiophen-7-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (4-6f) (0.43 g, 1.49 mmol,51.40 %) as off-white solid.1H NMR (400 MHz, DMSO-d6): δ 7.93-7.91 (dd, 1H), 7.72- 7.70 (dd, 1H), 7.43-7.38 (m, 2H), 2.85-2.79 (q, 2H), 1.34 (s, 9H), 1.32-1.27 (t, 3H). Intermediate 4-6g. 7-bromo-6-fluorobenzo[b]thiophene Step 1. ethyl 7-bromo-6-fluorobenzo[b]thiophene-2-carboxylate To a mixture of 3-bromo-2,4-difluorobenzaldehyde (932 mg, 4.22 mmol) and K2CO3 (874 mg, 6.33 mmol) in DMF (10 mL) was added dropwise ethyl 2-mercaptoacetate (0.557 mL, 4.64 mmol) and the resulting mixture was stirred at 70 °C for 16 hr. The reaction mixture was treated with water and stirred at ambient temperature for 15 min. The resulting suspension was filtered and the obtained solid was washed with water and dried under high vacuum at 60 deg to provide the desired product ethyl 7-bromo-6-fluorobenzo[b]thiophene-2-carboxylate (1200 mg, 3.96 mmol, 94 % yield) as a white solid. MS [M+H]+ = not clear ionization.1H NMR (400 MHz, DMSO- d6) δ 8.25 (s, 1H), 7.82 (dd, J = 8.5, 4.4 Hz, 1H), 7.33 (dd, J = 9.9, 8.4 Hz, 1H), 4.38 (dd, J = 7.1, 1.1 Hz, 2H), 1.37 - 1.34 (m, 3H). Step 2.7-bromo-6-fluorobenzo[b]thiophene-2-carboxylic acid To a vial containing ethyl 7-bromo-6-fluorobenzo[b]thiophene-2-carboxylate (From step 1, 1200 mg, 3.96 mmol) was added THF (10 mL, Ratio: 2), MeOH (5.00 mL, Ratio: 1.000) and then NaOH (4N) (2.97 mL, 11.88 mmol). The mixture was agitated at 60-Deg-C for 1 hr, at which time LCMS showed completion of the reaction. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was diluted with 40 mL water and acidified with aq. 1.0 N HCl (40 mL) to pH ~4. The resulting precipitates were collected by vacuum filtration (washed with water, 60 mL). Then the collected solid was dried under vacuum until the constant mass was achieved to provide 7-bromo-6- fluorobenzo[b]thiophene-2-carboxylic acid (1041 mg, 2.65 mmol, 66.9 % yield). MS [M-H]- = 274.9. 1H NMR (400 MHz, DMSO-d6) δ 13.79 (s, 1H), 8.17 (d, J = 2.1 Hz, 1H), 7.82 - 7.78 (m, 1H), 7.31 (dd, J = 9.9, 8.4 Hz, 1H). Step 3.7-bromo-6-fluorobenzo[b]thiophene To a suspension of 7-bromo-6-fluorobenzo[b]thiophene-2-carboxylic acid (From step 2, 1000 mg, 2.54 mmol), Ag2CO3 (210 mg, 0.763 mmol) in NMP (12 mL) in a 100 mL RB flask was added Acetic acid (0.044 mL, 0.763 mmol) and heated at 130 °C to reflux for 16 hours with air condenser under N2. The reaction mixture was filtered through celite pad, diluted with EtOAc and water. Aq. layer was extracted with EtOAc (2 X 100 mL), combined Organic layers were washed with brine dried over Na2SO4, filtered and concentrated in vacuo. The crude material was purified via silica gel column (40 gram, 0-100% EtOAc/Hept) to provide 7-bromo-6- fluorobenzo[b]thiophene (553 mg, 2.93mmol, 94 % yield). MS [M-H]- = 229.1.1H NMR (400 MHz, DMSO-d6) δ 8.00 (dt, J = 5.5, 0.7 Hz, 1H), 7.70 (d, J = 5.5 Hz, 1H), 7.66 (ddd, J = 8.4, 4.3, 0.6 Hz, 1H), 7.26 (dd, J = 10.0, 8.4 Hz, 1H). Intermediate 5-5a. 2-(6-methoxybenzo[b]thiophen-4-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane Step 1.2-bromo-6-fluoro-4-methoxybenzaldehyde To a solution of 1-bromo-3-fluoro-5-methoxybenzene (10.0 g, 48.7 mmol) in THF (100.00 ml) was added lithium diisopropylamine (2M in THF) (29.2 mL, 58.47 mmol) dropwise at -78oC and reaction mixture was allowed to stir at -78oC for 30 minutes. After 30 minutes added N,N- dimethyl formamide (4.29 g, 58.47 mmol) dropwise, and the reaction continued at same temperature for 1.5h. Progress of reaction was monitored by TLC using mobile phase 30% EtOAc in hexane. The reaction mixture was quenched with saturated water and acetic acid (7 mL) and extracted the product with ethyl acetate (100 mL). The organic layer was washed with brine solution (1 x 50.0 mL) and dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude was purified by combi flash, (40 g Silicycle cartridge column) using 20% ethyl acetate: hexane as eluent to afford 2-bromo-6-fluoro-4-methoxybenzaldehyde (1.4 g, 6.00 mmol, 12.3 %) as white solid.1H NMR (400 MHz, CDCl3): δ 10.22 (s, 1H), 7.00 (s, 1H), 6.44- 6.61 (m, 1H), 3.86 (s, 3H). Step 2.4-bromo-6-methoxybenzo[b]thiophene-2-carboxylate To a solution of 2-bromo-6-fluoro-4-methoxybenzaldehyde (From step 1, 0.5 g, 2.14 mmol) in DMF (5.0 mL) were added potassium carbonate (0.450 g, 3.21 mmol) and ethyl 2- mercaptoacetate (0.380 g, 3.21 mmol). The reaction mixture was heated to 70oC for 16h. The progress of the reaction was monitored by TLC, using 10% EtOAc in hexane. The reaction mixture was partitioned between water (50 mL) and EtOAc (25 mL), and the aqueous layer was extracted with EtOAc (20 mL x 2). The combined organic phases were washed with saturated brine solution, dried over anhy. Na2SO4 and concentrated under reduced pressure to furnish the desired compound. The crude was purified by combi flash using (4 g, Silicycle cartridge column) 10% EtOAc hexane as eluent to afford ethyl 4-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (0.360 g, 1.14mmol, 54%) as off-white solid.1H NMR (400 MHz, DMSO-d6): δ 7.90 (s, 1H), 7.71 (s, 1H), 7.39 (s, 1H), 4.37-4.31 (q, 2H), 3.86 (s, 3H), 1.35-1.30 (t, 3H). Step 3.4-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid To a solution of 4-bromo-6-methoxybenzo[b]thiophene-2-carboxylate (From step 2, 0.350 g, 1.11mmol) in THF (3.5 mL) was added 4N NaOH (3.5 mL) and the reaction mixture was heated to 70o C for 6h. The completion of the reaction was monitored by TLC using mobile phase: 20 % hexane in EtOAc. The reaction mixture was concentrated to remove the volatiles and acidified with cHCl at 0o C. The solid formed was filtered, washed with n-pentane and dried under vacuum to afford 4-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (0.300 g, 1.04 mmol, 96.0 %) as pale-yellow solid.1H NMR (300 MHz, DMSO-d6): δ 13.60 (s,1H), 7.85 (s, 1H),7.69 (s, 1H), 7.38 (s, 1H), 3.85 (s, 3H). LC-MS (ESI): m/z = 284.8 [M+H]+ Step 4.4-bromo-6-methoxybenzo[b]thiophene (5-4a) To a solution of 4-bromo-6-methoxybenzo[b]thiophene-2-carboxylic acid (From step 3, 0.300 g, 1.04 mmol) in Quinoline (5.0 ml), copper powder was added (0.140 g, 2.29 mmol) and the reaction mixture was heated to 150o C for 6h. The completion of the reaction was monitored by TLC using mobile phase: 5% hexane in EtOAc. The reaction mixture was quenched with conc. HCl and extracted to ethyl acetate. The organic layer was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by combi flash (4 g, Silicycle cartridge column) using 1% EtOAc in hexane as eluent to afford 4-bromo-6-methoxybenzo[b]thiophene (5-4a) (0.21 g, 0.730 mmol) as yellow oil.1H NMR (300 MHz, CDCl3): δ 7.36-7.34 (m, 1H), 7.31-7.20 (m, 2H), 7.20 (s, 1H), 3.85 (s, 3H); LC-MS (ESI): m/z = Not ionized. Step 5.2-(6-methoxybenzo[b]thiophen-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (5-5a) To a stirred solution of 4-bromo-6-methoxybenzo[b]thiophene (5-4a) (0.20 g, 0.820 mmol) in dioxane (2.0 mL, 10 vol) were added 4,4,4′,4′,5,5,5′,5′-Octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.410 g, 1.62 mmol), potassium acetate (0.240g, 2.4 mmol). The reaction mixture was purged with argon for 30 min. Then added Pd(dppf)Cl2.DCM (0.067 g, 0.08 mmol), and heated up to 50°C for 1h followed by 100°C for 16h. The completion of the reaction was monitored by TLC, using mobile phase 5% EtOAc in hexane. Cooled the reaction mixture to room temperature. The reaction mixture was partitioned between water and EtOAc. The organic layer was washed with brine solution, dried over Na2SO4 and concentrated under reduced pressure to get crude product. The crude product was purified by combi-flash (4 g, Silicycle cartridge column) eluted with 3% EtOAc in Hexanes to afford 2-(6-methoxybenzo[b]thiophen-4-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane (5-5a) (0.160 g, 0.547 mmol, 66.0 %) as white solid.1H NMR (300 MHz, DMSO- d6): δ 7.75-7.72 (m, 2H), 7.60-7.58 (m, 1H), 7.29 (s, 1H), 3.82 (s, 3H), 1.34 (s, 12H); LC-MS (ESI): m/z = Not ionized. Intermediate 5-5b.4-bromo-5-methoxybenzo[b]thiophene Step 1.2-bromo-6-fluoro-3-methoxybenzaldehyde To a solution of 2-bromo-4-fluoro-1-methoxybenzene (10.0 g, 49.02 mmol) in THF (100.0 mL) was added LDA (2M in THF, 24.51 mL, 49.02mmol) dropwise at -78oC, (color of the reaction mixture changed from colorless to yellowish) and reaction mixture was allowed to stir at -78oC for 30 minutes. After 30 minutes, added DMF (3.034 mL, 39.21mmol) in dropwise and the reaction was allowed to stir at same temperature for 4h. The completion of the reaction was monitored by TLC, using mobile phase: 10% EtOAc in hexane. The reaction mixture was quenched with saturated ammonium chloride solution (20.0 mL) and extracted the product with ethyl acetate (2 x 30 mL). The combined organic layer was washed with brine solution (10.0 mL) and dried over anhydrous Sodium sulphate and concentrated under reduced pressure. The crude was purified by combi flash using 24 g column using10% ethyl acetate: hexane as eluent to afford 2-bromo-6- fluoro-3-methoxybenzaldehyde (4.0 g, 17.16 mmol, 35.08 %) as white solid.1H NMR (400MHz, CDCl3): δ 10.37 (s, 1H),7.12-7.08 (m, 2H), 3.91 (s, 3H), the formation of desired product, Step 2. ethyl 4-bromo-5-methoxybenzo[b]thiophene-2-carboxylate To a solution of 2-bromo-6-fluoro-3-methoxybenzaldehyde (From step 1, 3.5 g, 15.01 mmol) in DMF (15.0 mL) were added potassium carbonate (2.071 g, 15.01mmol) and ethyl 2- mercaptoacetate (1.804mL, 1501mmol), (color of the reaction mixture changed from colorless to pale yellowish) and the reaction mixture was heated to 70oC for 12h. The completion of the reaction was monitored by TLC, using mobile phase:10% EtOAc in hexane. The reaction mixture was partitioned between water (50 mL) and EtOAc (25 mL) and the aqueous layer was extracted with EtOAc (2 x 20 mL). The combined organic phases were washed with saturated brine solution, dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure to furnish the crude. The crude was purified by combi flash using 24 g cartridge and eluted the product at 8% ethyl acetate in hexane to afford ethyl 4-bromo-5-methoxybenzo[b]thiophene-2-carboxylate (1.2 g, 3.80 mmol, 25.53 %).1H NMR: (400MHz, CDCl3): δ 8.13 (s, 1H),7.74-7.72 (d, J= 8.8Hz, 1H), 7.15-7.13 (d, J= 8.8Hz, 1H), 4.44-4.38 (q, 2H), 3.97 (s, 3H), 1.43-1.40 (t, 3H). Step 3.4-bromo-5-methoxybenzo[b]thiophene-2-carboxylic acid To a solution of ethyl 4-bromo-5-methoxybenzo[b]thiophene-2-carboxylate (From step 2, 1.00 g, 3.18 mmol) in THF (10.0 mL), was added 4N NaOH (12.0 mL) and reaction mixture was heated to 70oC for 12h. The completion of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Cooled the reaction mixture to room temperature. The reaction mixture was concentrated to remove the volatiles. Diluted the evaporated residue with water and acidified to pH=3 using cHCl at 0o C. The solid precipitated, filtered, washed with n-pentane and dried under vacuum to afford 4-bromo-5-methoxybenzo[b]thiophene-2-carboxylic acid (0.700 g, 2.44 mmol,76.92%) as white solid.1H NMR: (300MHz, DMSO-d6): δ 13.73 (s, 1H), 8.08-8.05 (d, J= 9.3 Hz, 1H), 7.90 (s, 1H), 7.44-7.42 (d, J= 8.4Hz, 1H), 3.93 (s, 3H). LC-MS (ESI): m/z = no ionization Step 4.4-bromo-5-methoxybenzo[b]thiophene (5-5b) To a solution of 4-bromo-5-methoxybenzo[b]thiophene-2-carboxylic acid (From step 3, 0.700 g, 2.44 mmol) in Quinoline (10.5 mL) was added copper powder (0.311 g, 4.89 mmol) and the reaction mixture was heated to 130 oC for 5h. The completion of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Cooled the reaction mixture to room temperature. The reaction mixture was diluted with water (10 mL) and extracted to ethyl acetate (2 x 15 mL). The organic layer was separated, washed with 2N HCl (2 x 10 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to get crude. The crude was purified by combi flash using 12 g cartridge and 100% hexane as eluent to afford 4-bromo-5- methoxybenzo[b]thiophene (5-5b) (0.300 g, 1.23 mmol, 50.67%) as pale yellow solid 1H NMR: (300MHz, CDCl3): δ 7.76-7.73 (m, 1H), 7.52-7.51 (d, J= 5.4Hz, 1H), 7.45-7.43 (d, J= 6Hz, 1H), 7.05-7.02 (d, J= 6Hz, 1H), 3.97 (s, 3H). LC-MS (ESI): m/z = no ionization. Intermediate 5-6c. 4,4,5,5-tetramethyl-2-(5-methylbenzo[b]thiophen-7-yl)-1,3,2- dioxaborolane Step 1. 2-bromo-6-fluoro-4-methylbenzaldehyde To a solution of 1-bromo-3-fluoro-5-methylbenzene (10.00 g, 52.90 mmol) in THF (100.00 mL) was added lithium diisopropylamine (2M in THF) (31.7 mL, 63.48 mmol) dropwise, (color of the reaction mixture changed from white to yellowish) at -78oC and reaction mixture was allowed to stir at -78oC for 30 minutes. After 30 minutes added N,N-dimethyl formamide (4.6 g, 63.48 mmol) dropwise, and the reaction continued at same temperature for 2h. Progress of reaction was monitored by TLC using mobile phase 30% EtOAc in hexane. The reaction mixture was quenched with water(100mL) and acetic acid (7.0 ml) and extracted the product with ethyl acetate (2x100 ml). The organic layer was washed with brine solution (1x50.0 ml) and dried over anhydrous sodium sulphate and concentrated under reduced pressure to get the crude. The crude was purified by combi flash (40 g Silicycle column) using 20% ethyl acetate: hexane as eluent to afford 2-bromo-6-fluoro-4-methylbenzaldehyde (2) (6.5 g, 30.09 mmol, 56.8 %) as off-white solid. 1H NMR: (300MHz, CDCl3); δ10.29 (s,1H), 7.29 (s, 1H), 6.95-6.91 (d, H=11.1, 1H), 2.38 (s, 3H) the formation of desired product. LC-MS (ESI): m/z = no ionization Step 2. ethyl 4-bromo-6-methylbenzo[b]thiophene-2-carboxylate To a solution of 1-bromo-3-fluoro-5-methylbenzene (From step 1, 4.0 g, 18.52 mmol) in DMF (40.0 mL) were added potassium carbonate (3.8 g, 27.8 mmol) and ethyl 2-mercaptoacetate (3.37 g, 27.78 mmol). The reaction mixture was heated to 70oC for 16h. The reaction mixture was partitioned between water (50 mL) and EtOAc (25 mL), and the aqueous layer was extracted with EtOAc (100 mL x 2). Progress of reaction was monitored by TLC using mobile phase 20% EtOAc in hexane. The combined organic phases were washed with saturated brine solution, dried over anhy. Na2SO4 and concentrated under reduced pressure to furnish the desired compound in its crude form. The crude was purified by combi flash (24 g, Silicycle column) using 5% EtOAc in hexane as eluent to afford ethyl 4-bromo-6-methylbenzo[b]thiophene-2-carboxylate (3.40 g, 11.36, 61.36%) as pale-yellow solid.1H NMR: (300MHz, DMSO-d6); δ 7.92 (s, 1H), 7.88 (s, 1H), 7.57 (s, 1H), 4.37-4.30 (q, 2H), 2.42 (s, 3H),1.34-1.29 (t, 3H) the formation of desired product. Step 3. 4-bromo-6-methylbenzo[b]thiophene-2-carboxylic acid To a solution of ethyl 4-bromo-6-methylbenzo[b]thiophene-2-carboxylate (From step 2, 3.20 g, 10.69 mmol) in THF (32.0 ml) was added 4N NaOH (32.0 ml) and the reaction mixture was heated to 70o C for 16h. The completion of the reaction was monitored by TLC using mobile phase: 20 % hexane in EtOAc. The reaction mixture was concentrated to remove the volatiles and acidified with cHCl at 0o C. The solid formed was filtered, washed with n-pentane and dried under vacuum to afford 4-bromo-6-methylbenzo[b]thiophene-2-carboxylic acid (2.70 g, 9.95 mmol, 93.15 %) as off-white solid.1H NMR: (400MHz, DMSO-d6); δ 7.79 (s, 1H), 7.71 (s, 1H), 7.50 (s, 1H), 2.42 (s, 3H). LC-MS (ESI): m/z = 268.6 [M+H]+ Step 4. 4-bromo-6-methylbenzo[b]thiophene (5-5c) To a solution of 4-bromo-5-methylbenzo[b]thiophene-2-carboxylic acid (From step 3, 1.0 g, 3.68 mmol) in Quinoline (15.0 mL) taken in sealed tube was added copper powder (0.467 g, 7.37 mmol) and the reaction mixture was heated to 150o C for 4h. The completion of the reaction was monitored by TLC using mobile phase: 5 % hexane in EtOAc. Solvents were removed under vacuum. The reaction mixture was quenched with conc HCl (2x10 mL) and extracted the product with EtOAc. The organic layer was washed with brine solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by combi flash (12 g, Silicycle column) using 1% EtOAc in hexane as eluent to afford 4-bromo-6- methylbenzo[b]thiophene (5-5c) (0.560 g, 2.46mmol, 67%) as pale-yellow oil.1H NMR: (400MHz, CDCl3); δ 7.59 (s, 1H), 7.40-7.38 (m, 3H), 2.45 (s, 3H). LC-MS (ESI): m/z = no ionization Step 5. 4,4,5,5-tetramethyl-2-(5-methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (5-6c) To a stirred solution of 4-bromo-6-methylbenzo[b]thiophene (5-5c) (0.400 g, 1.76 mmol) in dioxane (4.0 ml), were added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane (0.894 g, 3.52mmol) and potassium acetate (0.518 g, 5.28 mmol) at room temperature and the reaction mixture was degassed with argon gas for 20 min. Pd(dppf)Cl2.DCM (0.143 g, 0.176 mmol) was added to the reaction mixture at room temperature The reaction mixture was heated at 110°C for 5h. The completion of the reaction was monitored by TLC, using mobile phase 100% hexane. Reaction mass was cooled and added brine solution (10 ml), then extracted with EtOAc (2 x 10ml) and dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by combi-flash (12 g, Silicycle column) eluted with 3.0% EtOAc: Hexane using 3% EtOAc in hexane to afford 4,4,5,5-tetramethyl-2-(5-methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (5-6c) (0.320 g, 1.16 mmol, 66.31 %) as pale-yellow solid.1H NMR: 1H NMR: (400MHz, CDCl3); δ 7.95-7.93 (d, J=5.7Hz, 1H), 7.77 (s, 1H),7.70 (s, 1H), 7.40-7.38 (d, J=5.7 Hz,1H), 2.47 (s, 3H), 1.39 (s, 12H). LC-MS (ESI): m/z = no ionization Intermediate 5-5d. 4-bromo-5-methylbenzo[b]thiophene Step 1. ethyl 4-bromo-5-methylbenzo[b]thiophene-2-carboxylate To a stirred solution of 2-bromo-6-fluoro-3-methylbenzaldehyde (10.0 g, 46.07 mmol) in DMF (100 mL, 10.0 vol) under nitrogen atmosphere were added Ethyl 2-mercaptoacetate (6.09 mL, 55.28mmol) and K2CO3 (7.64 g, 55.28 mmol) the reaction mixture was heated at 70°C for 4 h. The completion of the reaction was monitored by TLC, 10% EtOAc in hexane. The reaction mixture was partitioned between water (50 mL) and EtOAc (3x100 mL), and the aqueous layer was extracted with EtOAc. The combined organic phases were washed with saturated brine solution, dried over anhy. Na2SO4 and concentrated under reduced pressure to furnish the desired compound in its crude form. The crude was purified by Column: LUNA Phenomenex (250mmx21.2mm), 5.0µ, Flow: 18ml/min, Mobile Phase: A= 0.1% HCOOH IN WATER, B= ACN Gradient Program: (Time/ %B); (0 70), (2 80), (6, 95); The obtained Prep-fractions are concentrated under vacuum to afford ethyl 4-bromo-5-methylbenzo[b]thiophene-2-carboxylate (8.2 g, 27.4 mmol, 57.5 %) as an off-white solid. (1H NMR:(400 MHz, CDCl3): δ 8.13 (bs, 1H), 7.63-7.61 (d, J= 8.0 Hz 1H), 7.27-7.25 (d, J= 8.0 Hz 1H, 4.43-4.38 (q, 4H), 2.50 (s, 3H), 1.43-1.40 (t, 3H), LCMS=(M+1) = 299.1 at RT = 2.04 min, HPLC=98.6 % at RT = 6.79 min. and unrequired isomer also isolated was ethyl 4-fluoro-7-methylbenzo[b]thiophene-2-carboxylate (4) (2.2 g) Pale yellow solid. The desired isomer was carried to the next step. Step 2. 4-bromo-5-methylbenzo[b]thiophene-2-carboxylic acid To a stirred solution of ethyl 4-bromo-5-methylbenzo[b]thiophene-2-carboxylate (From step 1, 3.00 g, 10.02 mmol) in THF (30 mL) was added 4N NaOH (36.0 mL) and the reaction mixture was heated to 70oC for 4h. The completion of the reaction was monitored by TLC, using mobile phase: 50 % EtOAc in hexane. Cooled the reaction mass to room temperature. The reaction mixture was concentrated to remove the volatiles and acidified using conc. HCl at 0oC. The solid formed was precipitated, washed with pentane, and dried under vacuum to afford 4- bromo-5-methylbenzo[b]thiophene-2-carboxylic acid (2.80 g, 10.32 mmol, 100 %) as white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.97-7.95 (d, J=8.0 Hz, 1H), 7.90 (s, 1H), 7.48-7.46 (d, J=8.4 Hz,1H), 2.50 (s, 3H, Merged with DMSO). LC-MS (ESI): m/z = 268.9 [M+H]+ Step 3. 4-bromo-5-methylbenzo[b]thiophene (5-5d) To a solution of 4-bromo-5-methylbenzo[b]thiophene-2-carboxylic acid (From step 2, 2.80 g, 10.32 mmol) in quinoline (42.0 mL) was added copper powder (1.31 g, 20.66 mmol) and the reaction mixture was heated to 130oC for 5h. The completion of the reaction was monitored by TLC, using 20% EtOAc in hexane. Cooled the reaction mass to room temperature. Diluted the reaction mass with water (10 mL) and extracted with ethyl acetate (2 x 15 mL). The combined organic layer was washed with 2N HCl (2 x 20 mL), dried over anhy. Na2SO4, filtered and concentrated the organic layer under reduced pressure. The crude was purified by combi flash using 12g cartridge, with mobile phase 10% EtOAc in hexane as eluent to afford 4-bromo-5- methylbenzo[b]thiophene (5-5d) (2.0 g) as pale-yellow solid. 1H NMR (300 MHz, DMSO-d6): δ7.94-7.82 (m, 1H), 7.79- 7.68 (d,1H) 7.44-7.43 (d, J=5.1 Hz,1H), 7.36-7.34 (d, =7.8 Hz,1H), 2.50 (s, 3H). LC-MS (ESI): m/z = no ionization Intermediate 5-5e. 3-bromo-4-methylbenzo[b]thiophene Step 1. ethyl 3-amino-4-methylbenzo[b]thiophene-2-carboxylate To a stirred solution of 2-bromo-6-methylbenzonitrile (5.00 g, 25.64 mmol) in DMF (50.0 mL) was added potassium carbonate (10.61 g, 76.93 mmol) and ethyl 2-mercaptoacetate (3.69 g, 30.77 mmol) at room temperature and reaction mixture was heated to 50 oC for 1h. Then 80 oC for 12h. The progress of reaction was monitored by TLC, using mobile phase 100% hexane. The reaction mixture was partitioned between water (30 mL) and EtOAc (2*30 mL), and the aqueous layer was extracted with EtOAc (2*20 mL). Combined organic phases were washed with saturated brine solution, dried over Na2SO4 and the solvent was removed under reduced pressure to get the crude. The crude was purified by combi-flash using 12g cartridge and product eluted with eluted with hexane to afford ethyl 3-amino-4-methylbenzo[b]thiophene-2-carboxylate (3) (4.00 g,17.01 mmol, 66.44 %) as Pale-yellow solid. LC-MS (ESI): m/z = 235.7 [M+H]+ complies. Step 2. ethyl 3-bromo-4-methylbenzo[b]thiophene-2-carboxylate Me Br CO2Et S To a stirred solution of tertiary butyl nitrite (2.27 mL, 19.14 mmol) and copper bromide (3.42 g, 15.31 mmol) in ACN (30.00 mL) was heated to 65 oC and after 10 minutes added ethyl 3-amino-4- methylbenzo[b]thiophene-2-carboxylate (From step 1, 3.00 g, 12.76 mmol) in ACN (30 mL) was added at the same temperature and continued the reaction for 2h. The progress of reaction was monitored by TLC, using mobile phase 30% EtOAc in hexane. The reaction mixture was partitioned between saturated ammonium chloride solution (10 mL) and EtOAc (2*10 mL), and the aqueous layer was extracted with EtOAc (2*10 mL). Combined organic phases were washed once with saturated brine solution, dried over Na2SO4 and the solvent was removed under reduced pressure to get the crude. The crude was purified by combi-flash 4 g cartridge and product eluted with hexane to afford ethyl 3-bromo-4- methylbenzo[b]thiophene-2-carboxylate (1.90 g, 6.37 mmol, 50.13 %) as Pale-yellow gummy liquid.1H NMR (300 MHz, DMSO-d6) δ 1.34 (t, J=7.09 Hz, 3 H), 2.96 (s, 3 H), 4.36 (q, J=7.14 Hz, 2 H), 7.27 - 7.37 (m, 1 H), 7.47 (t, J=7.74 Hz, 1 H), 8.03 (s, 1 H) ppm. LC-MS (ESI): m/z = not ionized. Step 3. 3-bromo-4-methylbenzo[b]thiophene-2-carboxylic acid To a stirred solution ethyl 3-bromo-4-methylbenzo[b]thiophene-2-carboxylate (From step 2, 1.90 g, 6.37 mmol) in THF (19.00 mL) was added 4N NaOH (22.80 mL) and reaction mixture was heated to 80o C for 16h. The completion of the reaction was monitored by TLC, using mobile phase: 50 % hexane in EtOAc. The reaction mixture was concentrated to remove the volatiles and acidified to pH-3 using conc. HCl at 0 oC. Solid was precipitated out was filtered, washed with pentane and dried under vacuum to afford 3-bromo-4-methylbenzo[b]thiophene-2-carboxylic acid (1.50 g, 5.55 mmol, 87.20 %) as white solid.1H NMR (300 MHz, DMSO-d6) δ 2.95 (s, 3 H), 7.24 - 7.33 (m, 1 H), 7.41 - 7.48 (m, 1 H), 7.93 (d, J=8.24 Hz, 1 H), 13.64 - 13.98 (m, 1 H) ppm. LC- MS (ESI): m/z = 271.0 [M+H]+ complies. Step 4. 3-bromo-4-methylbenzo[b]thiophene (5 – 5e) To a stirred solution of 3-bromo-4-methylbenzo[b]thiophene-2-carboxylic acid (From step 3, 1.00 g, 3.71 mmol) in DMA (10.00 mL) was added DBU (1.76 mL, 11.85 mmol) and heated to 200o C for 70 minutes under microwave radiation. The completion of the reaction was monitored by TLC, using mobile phase 50% EtOAc in hexane. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (2*20 mL). The organic layer was separated, dried over sodium sulfate, filtered, and concentrated under reduced pressure to get crude. The crude was purified by combi-flash 4 g cartridge and product eluted with 50% EtOAc in hexane to afford 3- bromo-4- methylbenzo[b]thiophene (5-5e) (0.780 g, 3.45 mmol, 93.97%) as colorless oil.1H NMR (300 MHz, DMSO-d6) δ 2.88 (s, 3 H), 7.20 - 7.23 (d, J=6.9 Hz, 1 H), 7.28 - 7.33 (t, J=7.5 Hz, 1 H), 7.89 - 7.93 (m, 2 H) ppm. LC-MS (ESI): m/z = not ionized. Intermediate 6-1a. tert-butyl (tert-butoxycarbonyl)(3-cyano-6-(2-methoxynaphthalen-1- yl)pyrazolo[1,5-a]pyrimidin-7-yl)carbamate To a stirred solution of tert-butyl(6-bromo-3-cyanopyrazolo[1,5-a]pyrimidine-7-yl) (tert- butoxycarbonyl)carbamate (1-4a, 0.5g, 1.14 mmol) and (2-methoxynaphthalen-1-yl)boronic acid (0.27g, 1.36 mmol) in THF (2.5 mL) were added 2% TPGS (5 mL, 10V) and triethylamine (0.62 mL, 4.56 mmol) and purged with argon for 10 min. Then added Pd(dtbpf)Cl2(0.150 g, 0.22 mmol) to this mixture and purged again with argon for 10 min. Then the reaction mixture was stirred at 70°C for 3h. The progress of the reaction was monitored by TLC, using mobile phase:20 % EtOAc in hexane. Cooled the reaction mixture to room temperature and was diluted with water and extracted with EtOAc. The organic layer was washed with brine solution and dried over anhy. Na2SO4 and concentrated under vacuum. The obtained product was dissolved in MeOH added silica-met-DMT and stirred for 12h and solvent was filtered over celite bed and washed the celite bed with MeOH. Evaporated the filtrate to get crude. The crude product was purified by flash chromatography, product eluted at 20% EtOAc in hexane to afford tert-butyl(tert-butoxycarbonyl) (3-cyano-6-(2-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)carbamate (0.25g, 0.48 mmol, 42.8%) as white solid. LC-MS (ESI): m/z = 516.2 [M+H]+ The following intermediates 6-1b to 6-1t (Table 17) are available in an analogous manner starting from core bromide intermediate 1-4a and appropriate biaryl boronate 3-3 (or 4-5, 5-5). The crude products contain various amount of mono Boc, the reaction mixtures are purified by chromatography and carried to the next step Table 17:
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Intermediate 6-2a. 6-(3-(tert-butyl)naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile 6-bromopyrazolo[1,5-a]pyrimidine-3-carbonitrile (2-2a) (835 mg, 3.75 mmol), Potassium phosphate tribasic (2271 mg, 10.70 mmol) and 2-(3-(tert-butyl)naphthalen-1-yl)-4,4,5,5- tetramethyl-1,3,2-dioxaborolane (830 mg, 2.68 mmol) were combined in Dioxane (1.4 mL) The mixture was degassed thoroughly refilling with nitrogen. PdCl2(dppf).CH2Cl2 adduct (306 mg, 0.375 mmol) was added and the mixture was degassed thoroughly refilling with nitrogen. The mixture was stirred at 110 oC for 48 hrs. Filtered through celite washing with ethyl acetate and evaporated to dryness. The residue was chromatographed on the ISCO 120g cartridge, eluting with 0-20% Ethyl Acetate in heptane. Obtained 6-(3-(tert-butyl)naphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carbonitrile (6-2a) (200 mg, 22% yield) 1H NMR (400 MHz, CDCl3) δ 8.88 – 8.80 (m, 2H), 8.40 (s, 1H), 7.92 – 7.84 (m, 2H), 7.66 – 7.60 (m, 1H), 7.54 – 7.46 (m, 2H), 7.46 – 7.38 (m, 1H), 5.23 (s, 0H), 4.05 (q, J = 7.1 Hz, 1H), 1.97 (s, 2H), 1.50 (s, 2H), 1.30 – 1.15 (m, 12H), 0.81 (t, J = 6.8 Hz, 4H). LC-MS (ESI): m/z = 327.5 [M+H]+ Intermediate 6-2b.6-(7-(tert-butyl)naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile The title compound was prepared analogously to Example 6-2a from intermediate 2-(7- (tert-butyl)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (800mg) and 2-2a. Yield: 14%. LC-MS (ESI): m/z = 310.0 [M+H]]+ Intermediate 6-3a. 2-amino-6-(2,6-dimethoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile To a stirred solution of 2-amino-6-bromopyrazolo[1,5-a]pyrimidine-3-carbonitrile(2-2b) (0.300g, 1.26 mmol) and 2-(2,6-dimethoxynaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2- dioxaborolane(3 – 3i) (0.475 g, 1.51 mmol in Dioxane (33.0 mL) was added sodium carbonate (0.400 g, 3.78 mmol) in water (1.0 mL) and degassed with argon gas for 20 minute. Then PdCl2(dppf).CH2Cl2 (1.13 g, 1.386 mmol) was added to the reaction mixture at room temperature and purged for another 10 minutes. The reaction mixture was heated at 100°C for 6h. The color changes from brown to black. The progress of the reaction checked by TLC with mobile phase 30% EtOAc in hexane. Reaction mass was cooled to room temperature and added brine solution (500 mL) and extracted the product with EtOAc (1000 mL). Added saturated NaHCO3 solution (300 mL) was added to organic layer and stirred for 15 minutes and extracted the product with EtOAc (800 mL). The aqueous layer was acidified with 1N HCl (pH~ 4.0), solid precipitated was filtered under vacuum to afford 2-amino-6-(2,6-dimethoxynaphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carbonitrile (6-3a) (0.40g, 1.15 mmol, 91.95 %) as off-white solid. HPLC: 90.45% at RT=6.65 min; LCMS(M+1) = 346.00 at RT= 1.493 min. Intermediate 6-3b. 2-amino-6-(3,4-dihydronaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile The title compound was prepared analogously to Example 6-3a from intermediates 2-(3,4- dihydronaphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.070 g, 1.51 mmol) and 2-2b. Yield: 75%.1H NMR: (400 MHz, DMSO-d6) δ 8.88 (s, 1H), 8.41 (s, 1H), 7.27-7.14 (m, 3H), 6.99- 6.97 (d, J=7.6Hz, 1H), 6.74 (bs, 2H), 6.32-6.29 (t, 1H), 2.84-2.80 (t, 2H), 2.42-2.36 (m, 2H). LC- MS (ESI): m/z = 287.7 [M+H]]+ Intermediate 6-3c.2-amino-6-(6-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile The title compound was prepared analogously to Example 6-3a from intermediate 4,4,5,5- tetramethyl-2-(6-methylbenzo[b]thiophen-7-yl)-1,3,2-dioxaborolane (4-6c) and 2-2b. Yield: 9%. LCMS (ESI): m/z = no ionization Intermediate 6-3d. 2-amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile The title compound was prepared analogously to Example 6-3a from intermediate (2- methylnaphthalen-1-yl)boronic acid and 2-2b. Yield: 40.83%. LC-MS (ESI): m/z = 300.0 [M+H]]+ Intermediate 7-1a. 6-(6-fluoro-2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile To a stirred solution of 1-bromo-6-fluoro-2-methylnaphthalene (3-2a) (0.300 g, 1.254 mmol) in THF (6.0 ml), were added (3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)boronic acid (2-3a) (0.259 g, 1.254 mmol), 2% TPGS-750-M (aq) solution (2.25 ml) and purged the reaction mixture with argon for 30 min. Then added Pd(dtbpf)Cl2 (0.163 g, 0.250 mmol) and triethylamine (0.698 ml, 5.016 mmol) and purged again with argon for 25min. Sealed the reaction mixture and was stirred at 85°C for 12h. The completion of the reaction was monitored by TLC, using mobile phase 40% EtOAc in hexane. The reaction mixture was cooled at room temperature and partitioned between water and ethyl acetate (5 mL x 3) and the organic layer was washed with brine solution (10mL) and dried with anhydrous Na2SO4 and concentrated under reduced pressure to afford the crude product. The crude product was purified by combi-flash, using 4g cartridge and eluted the product with 30-40% EtOAc: Hexane and evaporated the solvent. The obtained product was dissolved in EtOH, added silica-met-DMT and stirred for 12h and filtered over celite bed, washed the celite bed with EtOH. The filtrate was concentrated to afford 6-(6-fluoro-2-methylnaphthalen- 1 yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile(7-1a) (0.210 g, 0.694 mmol, 55.36%) as a yellow solid. 1H NMR (300 MHz, CDCl3): δ 8.73-8.67 (m, 2H),8.49 (s,1H),7.88-7.85 (d, J=8.4Hz, 1H),7.55-7.49(m,2H),7.36-7.33(m, 1H), 7.24-7.21 (m, 1H), 2.33 (s, 3H); 19FNMR (300 MHz, CDCl3); δ -115.11 δ ppm. LC-MS (ESI): m/z = 303.2 [M+H]]+ The following intermediates 7-1b to 7-1z and 7-1aa, 7-1ab (Table 18) are available in an analogous manner starting from core boronic acid intermediate 2-3a and appropriate biaryl bromides 3-2. The crude products were purified by chromatography Table 18
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
Figure imgf000258_0001
Figure imgf000259_0001
Figure imgf000260_0001
Figure imgf000261_0001
Intermediate 7-2a. 2-amino-6-(2-methoxy-6-methylnaphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carbonitrile A stirred solution of 1-bromo-2-methoxy-6-methylnaphthalene (3-2k) (0.300 g, 1.20 mmol), (2-amino-3-cyanopyrazolo[1,5-a]pyrimidin-6-yl)boronic acid (2-3b) (0.292 g, 1.44 mmol), 2% TPGS-750-M (3 mL) and triethylamine (0.67 mL, 4.80 mmol) in THF (3.0 mL) at room temperature was degassed with argon gas for 30 minutes, then [1,1′-Bis(di-tert- butylphosphino)ferrocene]dichloropalladium(II) (0.117 g, 0.18 mmol) was added to reaction mixture at the same temperature and the reaction mixture was heated to 70o C for 16h. The completion of the reaction was monitored by TLC, using mobile phase: 30% EtOAc in hexane. The reaction mixture was diluted with water (10 mL) and extracted to ethyl acetate (2 x 15 mL). The organic layer was separated, washed with 2N HCl (2 x 10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by combi flash using 4g cartridge and product eluted with eluted with 50% EtOAc: hexane as eluent. The obtained product was dissolved in MeOH and Silica DMT was added then stirred for 12h, then filtered over celite. Filtrate concentrated under vacuum to afford 2-amino-6-(2-methoxy-6- methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile (7-2a) (0.230 g, 0.698 mmol, 58.37%) as off-white solid.1H NMR (400 MHz, DMSO-d6): δ 8.98 (s, 1H),8.42(s, 1H), 8.00 -7.98 (d, J = 8.8 Hz,1H), 7.72 (s, 1H), 7.56-7.53 (d, J = 9.2 Hz, 1H), 7.48-7.45 (d, J = 8.8 Hz, 1H), 7.30- 7.28 (m, 1H), 6.76 (bs, 2H), 3.84 (s, 3H), 2.43 (s, 3H). LC-MS (ESI): m/z = 329.8 [M+H]]+ The following intermediates 7-2b to 7-2k (Table 19) are available in an analogous manner starting from core boronate intermediate 2-3b and appropriate biaryl bromides 3-2 (or commercially available bromides). The crude products were purified by chromatography Table 19
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Intermediate 8-1a. 2-(2-(difluoromethyl)naphthalen-1-yl)-3-hydroxyacrylonitrile To a stirred solution of 1-bromo-2-(difluoromethyl)naphthalene (3-2d) (0.700 g, 2.73 mmol) in THF (2.0 mL, 3 vol) were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (0.640 g, 3.28 mmol), 2% TPGS-750-M (8.0 mL, 13.0 vol), triethylamine (1.14 mL, 8.20 mmol) and purged with argon for 15 minutes. After 15 minutes, added [1,1′-Bis(di-tert- butylphosphino)ferrocene]dichloropalladium(II) (0.053 g, 0.082 mmol) and reaction mixture was purged again with argon for 25 min and heated up to 50°C for 12 h. The progress of the reaction was monitored by TLC, using mobile phase:30 % EtOAc in Hexane. The reaction mixture was diluted with water (10 mL) and extracted to ethyl acetate (2 x 15 mL). The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure to get the crude. The crude was purified by combi-flash using 4g cartridge and product eluted with eluted with 40% EtOAc: hexane. The obtained product was dissolved in methanol (15 vol), added Silica Met DMT resin and stirred for 16h. After 16h, the solvent was filtered and then washed with methanol and filtrate was concentrated to afford 2-(2-(difluoromethyl)naphthalen-1-yl)-3- hydroxyacrylonitrile (8-1a) (0.23g, 0.399 mmol) as off-white solid. LC-MS (ESI): m/z = 247.3 [M+H]]+ The following intermediates 8-1b to 8-1s (Table 20) are available in an analogous manner starting from biaryl bromide 3-2 and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole. The crude products were purified by chromatography if necessary Table 20
Figure imgf000265_0001
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Intermediate 8-2a.7-amino-6-(2-(difluoromethyl)naphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carbonitrile To a microwave vail was added 2-(2-(difluoromethyl)naphthalen-1-yl)-3- hydroxyacrylonitrile (8-1a) (0.185 g, 0.754 mmol) and 5-amino-1H-pyrazole-4-carbonitrile (0.081 g, 0.754 mmol) in toluene ( 2.0 mL) was added p-toluenesulfonic acid monohydrate (0.143 g, 0.754 mmol) and the reaction mixture was heated to 110°C for 16h.The progress of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Reaction mixture was concentrated directly to dryness to get the crude. The crude was purified by combi flash using 4g cartridge, eluting the product at 50% EtOAc in hexane to afford 7-amino-6-(2- (difluoromethyl)naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile (8-2a) (0.130g g, 0.387 mmol, 51.58%) as off-white solid. LC-MS (ESI): m/z = 335.7 [M+H]]+ The following intermediates 8-2b to 8-2p (Table 21) are available in an analogous manner starting from corresponding enol intermediate 8-1 and 5-amino-1H-pyrazole-4-carbonitrile. The crude products were purified by chromatography Table 21
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
Example I – 1: 6-(benzo[b]thiophen-3-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7- amine
Figure imgf000274_0001
Step 1. 2-(benzo[b]thiophen-3-yl) acetamide To a stirred solution of 2-(benzo[b]thiophen-3-yl)acetic acid (3.0 g, 15.60 mmol) in DMF (30.0 mL), were added EDCI. HCl (4.49g, 23.41 mmol), HOBt.H2O (3.58g, 23.41mmol), NH4Cl(2.50g, 46.82 mmol), DMAP(0.19g, 1.56mmol) and DIPEA (10.87mL, 62.43mmol) and the reaction mixture was allowed to stir at room temperature for 16 h. The completion of the reaction was monitored by TLC, using mobile phase 5% MeOH in DCM. Poured the reaction mixture to ice cold water and extracted with EtOAc. The organic layer was washed with brine solution, dried over anhydrous Na2SO4 and concentrated under vacuum to afford 2-(benzo[b]thiophen-3-yl) acetamide (12-1) (2.7 g, crude) as amber coloured oil. 1H NMR the formation of desired product,1H NMR (300 MHz, DMSO-d6): δ 7.98-7.95(m, 1H), 7.85-7.82 (m, 1H), 7.56 (bs, 1H),7.50 (s, 1H), 7.42-7.35 (m, 2H), 7.00 (bs, 1H), 3.65 (s, 2H). LC-MS (ESI): m/z = 192.0 [M+H]]+ Step 2. 2-(benzo[b]thiophen-3-yl)acetonitrile To a stirred solution of 2-(benzo[b]thiophen-3-yl)acetamide (12-1) (2.70 g, 14.116 mmol) and triethylamine (4.28g, 42.35mmol) in 1,4-Dioxane (30 mL) was added TFAA (4.45 mL, 21.17 mmol) at 0°C.The reaction mixture was stirred at room temperature for 16h. The completion of the reaction was monitored by TLC, using mobile phase: 5% MeOH in DCM. water and extracted with EtOAc. The organic layer was washed with brine solution, dried over anhydrous Na2SO4 and concentrated under vacuum. The crude was washed with pentane and dried under vacuum to afford 2-(benzo[b]thiophen-3-yl)acetonitrile (12-2) (2.2g,12.696mmol, 89.8 %) as light brown solid. 1H NMR (600 MHz, CDCl3): δ 7.90-7.89(m, 1H), 7.71-7.70 (d, J=7.8 Hz, 1H), 7.49-7.41 (m, 3H), 3.91 (s, 2H), the formation of desired product. Step 3. 2-(benzo[b]thiophen-3-yl)-3-oxopropanenitrile To a stirred solution of 2-(benzo[b]thiophen-3-yl)acetonitrile (12-2) (1.0g, 5.77mmol) in THF (10.0mL), were added Ethyl Formate (0.64g, 8.65mmol) and sodium hydride (60% in mineral oil, 0.30g, 7.50mmol) at room temperature. The reaction mixture heated at 50°C for 30 minutes. The completion of the reaction was monitored by TLC using mobile phase: 20 % EtOAc in hexane. Reaction mixture was cooled to room temperature and quenched with dilute HCl and extracted with EtOAc. The organic layer washed with brine solution and dried over Na2SO4 and concentrated in vacuum to afford 2-(benzo[b]thiophen-3-yl)-3-oxopropanenitrile (12-3) (1.1 g, crude) as pale-yellow solid. LCMS; No ionization. The obtained crude product was taken to the next step. Step 4. 7-amino-6-(benzo[b]thiophen-3-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile To a stirred solution of 2-(benzo[b]thiophen-3-yl)-3-oxopropanenitrile (12-3) (1.0 g, 4.96 mmol) in EtOH: AcOH (20mL:20mL), was added 5-amino-1H-pyrazole-4-carbonitrilem (0.59g, 5.46 mmol) and then refluxed the reaction mixture at 85°C for 16h. The completion of the reaction was monitored by TLC, using mobile phase 40% EtOAc in hexane. The reaction mass was cooled and evaporated the solvents under vacuum to dryness. The residue was quenched the reaction mass with saturated NaHCO3 solution, precipitated solid filtered and dried under vacuum to get the crude. The crude was purified by combi flash (12 g Silicycle column cartridge) and eluted with 40-70% in EtOAc: hexane to afford 7-amino-6-(benzo[b]thiophen-3-yl)pyrazolo[1,5-a]pyrimidine- 3-carbonitrile (12-4) (0.650 g, 2.23mmol, 45.13%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6): δ 8.72 (s, 1H), 8.26 (s, 1H), 8.10-8.08 (m, 3H), 7.93 (s, 1H), 7.57-7.55 (d, J=7.2Hz, 1H), 7.44-7.39 (m, 2H). LC-MS (ESI): m/z = 292.3 [M+H]]+ Step 5. 6-(benzo[b]thiophen-3-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine To a stirred solution of 7-amino-6-(benzo[b]thiophen-3-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile (12-4) (0.30g, 1.56mmol) in DMF (5.0 mL) was added sodium azide (0.682 g, 10.5 mmol) and ammonium chloride (0.838 g, 15.67 mmol) at room temperature. The reaction mixture was heated at 100°C for 16h. The progress of the reaction was monitored by TLC, using 5% MeOH in DCM. Evaporated the reaction mixture to dryness. The residue was diluted with saturated NaHCO3 solution, precipitated solid filtered and dried under vacuum to get the crude. The crude was purified by Column: Column: KINETEX EVO (150mmx21.2mm), 5.0µ, Flow: 20ML, Mobile Phase: A= 0.1% HCOOH in water, B = MeCN, Gradient Program: (Time %B) :(0,20), (, 25), (7,60), followed by the lyophilization of the purified sample for 4 days to afford 6- (benzo[b]thiophen-3-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine (I-1) (0.070g, 0.20mmol, 20.58%) as light brown solid.1H NMR: (300 MHz, DMSO-d6): δ 8.75 (s, 1H), 8.30 (s, 1H), 8.13-8.11 (d, J=8.0Hz, 1H), 7.97-7.96 (m, 2H), 7.60-7.58 (d, J=7.2Hz, 1H), 7.46-7.39 (m, 2H). LC-MS (ESI): m/z = 335.1 [M+H]]+. HPLC: 99.54 % at RT = 4.917 min. (method 11) Example I – 2: 6-(5-methoxybenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5- a]pyrimidin-7-amine Step 1. 2-(5-methoxybenzo[b]thiophen-4-yl)acetonitrile To a stirred solution of 4-bromo-5-methoxybenzo[b]thiophene (5-5b) (0.300 g, 1.23 mmol) in THF (3.0 mL, 10 vol) were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (0.290 g, 1.48 mmol), TPGS-750-M (3.0 mL, 10.0 vol) and triethylamine (0.68 mL, 4.95 mmol).The reaction mixture was purged with argon for 30 minutes, followed by the addition of [1,1′-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II)(0.121 g, 0.185 mmol) and heated the reaction mixture to 50°C for 1h followed by 80°C for 12h. The completion of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Cooled the reaction mixture to room temperature. The reaction mixture was diluted with water (10 mL) and extracted to ethyl acetate (2 x 15 mL). The organic layer was separated, washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to get the crude. The crude product was purified by combi-flash eluted with 20% EtOAc in hexane. The obtained product was dissolved in methanol (15 vol), added Silica Met DMT resin and stirred for 16h. After 16h, the solvent was filtered and washed the celite bed with methanol and filtrate was concentrated to afford 2-(5-methoxybenzo[b]thiophen-4-yl)acetonitrile (13-1) (0.190 g, 0.935 mmol,75.69 %) as pale yellow colored solid. 1H NMR: (300MHz, DMSO-d6): δ 8.00-7.98 (d, J= 8.7Hz, 1H), 7.89- 7.87 (m, 1H), 7.63-7.61 (d, J= 5.7 Hz, 1H), 7.26-7.23 (d, J= 8.7 Hz, 1H), 4.17 (s, 2H), 3.92 (s, 3H). LC-MS (ESI): m/z = no ionization. Step 2. 4-amino-3-(5-methoxybenzo[b]thiophen-4-yl)pyrrolo[1,2-a]pyrimidine-8- carbonitrile To a stirred solution of 2-(5-methoxybenzo[b]thiophen-4-yl)acetonitrile (13-1) (0.190 g, 0.935 mmol) in toluene (1.9 mL, 10 vol) under nitrogen atmosphere, was added 1-tert-butoxy- N,N,N',N'-tetramethylmethanediamine (0.326, 1.87 mmol) and stirred the reaction mixture for 120°C for 1h. Concentrated the reaction mixture and added toluene (1.9 mL, 10 vol), followed by acetic acid (1.9 mL,10.0 vol) and 5-amino-1H-pyrazole-4-carbonitrile (0.101 g, 0.935 mmol) and heated the reaction mixture at 130°C for 16h. The completion of the reaction was monitored by TLC, using mobile phase:50% EtOAc in hexane. Cooled the reaction to room temperature. Added saturated NaHCO3 solution to reaction mixture and extracted with EtOAc (20 x 3 mL) and the organic layer was dried over anhy. Na2SO4 and concentrated under vacuum to get crude. The crude product was purified by combi-flash eluted with 20% EtOAc in hexane to afford 4-amino-3- (5-methoxybenzo[b]thiophen-4-yl)pyrrolo[1,2-a]pyrimidine-8-carbonitrile (13-2) (0.130 g,0.404 mmol,43.33%) as pale-yellow solid.1H NMR: (300MHz, DMSO-d6): δ 8.70 (s, 1H), 8.13 (s, 1H), 8.09-8.06 (d, J= 8.7 Hz, 1H), 7.91 (bs, 2H), 7.75-7.73 (d, J= 5.4 Hz, 1H), 7.32-7.29 (d, J= 9 Hz, 1H), 7.03-7.01 (d, J= 5.4 Hz, 1H), 3.81 (s, 3H). LC-MS (ESI): m/z = 321.7 [M+H]]+ Step 3. 6-(5-methoxybenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7- amine To a stirred solution 4-amino-3-(5-methoxybenzo[b]thiophen-4-yl)pyrrolo[1,2- a]pyrimidine-8-carbonitrile (13-4) (0.130 g, 0.404 mmol) in DMF (2 mL) under nitrogen atmosphere were added sodium azide (0.263 g, 4.048 mmol) and ammonium chloride (0.218 g, 4.048 mmol) and the reaction mixture was heated at 130oC for 16h. The completion of the reaction was monitored by TLC, using mobile phase: 50% EtOAc in hexane. Cooled the reaction mixture to room temperature. Reaction mixture was quenched with distilled water (20 mL) and acidified with 15% citric acid solution, the solid was precipitated out was filtered and dried under vacuum to afford 6-(5-methoxybenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7- amine (I - 2) (0.085g, 0.233 mmol, 57.82 %) as off-white solid.1H NMR: (400MHz, DMSO-d6): δ 8.73 (s, 1H), 8.20 (s, 1H), 8.09-8.06 (d, J= 8.8 Hz, 1H), 7.77 (bs, 2H), 7.75-7.74 (d, J= 5.2 Hz, 1H), 7.33-7.31 (d, J= 9.2 Hz, 1H), 7.05-7.03 (d, J= 5.6 Hz, 1H), 3.82 (s, 3H). LC-MS (ESI): m/z = 364.8 [M+H]]+. HPLC: 92.06% at RT = 6.175 min. (method 1). Example I - 3: 6-(5-methylbenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5- a]pyrimidin-7-amine Step 1. 2-(5-methylbenzo[b]thiophen-4-yl)acetonitrile To a stirred solution of 4-bromo-5-methylbenzo[b]thiophene (5-5d) (1.800 g, 7.92 mmol) in THF (20.0 mL, 10 vol) were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (1.85 g, 9.51 mmol), followed by TPGS-750-M (20.0 mL, 10.0 vol) and triethylamine (4.41 mL, 31.70 mmol). The reaction mixture was purged with argon for 30 minutes. After 30 minutes, added [1,1′-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) (0.774 g, 1.18 mmol), and heated up to 50°C for 1h, followed by further heating to 80°C for 12 h. The progress of the reaction was monitored by TLC, using 30 % EtOAc in hexane. Reaction mass was cooled, and brine solution added (10 mL), then extracted with EtOAc (2 x 10mL) and dried over Na2SO4 and concentrated under vacuum. The crude was purified by combi-flash (24 g cartridge) eluted with 20% EtOAc: hexane. Then obtained product was dissolved in methanol (15 vol), added Silica Met DMT resin and stirred for 16h to afford 2-(5-methylbenzo[b]thiophen-4-yl)acetonitrile (14-1) (0.700 g, 3.73 mmol, 47.23%) as off-white solid.1H NMR (300 MHz, DMSO-d6): δ 7.91-7.89 (d, J=8.1 Hz,1H), 7.85-7.84 (d, J=5.1 Hz,1H), 7.70-7.68 (d, J=5.4 Hz, 1H), 7.29-7.26 (d, J=8.4 Hz,1H),4.31 (s,2H), 2.46 (s,3H). LC-MS (ESI): m/z = no ionization Step 2. 7-amino-6-(5-methylbenzo[b]thiophen-4-yl)pyrazolo[1,5-a]pyrimidine -3- carbonitrile To a stirred solution of 2-(5-methylbenzo[b]thiophen-4-yl)acetonitrile (14-1) (0.250 g, 1.336 mmol) in toluene (2.5 mL, 10 vol) under nitrogen atmosphere was added 1-tert-butoxy- N,N,N',N'-tetramethylmethanediamine (0.465, 2.67 mmol) and stirred the reaction mixture for 120°C for 1h. Concentrated the reaction mixture and added toluene (2.5 mL, 10 vol), acetic acid (2.5 mL, 10.0 vol) and 5-amino-1H-pyrazole-4-carbonitrile (0.144 g, 1.34 mmol). Then the reaction mixture was stirred at 130°C for 12h. The completion of the reaction was monitored by TLC, using mobile phase: 30% EtOAc in hexane. Reaction mixture was basified with saturated NaHCO3 solution and extracted the product with EtOAc (20 x 3 mL). The combined organic layer was washed with brine solution and dried over anhy. Na2SO4 and concentrated under vacuum to get crude. The crude product was purified by combi-flash, (4 g, Silicycle cartridge) eluting the product with 20% EtOAc: Hexane. Then obtained product was dissolved in methanol (15 vol), added Silica Met DMT (10 eq) resin and stirred for 16h. After 16h, the solvent was filtered and then washed with methanol and filtrate was concentrated to afford 7-amino-6-(5-methylbenzo[b]thiophen-4- yl)pyrazolo[1,5-a]pyrimidine -3-carbonitrile (14-2) (0.125 g, 0.410 mmol, 30.71 %) as off-white solid. LC-MS (ESI): m/z = 305.7 [M+H]]+ Step 3. 6-(5-methylbenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7- amine To a stirred solution of 7-amino-6-(5-methylbenzo[b]thiophen-4-yl)pyrazolo[1,5- a]pyrimidine-3-carbonitrile (14-2) (0.120 g, 0.393 mmol) in DMF(1.2 mL) under nitrogen atmosphere were added sodium azide (0.255 g, 3.93 mmol), followed by ammonium chloride (0.212 g, 3.93 mmol), the reaction mixture was heated at 130oC for 16h. The progress of the reaction was monitored by TLC, using mobile phase 50% EtOAc in hexane. The solvent from reaction mixture was removed under vacuum and distilled water added. Acidified with 15% acetic acid, precipitated solid was filtered and dried under vacuum to get crude. The crude compound was purified by Column: LUNA Phenomenex (250mmx21.2mm), 5.0µ Flow: 18mL/min, mobile phase: A= 0.1% HCOOH in water. B= ACN and Gradient Program: (Time %B): (0,50), (2,60), (8, 70), followed by the lyophilization of the purified sample for 2 days to afford 6-(5- methylbenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine ( I – 3) (0.030 g, 0.086 mmol, 22.05 %) as white solid.1H NMR (300 MHz, DMSO-d6): δ 8.75 (s ,1H), 8.18 (s, 1H), 8.02-7.99 (d, J=9.0 Hz,1H), 7.80 (bs, 2H), 7.70-7.68 (d, J=5.4 Hz, 1H), 7.42-7.39 (d, J=8.7 Hz, 1H), 7.03-7.01 (d, J=5.1 Hz,1H), 2.25 (s, 3H). LC-MS (ESI): m/z = 348.8 [M+H]]+. HPLC: 96.7 % at RT=4.77 min. (method 4). Example I - 4: 6-(8-chloronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7- amine Step 1. 2-(8-chloronaphthalen-1-yl)acetonitrile To a stirred solution of 1-bromo-8-chloronaphthalene (0.50 g, 2.070 mmol) and 4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (0.40 g, 2.070 mmol) in THF(2.5 mL) were added 2% TPGS-750-M (5.0 mL) and Et3N (1.16 mL, 8.250 mmol) at room temperature and the reaction mixture was degassed with argon gas for 20 minute. Then added Pd(dtbpf)Cl2 (0.13 g, 0.207 mmol) at room temperature and heated at 50°C for 1h, then after heated at 80°C for 5 h. The color of the reaction mixture changes from brown to black upon heating. The progress of the reaction was monitored by TLC, using mobile phase:30% EtOAc in hexane. Reaction mixture was cooled and added brine solution (100 mL), then extracted the product with DCM (200 mL), then dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by combi-flash (12 g cartridge) eluted with 10% EtOAc: Hexane. The obtained product was dissolved in MeOH (100 mL) and Silia DMT was added then stirred for 12 h, then filtered over celite. Filtrate concentrated under vacuum to afford 2-(8-chloronaphthalen-1-yl)acetonitrile (15-1) (0.25g, 1.237 mmol, 61.2%) as yellow liquid. LC-MS (ESI): m/z = 202.5 [M+H]]+ Step 2. 2-(8-chloronaphthalen-1-yl)-3-oxopropanenitrile To a stirred solution sodium hydride (0.075 g, 1.855 mmol) in THF (5.0 mL) at 0°C under nitrogen atmosphere, was added 2-(8-chloronaphthalen-1-yl) acetonitrile (15-1) (0.25 g, 1.237 mmol) solution in THF (5.0 mL) and stirred for 15 min. Then added Ethyl Formate (0.49 mL, 6.188 mmol) in dropwise to the reaction mixture at 0°C and heated at 50°C for 2h. The color of the reaction mixture changes from yellow to brown. The progress of the reaction was monitored by TLC, using mobile phase:40% EtOAc in hexane. Reaction mixture was cooled to room temperature and quenched with 1N HCl solution (100 mL), extracted with EtOAc (200 mL). The organic layer was washed with brine solution, dried anhydrous over Na2SO4 and concentrated under vacuum. The crude was purified by combi-flash (12 g cartridge), eluted the product with 60% EtOAc: Hexane to afford 2-(8-chloronaphthalen-1-yl)-3-oxopropanenitrile (15-2) (0.110g, 0.480mmol, 38.8%) as yellow solid. LC-MS (ESI): m/z = 227.6 [M+H]]+ Step 3.7-amino-6-(8-chloronaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile To a stirred solution of 2-(8-chloronaphthalen-1-yl)-3-oxopropanenitrile (15-2) (0.10 g, 0.436 mmol) in AcOH (1.0 mL) and EtOH (1.0 mL) at room temperature was added 5-amino-1H- pyrazole-4-carbonitrile (0.06 g, 0.567 mmol) and reaction mixture was heated at 110°C for 12h. The color of the reaction mixture changes from yellow to brown. The progress of the reaction was monitored by TLC, using mobile phase: 40% EtOAc in hexane. Cooled the reaction mixture to room temperature and evaporated under vacuum to dryness to get the residue. The residue was basified with saturated sodium bicarbonate solution, the solid precipitated, filtered out under vacuum and washed with hexane to afford 7-amino-6-(8-chloronaphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carbonitrile (15-3) (0.125 g, 0.391mmol, 89.6%) as light brown solid. 1H NMR: (300MHz, DMSO-d6): δ 8.71-8.69 (d, J= 8.4 Hz, 1H), 8.18-8.13 (m, 1H), 8.07-8.02 (m, 2H), 7.92 (bs, 2H), 7.69-7.47 (m, 4H). LC-MS (ESI): m/z = 319.7 [M+H]]+ Step 4.6-(8-chloronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine To a stirred solution of 7-amino-6-(8-chloronaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile (15-3) (0.110 g, 0.344 mmol) in DMF (2.2 mL) at room temperature, were added NaN3 (0.111 g, 1.720 mmol) and (NH4)2Ce(NO3)6 (0.188 g, 0.344 mmol). The reaction mixture was heated at 100°C for 12h. The color of the reaction mixture changes from white to brown. The progress of the reaction was monitored by TLC, using mobile phase:5% MeOH in DCM. Cooled the reaction mixture to room temperature and the reaction mixture was poured to ice cold water, the solid precipitated. Filtered the solid, dried under vacuum, washed with pentane to get the crude. The crude was purified by reverse phase HPLC using Column: KINETEX (250mm x 21.2mm), 5.0μ, Flow: 18 mL, Mobile Phase: A= 0.1% HCOOH in water, B= ACN, Gradient Program:(Time, %B) :(0,15), (2,25), (8,50), followed by the lyophilization of the purified sample for 3 days to afford 6-(8-chloronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7- amine (I – 4) (0.030 g, 0.0828 mmol, 23.8%) as off-white solid.1H NMR (300 MHz, DMSO-d6): δ 8.64(s, 1H), 8.17-8.15 (d, J=7.8 Hz, 1H), 8.09-8.06 (d, J=8.7 Hz, 2H),7.71-7.50 (m, 6H). LC-MS (ESI): m/z = 362.7 [M+H]]+. HPLC:95.0%, at RT = 6.13 min. (method 9). Example I - 5: 6-(naphthalen-1-yl)-3-(2H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine Step 1. 2-(naphthalen-1-yl)-3-oxopropanenitrile To a stirred solution 2-(naphthalen-1-yl)acetonitrile (2.0 g,11.95 mmol) in THF (30.0 mL) was added ethyl formate (1.32g, 17.93 mmol) and sodium hydride (60% in mineral oil, 0.621g, 15.53 mmol) and heated for 50°C for 30min. The completion of the reaction was monitored by TLC using mobile phase: 20% EtOAc in hexane. Cooled the reaction mass to room temperature. Quenched this reaction mass with dil. HCl and extracted with ethyl acetate. The organic layer was washed with brine solution and dried with anhy. Na2SO4 and concentrated under reduced under vacuum to afford 2-(naphthalen-1-yl)-3-oxopropanenitrile (16-1) (2.2 g, 97%) as pale-yellow solid. LC-MS (ESI): m/z = 194.0 [M+H]]+ Step-2. 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carbonitrile To a stirred solution of 2-(naphthalen-1-yl)-3-oxopropanenitrile (16-1) (0.6 g, 3.07mmol) in EtOH/AcOH (1:1) (12 mL) was added 5-amino-1H-pyrazole-4-carbonitrile (0.364 g, 3.37mmol) and stirred at 85°C for 16h. The completion of the reaction was monitored by TLC using mobile phase: 40% EtOAc in hexane. Solvents were removed under rotavapor, and reaction mixture was diluted with saturated NaHCO3 solution, solid was precipitated out was filtered and dried under vacuum to get the crude. The crude was recrystalised by EtOAc: EtOH (10:5 mL), heated at 85oC for 30min and filtered and dried under vacuum to afford 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carbonitrile (16-2) (0.366 g,1.28 mmol,42.02%) as off-white solid. 1H NMR (300 MHz, DMSO-d6): δ 8.73 (s, 1H), 8.19 (s, 1H), 8.06-8.03 (m, 2H),7.92 (bs, 2H), 7.65-7.46 (m, 5H). LC-MS (ESI): m/z = 286.0 [M+H]]+ Step 3. 6-(naphthalen-1-yl)-3-(2H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine To a stirred solution of 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carbonitrile (16-2) (0.155 g ,0.143 mmol) in dimethyl formamide (2.0 mL) under nitrogen atmosphere were added sodium azide (0.353 g, 5.43 mmol) and ammonium chloride (0.290 g, 5.43 mmol), the reaction mixture was heated at 100oC for 16h. The completion of the reaction was monitored by TLC, using mobile phase: 40% EtOAc in hexane. Evaporated the reaction mass under vacuum to dryness. Diluted the solid with distilled water, the solid was precipitated, filtered the solid, dried under vacuum to get crude. The crude was purified by reverse phase HPLC using Column: KINETEX , Flow: 20 ml/min, mobile phase: A= 0.1% HCOOH in water B= MeCN, Gradient Program:(Time,%B): (0,15), (2,25), (9,70), followed by the lyophilization of the purified sample for 3 days to afford 6-(naphthalen-1-yl)-3-(2H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7- amine (I – 5) (0.026 g, 0.079 mmol, 14.6 %) as off-white solid. 1H NMR (400 MHz, DMSO-d6): δ 8.67(s,1H),8.18(s,1H),8.03-8.01(d, J=8.4 Hz,2H),7.63 (bs, 2H), 7.62-7.56 (m, 5H). LC-MS (ESI): m/z = 329.3 [M+H]]+. HPLC:97.9%, at RT = 6.39 min. (method 7). The following compounds were prepared according to the above reaction protocol Step 3 of the above Examples I - 5 using appropriate cyano intermediates (from Table 17 - 19 and 21) and sodium azide. Table 22
Figure imgf000284_0001
Figure imgf000285_0001
Figure imgf000286_0001
Figure imgf000287_0001
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000290_0001
Figure imgf000291_0001
Figure imgf000292_0001
Figure imgf000293_0001
Figure imgf000294_0001
Figure imgf000295_0001
Figure imgf000296_0001
Figure imgf000297_0001
Figure imgf000298_0001
Figure imgf000299_0001
Figure imgf000300_0001
Figure imgf000301_0001
Figure imgf000302_0001
Figure imgf000303_0001
Figure imgf000304_0001
Intermediate 6-1u. tert-butyl (tert-butoxycarbonyl)(3-cyano-6-(2-methylnaphthalen-1- yl)pyrazolo[1,5-a]pyrimidin-7-yl)carbamate To a vial containing 8-2g (78 mg, 0.261 mmol), Boc2O (0.151 mL, 0.651 mmol), TEA (0.073 mL, 0.521 mmol) and DMAP (3.18 mg, 0.026 mmol) was added DCM (1 mL) and stirred overnight at 35 °C at which time LCMS indicated formation of desired product. The reaction was diluted with EtOAc and washed with water, dried (Na2SO4), and concentrated under reduced pressure to provide crude product tert-butyl (tert-butoxycarbonyl)(3-cyano-6-(2- methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)carbamate (6-1u), which was used as is for next step. LC-MS (ESI): m/z = 500.1 [M+H]]+ Intermediate 9-2a. tert-butyl (Z)-(3-(N'-hydroxycarbamimidoyl)-6-(2-methylnaphthalen-1- yl)pyrazolo[1,5-a]pyrimidin-7-yl)carbamate To a solution of 6 – 1u (130 mg, 0.260 mmol) in DMSO (1 mL) in a microwave vial were added hydroxylamine hydrochloride (90 mg, 1.301 mmol) and Na2CO3 (138 mg, 1.301 mmol). The resulting mixture was stirred at 60 °C for 4 h. Water was added and the precipitated was filtered to afford the mono-boc protected tert-butyl (Z)-(3-(N'-hydroxycarbamimidoyl)-6-(2- methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)carbamate (9-2a) (91 mg, 0.210 mmol, 81 % yield). LC-MS (ESI): m/z = 433.3 [M+H]]+ Intermediate 9 – 3a. (Z)-7-amino-N'-hydroxy-6-(5-methylbenzo[b]thiophen-7- yl)pyrazolo[1,5-a]pyrimidine-3-carboximidamide
Figure imgf000306_0001
To a stirred solution of 6-1c (0.620 g, 2.063 mml) in DMSO (6.2 ml) was added NH2OH.HCl (0.716 g,10.31 mmol), followed by addition of Na2CO3 (1.090 g, 10.31 mmol) and the reaction mixture was stirred at 60° C for 4h. The completion of the reaction was monitored by TLC, 30% EtOAc in hexane. The reaction mixture was cooled to room temperature and added water to reaction mass. The precipitated solid was filtered and dried to get the crude. The crude was triturated with n-pentane to afford (Z)-7-amino-N'-hydroxy-6-(5-methylbenzo[b]thiophen-7- yl)pyrazolo[1,5-a]pyrimidine-3-carboximidamide (9-3a) (0.510 g, 1.510 mmol, 73.05 %) as yellow solid. LC-MS (ESI): m/z = 339.2 [M+H]]+ The following intermediates 9-2b to 9-2e and 9-3b to 9-3e (Table 23) are available in an analogous manner starting from core cyano intermediate 6-1 and hydroxylamine hydrochloride. The crude products were purified by chromatography Table 23
Figure imgf000306_0002
Figure imgf000307_0001
Figure imgf000308_0001
Example II - 1: 3-(7-amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)- 1,2,4-oxadiazol-5(4H)-one To a solution of 9-2a (90 mg, 0.208 mmol) in THF (1 mL) at 0 °C, pyridine (0.020 mL, 0.250 mmol) and 2-ethylhexyl carbonochloridate (0.040 mL, 0.208 mmol) were added. The resulting mixture was stirred at 0 °C for 5 min and then at rt for 20 min at which time LCMS indicated formation of desired intermediate. THF was removed and dioxane (1.5 mL) was added followed by pyridine (0.020 mL, 0.250 mmol). The reaction mixture was heated in the microwave at 130 °C for 40 min at which time LCMS indicated desired mass peak. Dioxane was removed under reduced pressure. The crude material was dissolved in MeCN and water purified via C18 Acidic Reverse Phase silica gel column (50 g, 0.1% Formic acid modifier, 0-30% ACN / H2O, 25 min). Desired fractions were combined and lyophilized to provide with desired product 3-(7- amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)-one (I-3) (7.5 mg, 0.019 mmol, 9.35 % yield).1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.03 (s, 1H), 7.91 (dd, J = 8.3, 2.1 Hz, 2H), 7.65 (s, 2H), 7.49 (d, J = 8.5 Hz, 1H), 7.42 (dt, J = 8.1, 4.1 Hz, 1H), 7.35 (d, J = 3.9 Hz, 2H), 2.20 (s, 3H). LC-MS (ESI): m/z = 359.0 [M+H]]+ The following compounds II – 2 to II – 10 were prepared according to the above reaction protocol as in the Examples II - 1 using appropriate oxime intermediates (From Table 23). The crude product (II) is purified by chromatography.
Figure imgf000310_0001
Figure imgf000311_0001
Figure imgf000312_0001
Example III - 1: 7-amino-6-(2-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylic acid Step 1. ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(2-methoxynaphthalen-1- yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (17-1) To a stirred solution of ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-bromopyrazolo[1,5- a]pyrimidine-3-carboxylate (1-4b) (0.600 gm,1.236 mml) in THF (6.0 ml) was added (2- methoxynaphthalen-1-yl)boronic acid (0.274 gm,1.359 mmol), 2% TPGS-750-M (aq) solution (4.5 ml) and purged with argon for 30 min. Pd (dtbpf)Cl2 (0.161gm, 0.247 mmol) and triethylamine (0.516 ml, 3.708 mmol) was added and further purged with argon for 25 min. Then the reaction mixture was stirred at 85°C for 12h. The completion of the reaction was monitored by TLC, using mobile phase 30 % EtOAc in hexane. Cooled the reaction mixture to room temperature and diluted with water (25mL) and extracted the product with ethyl acetate (40 mL) twice. The combined organic layer was washed with brine solution (10mL x 3) and dried with anhy. Na2SO4 and concentrated under reduced pressure to afford the crude. The crude was purified by flash chromatography, eluting the product at 30-40% EtOAc in hexane, obtained product was dissolved in EtOH added Silia-met-DMT and stirred for 12h, then filtered over celite bed and washed with EtOH Concentrated the filtrate to afford the ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(2- methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (17-1) (0.360 g, 0.639 mmol, 51.76% ) as yellow solid. The obtained mixture of products was taken to the next step based on TLC. Step 2. ethyl 7-amino-6-(2-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylate (17-2) To a stirred solution of mixture of product obtained from Step 1 (0.350 gm,0.622 mml) in DCM (1.75 ml) was added Dioxane in HCl (4N, 7.0 ml) at 0°C. The reaction mixture was stirred at room temperature for 8h. The progress of the reaction was monitored by TLC, using mobile phase:50 % EtOAc in hexane. The reaction mixture was evaporated in rotavapor under nitrogen. The crude was triturated with n-pentane to afford ethyl 7-amino-6-(2-methoxynaphthalen-1- yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (17-2) (0.280g, 0.808 mmol, 98.19%) as pale-yellow solid. LC-MS (ESI): m/z = 363.0 [M+H]]+ Step 3. 7-amino-6-(2-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (III - 1) To a stirred solution of ethyl 7-amino-6-(2-methoxynaphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carboxylate (17-2) ( 0.210 gm,0.579 mml) in THF: EtOH (1:1) (4.2mL) was added 4N aq. NaOH solution (2.1 mL) at 0°C. The reaction mixture was stirred at 100°C for 12h. The progress of the reaction was monitored by TLC, using mobile phase:10% MeOH in DCM. The reaction mixture was evaporated under vacuum to get the residue. The residue was diluted with water and aqueous layer was acidified with AcOH to pH=7. The obtained precipitated solid filtered, washed with n-pentane, decanted and dried under vacuum. The crude was purified by reverse phase preparative HPLC: Column: LUNA (C18, 21.2mm X 250mm) Flow: 18ml/min; Mobile Phase: A=0.1% HCOOH in water, B= MeCN; Gradient Program (Time, %B): (0,20), (2,20), (10, 55). The purified HPLC samples were lyophilized for 3 days to afford 7-amino-6-(2- methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (III - 1) (0.03g, 0.101 mmol, 17.55%) as off-white solid.1H NMR(300 MHz, DMSO-d6):δ 8.47 (s, 1H), 8.10-8.04 (m, 2H), 8.00 (m, 1H), 7.59-7.56 (m, 3H), 7.39 (m, 3H), 3.85 (s, 3H). LC-MS (ESI): m/z = 335.2 [M+H]]+. HPLC: 99.12 % at RT = 7.955 min. (method 10)
Example III - 2: 7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylic acid Step 1. ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carboxylate (18-1) To a stirred solution of ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-bromopyrazolo[1,5- a]pyrimidine-3-carboxylate (1-4b) (0.600 gm,1.236 mml) in THF (6.0 mL), was added 4,4,5,5- tetramethyl-2-(3-methylnaphthalen-1-yl)-1,3,2-dioxaborolane (0.397 gm,1.483) followed by 2% TPGS-750-M (aq) solution (4.5 mL) and purged the reaction mass with argon for 30 min. Pd (dtbpf)Cl2 (0.161 gm, 0.247 mmol) and triethylamine (0.668 ml, 4.944 mmol) was added to the reaction mixture and further purged with argon for 25 min. Then the reaction mixture was stirred at 85°C for 12 h. The completion of the reaction was monitored by TLC, using mobile phase 30 % EtOAc in hexane. Cooled the reaction mass to room temperature and diluted with water (25mL) and extracted the product with ethyl acetate (40 mL) twice. The combined organic layer was washed with brine solution (10mL x 3) and dried with anhy. Na2SO4 and concentrated under reduced pressure. The crude was purified by flash chromatography (silica column 24 gm), eluting the product at 30-40% EtOAc in hexane, obtained product was dissolved in EtOH added silica- met-DMT and stirred for 12h and then filtered over celite bed and washed with EtOH. Then concentrated the filtrate to afford the ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(3- methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (18-1) (0.460 g, 0.841 mmol, 68.07%) as yellow solid. LC-MS (ESI): m/z = 547.8 [M+H]]+. Along with above titled product, Monoboc (M-100H) = 447.1 and free amine products also observed, (M-200H) =347.05. The mixture of products was taken to next step. Step 2. ethyl 7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (18-2) To a stirred solution of mixture of product 18-1 obtained (From step 1, 0.450 gm,0.823 mml) in dioxane (2.25 mL) was added Dioxane in HCl (4N, 4.5 ml) at 0°C. The reaction mixture was stirred at room temperature for 2h. The progress of the reaction was monitored by TLC, using mobile phase:50 % EtOAc in hexane. The reaction mixture was evaporated in rotavapor under nitrogen. The crude was triturated with n-pentane to afford ethyl 7-amino-6-(3-methylnaphthalen- 1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate(18-2) (0.280g, 0.808 mmol, 98.19%) as pale-yellow solid.1H NMR (300 MHz, DMSO-d6): δ 9.20 (bs, 2H), 8.74 (s, 1H), 8.23 (s, 1H), 7.96-7.94 (d, J= 8.4 Hz, 1H), 7.87(s, 1H), 7.74-7.72 (d, J= 8.1 Hz, 1H), 7.57-7.52 (t, J= 7.2 Hz, 1H), 7.44-7.40 (m, 2H),4.42-4.34 (q, 2H), 2.52 (s, 3H), 1.37-1.32 (t, 3H). LC-MS (ESI): m/z = 347.0 [M+H]]+ Step 3. 7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (III-2) To a stirred solution of ethyl 7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5- a]pyrimidine-3-carboxylate (18-2) (0.340 gm,0.981 mml) in THF: EtOH (3.4mL) was added 4N aq. NaOH solution (3.4 mL) at 0°C. The reaction mixture was stirred at 100°C for 12h. The progress of the reaction was monitored by TLC, using mobile phase:10% MeOH in DCM. The reaction mixture was evaporated under vacuum to get residue. The residue was diluted with water and aqueous layer was acidified with AcOH to pH=7, the solid precipitated. Filtered the solid, dried under vacuum. The crude product was purified by reverse phase preparative HPLC: Column LUNA (150mm X 21.2mm), 5.0µ Flow: 18mL/min; Mobile Phase: A= 0.1% HCOOH in water, B= ACN; Gradient program: (Time %B); (0, 20), (2, 30), (8, 50). The purified HPLC samples were lyophilized for 7 days to afford 7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylic acid (III - 2) (0.030g, 0.094 mmol, 9.60%) as off-white solid (S0-EE-SH3J).1H NMR (300 MHz, DMSO-d6): δ 12.05 (bs, 1H), 8.52 (s, 1H), 8.17 (s, 1H), 7.94-7.91 (m, 1H), 7.81 (s, 1H), 7.69 (bs, 2H), 7.52-7.49 (m, 2H), 7.42-7.40 (m, 2H), 2.52 (s, 3H). LC-MS (ESI): m/z = 319.1 [M+H]]+. HPLC=97.10 %; RT= 9.05 min. (method 10) Example III - 3: 7-amino-6-(5-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylic acid Step 1. ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(5-methylbenzo[b]thiophen-7- yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (19-1) To a stirred solution of 4,4,5,5-tetramethyl-2-(5-methylbenzo[b]thiophen-4-yl)-1,3,2- dioxaborolane (4-6b) (0.180 g, 0.49mmol) and ethyl 7-(bis(tert-butoxycarbonyl)amino)-6- bromopyrazolo[1,5-a]pyrimidine-3-carboxylate (1-4b) (0.265 g, 0.54mmol) in THF (5.0 mL), were added 2%TPGS(1.8 mL) and triethylamine (0.25 ml,1.96 mmol) at room temperature. Reaction mixture degassed for 20 min. Then added [1,1′-Bis(di-tert- butylphosphino)ferrocene]dichloropalladium(II) (47mg, 0.0735mmol) at room temperature and repeated the purging for another 10 minutes. The reaction was heated at 70°C for 12h, Color changed from colorless to black. The progress of the reaction was monitored by TLC, using 40% EtOAc in hexane. The reaction mixture was cooled to room temperature and diluted with water and extracted with DCM. The combined organic layer was washed with brine solution and dried with anhydrous Na2SO4 and concentrated under reduced pressure to get crude product. Crude product was purified by flash chromatography (silica column 24 g), product eluted at 25 % EtOAc in hexane. The obtained product was dissolved in MeOH, added Silia-met-DMT and stirred for 12h and then filtered over celite bed and washed with MeOH. The filtrate was concentrated to afford ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(5-methylbenzo[b]thiophen -4-yl)pyrazolo[1,5- a]pyrimidine-3-carboxylate (19-1) (0.140 g, 0.253 mmol, 51.09 %) as colorless oil. LC-MS (ESI): m/z = 453.3 [M-100H]]+. The crude products was taken to next step. Step 2. ethyl 7-amino-6-(5-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylate (19-2) To a stirred solution of ethyl 7-(bis(tert-butoxycarbonyl)amino)-6-(5- methylbenzo[b]thiophen -4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (19-1) (From step 1, 0.130 g, 0.235mmol), in DCM (5.0ml) then added TFA (1.14ml, 10v) at 00 C the reaction was maintained for 2h at room temperature. The progress of the reaction was monitored by TLC, using 50% EtOAc in hexane. The reaction mixture was concentrated under vacuum in the presence of nitrogen atmosphere to afford ethyl 7-amino-6-(5-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine- 3-carboxylate (19-2) (0.110 g, crude) as colorless oil. LC-MS (ESI): m/z = 353.1 [M+H]]+ Step 3. 7-amino-6-(5-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (III - 3) To a stirred solution of ethyl 7-amino-6-(5-methylbenzo[b]thiophen-7-yl) pyrazolo[1,5-a] pyrimidine-3-carboxylate (19-2) (0.100 g,0.28 mml) in THF: EtOH (1.0 ml) was added Aq NaOH solution (1.0 ml) and stirred the reaction mixture at 100°C for 12h. The progress of the reaction was monitored by TLC, using 10% MeOH in DCM. The reaction mixture was concentrated under vacuum, and the residue was acidified with AcOH (pH=5-6), the solid precipitated, filtered, washed with n-pentane, dried under vacuum to get crude. The crude was purified by reverse phase HPLC using Column: KINETEX (150mm x 21.2mm), 5.0µL Flow: 20ml/min, Mobile Phase: A= 0.1% HCOOH in water, B= ACN Gradient Program: (Time %B): (020), (230), (840), followed by the lyophilization of the purified sample for 48h to afford 7-amino-6-(5-methylbenzo[b]thiophen- 7-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (III - 3) (0.009 g, 0.027 mmol, 10.0%) as off-white solid.1H NMR (300 MHz, CD3OD): δ 8.51 (s, 1H), 8.31 (s, 1H) 7.77 (s, 1H), 7.55 (m, 1H).7.42- 7.40 (m, 1H), 7.29 (s, 1H), 2.54 (s, 3H). LC-MS (ESI): m/z = 325.0 [M+H]]+. HPLC: 98.49%, at RT= 3.81 min. (method 4) Intermediate 11-1a. ethyl 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylate To a stirred solution of 2-(naphthalen-1-yl)-3-oxopropanenitrile(16-1) (0.3g, 1.53mmol) in EtOH: AcOH(1:1)(8 mL) was added ethyl 5-amino-1H-pyrazole-4-carboxylate (0.262g, 1.69mmol) and stirred at 85°C for 16h. The completion of the reaction was monitored by TLC using mobile phase: 40% EtOAc in hexane. Solvents were removed under vacuum and reaction mixture was diluted with saturated NaHCO3 solution. Obtained solid was filtered and dried under vacuum to get the crude. The crude was recrystalised by using EtOAc and EtOH (10 :5 mL), heated at 85 oC for 30min. After 30 min reaction mixture was cooled and obtained solid was filtered and dried under vacuum to afford ethyl 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (11-1a) (0.255g, 0.767 mmol, 50.0%) as white solid.1H NMR: (300 MHz, DMSO-d6): δ 9.30-9.20 (bs, 2H), 8.74 (s, 1H), 8.24 (s, 1H), 8.12-8.05 (m, 2H), 7.82-7.79 (d, J=8.4 Hz,1H),7.68-7.51 (m, 4H), 4.42-4.35 (q, 2H), 1.38-1.33 (t, 3H). LC-MS (ESI): m/z = 333.2 [M+H]]+ The following intermediates 11-1b to 11-1f (Table 25) are available in an analogous manner starting from biaryl hydroxyacrylonitrile 8-1 (From Table 20) and ethyl 5-amino-1H-pyrazole-4- carboxylate. The crude products were purified by chromatography Table 25
Figure imgf000319_0001
Figure imgf000320_0001
Example III – 4. 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid To a stirred solution of ethyl 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3- carboxylate (11-1a) (0.130 g ,0.391 mmol) in EtOH: THF(1:1, 10.0 mL) was added Aq. NaOH(4M, 0.8mL), the reaction mixture was heated at 100oC for 16h. The completion of the reaction was monitored by TLC, using mobile phase: 20% EtOAc in hexane. Evaporated the reaction mixture under vacuum to dryness. Acidified with acetic acid and precipitated solid was filtered. The solid was taken in THF: MeCN and concentrated multiple times to remove residual acetic acid and dried under vacuum to afford 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (III – 4) (0.026 g, 0.079 mmol, 14.6 %) as pale-yellow solid. 1H NMR: (300 MHz, CD3OD): δ 8.50 (s, 1H), 8.20 (s, 1H), 8.03-7.97 (t, 2H), 7.65-7.47 (m, 5H). LC-MS (ESI): m/z = 305.2 [M+H]]+. HPLC: 95.35 % at RT = 5.161 min. (method 7) The following compounds III - 5 to III – 9 were prepared according to the above reaction protocol as in the Examples III - 4 using appropriate ester intermediates (From Table 25). The crude product is purified by chromatography. Table 26
Figure imgf000321_0001
Figure imgf000322_0001
Figure imgf000323_0002
Biological Methods The activity of a compound according to the present disclosure can be assessed by the following in vitro methods. Example 1: HTRF assay to measure inhibition of TET2 enzymatic activity A homogeneous time resolved energy transfer system was used to develop high- throughput and quantitative assays to measure changes in TET2-induced 5hmC levels in response to compounds. Assays were developed to assess the ability of compounds to inhibit recombinant human TET2 and TET3. Production of recombinant catalytic domain of human TET2 protein Cloning of GST-TEV-human TET2 (1129-1459-(GS)x3-1844-1925) DNA fragment encoding human TET2 (1129-1459-(GS)x3-1844-1925) was first synthesized then sub-cloned into a modified pFastBac vector that contains Glutathione-S- transferase (GST) tag and Tobacco Etch Virus Protease (TEV) cleavage site using restriction sites BamHI and XhoI (GenScript). Sequence of the synthesized DNA
Figure imgf000323_0001
Figure imgf000324_0001
Expression of GST-TEV-human TET2 (1129-1459-(GS)x3-1844-1925) The recombinant vector generated above was used to make recombinant bacmid by transforming to DH10Bac cells using standard protocols as detailed by the manufacturer (Life Technologies). High titer P3 virus was generated by transfecting the bacmid to Spodoptera frugiperda 9 (Sf9) cells and amplifying the virus using standard methods as detailed by Life Technologies. Human TET2 was expressed from Sf9 cells in log phase growth (2-2.5x106 cells/ml). The infection was allowed to proceed on the rocking incubator at 27 °C and harvested three days post infection after cell viability had dropped to 80% with an increase in the overall cell diameter consistent with infection. Cells were harvested at 4,000xg for 20 min, flash frozen and stored at –80 °C until use. Purification of human TET2 (1129-1459-(GS)x3-1844-1925) (scaled for 1L cell culture) Sf9 cells expressing recombinant human TET2 (1129-1459-(GS)x3-1844-1925) were lysed in 50mM Hepes (pH 7.5), 300mM NaCl, 1mM TCEP, 2mM MgCl2 and 200ug/ml DNAse, supplemented with protease inhibitor cocktail (Roche cOmplete EDTA-free protease inhibitor tablets, 1 tablet per 50 mL of buffer), using 40 ml lysis buffer. Cells were lysed upon thawing, homogenized and subsequently clarified by centrifugation in a JA25.50 rotor at 50,000xg for 1 hour. The clarified lysate was mixed with 2 ml Glutathione Agarose Beads (G-Biosciences) at 4oC for 1.5 hours. Bound beads were washed with ten column volume of wash buffer (50mM Hepes (pH 7.5), 300mM NaCl, 1mM TCEP) using gravity column. Then 5ml of wash buffer supplemented with 0.4 mg of His-TEV protease (produced in-house) was added to the resins and the mixture was placed in a tube and incubated overnight at 4oC for on-beads cleavage of GST tag. On the next day, the mixture was placed in a gravity column and drained, then washed three times with 2.5 ml of wash buffer each time. Flow through and washes (containing His-TEV cleaved sample) were collected and mixed with 0.25 ml of equilibrated Talon resins (Takara Bio) for 1 hour at 4oC to bind His-TEV protease. Flow through from Talon binding and subsequent washes (three washes with 0.5ml of wash buffer each time), which contains tag less human TET2 (1129-1459- (GS)x3-1844-1925), were pooled and concentrated to 3ml and loaded onto a Superdex 200, 16/600 column equilibrated in 25mM Hepes pH7.5, 150mM NaCl, 1mM TCEP. Fractions containing human TET2 (1129-1459-(GS)x3-1844-1925) were pooled and concentrated to 2-4 mg/ml, flash frozen and stored at –80 °C until used in downstream assays. Production of recombinant catalytic domain of human TET3 protein: Cloning of His-TEV-human TET3 (688-1019-(GS)x3-1501-1582) DNA fragment encoding human TET3 (688-1019-(GS)x3-1501-1582) bearing a N-terminal 6xHis tag and Tobacco Etch Virus Protease (TEV) cleavage site was synthesized and sub-cloned into pET-47b(+) vector using restriction sites NdeI and XhoI (GenScript). Sequence of the synthesized DNA
Figure imgf000325_0001
Figure imgf000326_0001
Expression of His-TEV-human TET3 (688-1019-(GS)x3-1501-1582) Human TET3 plasmid was transformed into competent BL21 (DE3) (star) cells using standard transformation protocol. E Coli strain BL21 Star™ (DE3) (ThermoFisher) cells transformed with His-TEV-human TET3 (688-1019-(GS)x3-1501-1582) plasmid were grown at 37°C in shaker flasks to an OD600 of 1.5 to 2.0 in Terrific Broth (Teknova) with 50 μg/ml of kanamycin, then lowered temperature to 16oC. Protein expression was induced by addition of isopropyl-β-D- thiogalactopyranoside (IPTG) to 0.5mM. The cells were subsequently grown overnight (14-18 hours) at 16oC, then harvested at 4000xG for 15min, flash frozen and stored at -80oC until ready for purification. Purification of human TET3 (688-1019-(GS)x3-1501-1582) The cells were thawed and resuspended in lysis buffer (800mL per cells from 12L culture) 50mM HEPES (pH 8.0), 300mM NaCl, 1mM TCEP, 100ug/ml lysozyme, 200ug/ml DNase, 2mM MgCl2, supplemented with protease inhibitor cocktail (Roche cOmplete EDTA-free protease inhibitor tablets, 1 tablet per 50 mL of buffer), then lysed by passing through a microfluidizer (M-110L, Microfluidics) once at 15k psi on ice. The lysate was cleared by centrifugation in a JA25.50 rotor at 50,000xG for 1 hour. The clarified lysate was mixed with 40ml of Talon beads (Takara Bio) equilibrated in equilibration buffer 50mM HEPES (pH 8.0), 300mM NaCl, 1mM TCEP, and 20mM Imidazole and rocked at 4oC for 1 hour. The bound beads were washed with five column volumes of equilibration buffer using a gravity column. Then the bound material (containing His-TEV- human TET3 (688-1019-(GS)3-1501-1582)) was eluted with five column volumes of elution buffer 50mM HEPES (pH 8.0), 300mM NaCl, 1mM TCEP, and 300mM Imidazole. To remove His tag, TEV protease was added to the Talon eluate at a ratio of 1mg TEV to 50mg fusion protein. The mixture was transferred to a dialysis tubing with 3500 Da molecular weight cutoff, then dialyzed against 4L of S-0 buffer 50mM Hepes (pH8.0), 1mM TCEP at 4 oC overnight. The dialyzed mixture was filtered using a 0.22 mm filter and loaded onto a HiTrap Q (GE Healthcare) equilibrated in S- 0 buffer. Following capture, the bound protein was eluted by a linear gradient elution using the same buffer supplemented with 1M NaCl (3mL/min, over 20 column volumes). Fractions containing human TET3 (688-1019-(GS)x3-1501-1582) were pooled and loaded onto a HiLoad 26/600 Superdex 200 column (GE Healthcare) equilibrated in 25 mM HEPES (pH 8.0), 150 mM NaCl, 1 mM TCEP. Fractions containing monomeric human TET3 (688-1019-(GS)x3-1501-1582) were pooled and concentrated to 1.5 mg/ml, flash frozen and stored at -80oC until used in downstream assays. Biochemical assay to assess TET2 inhibitors To assess the ability of compounds to inhibit the enzymatic function of TET2 and TET3, recombinant human TET2 protein (0.375nM) or human TET3 protein (1.56 nM) was pre-incubated with different concentrations of compound for 30 minutes (10 µM – 0.0135µM, 3-fold serial dilutions, in 384 DSF 4TiTude plates), then incubated with 25 nM substrate annealed oligonucleotides containing 5mC (Oligo#1:5’-AGACCGGAGCAAGCGAACGAGGCACTAAG- 3’forward, unmodified; Oligo#9:Biotin-CTTAGTGCCTCGTTmCGCTTGCTCCGGTCT-3’, reverse,) at 37 degrees C for 30 minutes, in buffer containing 20mM HEPES pH 6.5, 50mM NaCL, 37.5uM (NH4)2Fe(SO4)2, 15uM α-KG, 125uM Ascorbate, 1mM TCEP, 0.01% Tween. The reaction was quenched by adding solution containing EDTA (final concentration 66.6 µM), and scavenger DNA (5’- CTTAGTGCCTCGTTCGCTTGCTCCGGTCT-3’, final concentration 125µM) and DNA was denatured at 95 degrees C for 5 minutes and allowed to cool to room temperature. Conversion of 5mC to 5hmC was measured by adding HTRF detection mix containing Europium coupled anti-5hmC antibody (final concentration 0.5 nM in PBS 0.5%BSA) and streptavidin labeled with XL665 (10 nM final concentration). After 1.5-hour incubation, fluorescence was read on a Pherastar reader. Substrate solution and assay buffer controls were also read. Inhibition curves were plotted using internal software (Helios) or GraphPad Prism, and IC50 values were calculated for each compound (Representative compound inhibition curves are depicted in Figure 1). Compounds were tested in this assay for ability to inhibit enzymatic function of human TET2 and TET3. AC50 values for these compounds are shown in Table 27. Example 2: Cellular assay system to quantify effect of TET2 inhibitor compounds on TET2 enzymatic activity To assess the activity of TET2 inhibitor compounds in cells we utilized a HeLa cell line engineered to overexpress TET2 catalytic domain when treated with doxycycline. The cell line was generated using parental HeLa cells (ATCC CCL2) infected with lentivirus expressing the TET2C delta construct (human TET21130-1459 (GS)31844-1925-HA) cloned into the Lenti-X Tet-One Inducible Expression System (Puro) (Takara Bio Cat #634847). To generate the cell line, HeLa cells were seeded at 150,000 per well in 6 well cell culture plates. Lentivirus expressing the TET2 construct was added with polybrene, final concentration 10 µg/ml. Cells were then split into T 75 flasks and puromycin was added at a final concentration of 1 µg/ml. Cells were pooled and cloned under puromycin selection. pCR148 clone 12D was identified as expressing high levels of TET2 after doxycycline treatment, as measured by Western blot using anti-HA antibody (Cell Signaling Technologies). Cells were expanded in culture and frozen down in liquid nitrogen for use in cellular assay to assess TET2 inhibitor compounds. To assess the cellular activity of TET2 inhibitor compounds, on day 0, HeLa cells (pCR148 clone 12D) were seeded in 384 well plates (black, clear bottom GreinerBio cat #781091), 1000 cells per well in 30 ul of culture medium (DMEM/10%FBS/Penicillin/Streptomycin). Cells were incubated overnight at 37 degrees Celsius in the presence of 5% CO2. On day 1, TET2 overexpression was induced by adding 30 ul of fresh media containing doxycycline (final concentration 1.5 µg/ml). Control cells were mock induced by adding only media with no doxycycline. Compounds were then added to wells in triplicate in serial dilutions (3X) starting at a high dose of 30 µM, down to a low dose of 0.04 µM. Cells and compounds were incubated for 48 hours at 37 degrees C in the presence of 5% CO2. On day 3 cells were cooled to room temperature, then fixed with paraformaldehyde (final concentration 4%) for 15 minutes. Cells were then washed twice with PBS (80µl per well) and permeabilized with PBS containing 0.5% Triton-X100 for 15 minutes. Cells were again washed (1X) with PBS, then denatured with 50 ul per well of 2N HCL for 30 minutes at room temperature. After 2 additional PBS washes, 70µl per well of Tris-HCL buffer (100 mM, pH8.5) was added and incubated with cells for 10 minutes at room temperature. Cells were again washed with PBS (3X) and blocked with PBS/0.05%Tween 20/5% goat serum for 1 hour at room temperature. After removing block from all wells, 50µl per well anti-5hmC antibody (Active Motif #39791) was added, diluted 1:3000 in PBS/5% goat serum, and incubated overnight at 4 degrees Celsius. On day 4, all wells were washed 6X with PBS/0.05%Tween 20. Secondary antibody (anti- rabbit Alex487, Invitrogen #A21244) was added to all wells, diluted 1:1000 in PBS/5% goat serum, containing DAPI stain, and incubated for 1 hour at room temperature. Wells were then washed 6X with PBS/0.05% Tween20, resuspended in 50 µl PBS, and analyzed using a Phenix immunofluorescence imager. AC50 values were calculated using internal software (Helios). Values were calculated for both 5hmC detection and DAPI nuclei count (to assess viability of the cells). Representative inhibition curves are depicted in Figure 2. AC50 and qualified AC50 values for each compound were calculated using internal software and are depicted in Table 27. Example 3: Primary human T cell assay for assessment of compounds Given the effects of TET2 genetic disruption on CART cells observed in Patient 10, including enhanced memory cell and T cell stemness phenotypes, we treated primary, in vitro activated human T cells from normal healthy donors with TET2 inhibitors and assessed the impact on T cell activation, exhaustion, and memory phenotypes. We utilized a short-term activation/re-stimulation system in which T cells isolated were stimulated with anti-CD3/anti-CD28 coated beads for 4 days, then re-stimulated for an additional 3 days. Flow cytometry was performed to assess the ability of compounds to modulate expression of TIGIT, FOXP3, and TCF7 by stimulated T cells. On day 0, PBMCs were isolated from blood taken from normal healthy donors and collected into CPT tubes (BD #02-685-125). Tubes were centrifuged at 1800xG for 20 minutes at room temperature and inverted several times to mix cells. Supernatants were collected and washed once in sterile PBS. Cells were counted, and CD3+ T cells were enriched using Miltenyi Pan T cells isolation kit (#130-096-535) following the manufacturer’s instructions. Isolated T cells were resuspended in RPMI/10% FBS and added to 96-well round bottomed tissue culture plates, 80,000 cells per well cells per well in 200 µl media. Cells were stimulated with anti- CD3/28 Dynabeads (Thermo Fisher # 11132D, 1:1 bead to cell ratio) and incubated overnight at 37 degrees C, 5% CO2. On day 1, compounds were added to cells at doses 3, 1, or 0.3µM. DMSO was added to control cells. Cells were incubated an additional 3 days at 37 degrees C, 5% CO2. On day 4, cells were harvested from plates and centrifuged at 1500 rpm for 5 minutes. Cell pellets were resuspended in 10 ml PBS, and beads were removed using a magnet. Cells were then counted and resuspended in RPMI/10%FBS containing fresh compound at 3, 1, or 0.3 µM. Cells were restimulated with anti-CD3/38 beads at a 1:1 bead to cell ratio, 1,000,000 cells per well in 24 flat bottoms well plates. On day 7, cells were harvested from plates, washed in PBS, and stained for FACS analysis. Cells were pre-blocked with 20 µl of human Fc receptor binding inhibitor per well (Biolegend #422301) for 20 minutes at 4 degrees Celsius. Cells were then stained with fluorescently labeled antibodies to human CD3, CD4, CD8, TIGIT for 30 minutes at 4 degrees Celsius in FACS buffer (PBS/0.5%BSA). Cells were then washed twice with cold FACS buffer, and permeabilized with fixation/permeabilization buffer (Ebioscience #00-05523), 200 µl per sample, 45 minutes at 4 degrees Celsius. Cells were then washed twice with cold FACS buffer and stained with fluorescently labeled anti-FOXP3 and anti-TCF7 antibodies in fixation/permeabilization buffer for 45 minutes at room temperature. Cells were then washed twice with 200 µl of fixation/permeabilization buffer, resuspended in 200 µl FACS buffer, and read on an LSR Fortessa flow cytometer. Data was analyzed using Flow Jo software (TreeStar). As shown in Figures 3-8, compounds modulated expression of TIGIT, FOXP3, and TCF7 by activated human T cells. Specifically, compounds reduced the expression of TIGIT and FOXP3, and enhanced expression of TCF7, by the cells. TIGIT is a marker of exhausted T cells, while TFC7 is required for the formation of memory T cells, and its expression by tumoral T cells is associated with response to immune checkpoint blockade. FOXP3 is a marker of regulatory T cells and is known to be regulated by TET2 via demethylation of cis-regulatory elements in the Foxp3 locus (Yue, X., (2016), J Exp Med. 213: 377-97). Thus, our compounds were able to modulate human T cells in vitro to a less exhausted and terminally differentiated state and reduce the expression of a gene known to be regulated by TET2. We observed these effects in T cells isolated from the blood of multiple normal, healthy donors.
Figure imgf000331_0001
Figure imgf000332_0001
Figure imgf000333_0001
Figure imgf000334_0001
Figure imgf000335_0001
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

CLAIMS 1. A compound of Formula AA: (AA) or a pharmaceutically acceptable salt thereof, wherein Ring A is selected from a 6-10 membered aryl, 6-10 membered heteroaryl, and 6-10 membered partially saturated carbocyclyl, wherein the aryl, heteroaryl and carbocyclyl are each independently unsubstituted or substituted with 0 to 5 substituents represented by R2, R3, R4, R5, R6, R7, or R8; R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(CH2)1-6R11, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O which is unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6 alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S,which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; E is S or CR3; U is C or CR4; W is C or CR7; m is 0 or 1; and n is 0 or 1. 2. A compound of Formula I: (I) or a pharmaceutically acceptable salt thereof , wherein is a single or a double bond; R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(CH2)1-6R11, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O which is unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6 alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S,which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; E is S or CR3; U is C or CR4; W is C or CR7; m is 0 or 1; and n is 0 or 1. 3. A compound of Formula II (II) or a pharmaceutically acceptable salt thereof, wherein: ------- is a single or a double bond; R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(CH2)1-6R11, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O which is unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, or N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy (C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), CN, or a 5-membered heterocycle comprising 1,
2,
3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; W is C or CR7; and m is 0 or 1.
4. A compound according to claim 3 of Formula IIa (IIa) or a pharmaceutically acceptable salt thereof, wherein: R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, -OH, halogen, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(C1-6alkyl)R11, or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which is unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which are unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; and G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5.
5. A compound according to claim 3 of Formula IIb
(IIb) or a pharmaceutically acceptable salt thereof, wherein: R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(C1-6alkyl)R11, or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy (C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which are unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; and G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5.
6. A compound of Formula III (III) or a pharmaceutically acceptable salt thereof, wherein: ------- is a single or a double bond; R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(C1-6alkyl)R11, or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl; haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which are unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; E is S or CR3; U is C or CR4; and n is 0 or 1.
7. A compound according to claim 6 of Formula IIIa (IIIa) or a pharmaceutically acceptable salt thereof, wherein: R1 is H, NH2, or NH(CH2)2N(CH3)2; R2 is H, halogen, -OH, C1-6alkyl, haloC1-6alkyl, CH2R11, C(R11)2, O(C1-6alkyl)R11, or a 5- membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, and O, which are unsubstituted or substituted with one or more R11; R3 is H, halogen, C1-6alkyl, haloC1-6alkyl, OH, hydroxy(C1-6alkyl), a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, (C1-6alkyl)(R11)2, or OR11; R4 is H, halogen, or C1-6alkyl; R5 is H, halogen, C1-6alkyl, N(R11)2, or absent; R6 is H, Halogen, C1-6alkyl, N(R11)2, COOH, hydroxy(C1-6alkyl), or O(C1-6alkyl)R11; R7 is H, C1-6alkyl, or O(C1-6alkyl); R8 is H, C1-6alkyl, Halogen, or absent; R9 is H, COOH, C1-6alkyl, or N(R11)2; R10 is H, CN, COOH, CON(R11)2, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which are unsubstituted or substituted with one or more R11; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, O, OH, COOH, O(C1-6alkyl), or CN; each R11 is independently selected from H, Halogen, C1-6alkyl, haloC1-6alkyl, oxo, OH, COOH, O(C1-6alkyl), N(CH3)2, CN, or a 5-membered heterocycle comprising 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which is unsubstitued or substituted with oxo; A is S, N, NR8, or CR8; provided that when G is S, A is N, NR8, or CR8; G is S, N, NR5, or CR5, provided that when A is S, G is N, NR5, or CR5; and E is S or CR3.
8. The compound of Formula AA, wherein Ring A is selected from naphthyl, benzothiophenyl, tetrahydronaphthyl, or indane.
9. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein R1 is H or NH2.
10. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein R2 is H or CH3.
11. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein R9 is H or NH2.
12. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein R10 is COOH, ; ; ; ; ; ; ; ; ; and .
13. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein A is CR8.
14. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein A is S, and , G is N, NR5, or CR5.
15. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein G is CR5.
16. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein G is S, and A is N, NR8, or CR8.
17. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein E is CR3.
18. The compound of Formula (AA), (I), (II), or (III), according to any claims 2-7, wherein E is S.
19. A compound which is: 6-(benzo[b]thiophen-3-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(5-methoxybenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(5- methylbenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(8-chloronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(naphthalen-1-yl)-3-(2H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-methoxybenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(5-methylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(7-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(4-fluoronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-methylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-methylbenzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-methylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(benzo[b]thiophen-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-ethylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(7-methoxy-6-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-isopropylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(7-ethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(5,6,7,8-tetrahydronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-ethoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 4-(7-amino-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-ol; 6-(2,6-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6,7-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(3-(tert-butyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(7-(tert-butyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2,6-dimethoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(3,4-dihydronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(6-methylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(6-fluoro-2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; (1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)methanol; 1-(1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)-N,N- dimethylmethanamine; 6-(2,7-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(6-methylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 7-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)-N,N-dimethylbenzo[b]thiophen-3-amine; 6-(7-isopropylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2,6-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 2-(4-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)-3-methylnaphthalen-2-yl)-1,3,4- oxadiazole; 6-(3-((1H-pyrazol-1-yl)methyl)-2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5- a]pyrimidine; 6-(6-(tert-butyl)naphthalen-2-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(5-fluoro-2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(3-(difluoromethyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 2-((1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)oxy)acetic acid; 3-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)benzo[b]thiophene-2-carboxylic acid; 6-(3-(1H-tetrazol-5-yl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 4-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-ol; 6-(3-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-methylbenzo[b]thiophen-3-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-(methoxymethyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; (4-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)methanol; 1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-ol; 6-(4-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 1-((1-(3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)naphthalen-2-yl)methyl)pyrrolidin-2-one; 6-(5-fluoronaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-methoxy-6-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2,7-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2-methoxy-7-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2,3-dihydro-1H-inden-4-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(3-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(5-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(3-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2-amine; 6-(2-(1H-tetrazol-5-yl)benzo[b]thiophen-3-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-2- amine; 6-(2-(difluoromethyl)naphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(5-ethylbenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methoxy-7-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-fluoro-2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2,7-dimethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(8-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(benzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 7-(7-amino-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-6-yl)-6-methylbenzo[b]thiophene-2- carboxylic acid; 6-(6-fluorobenzo[b]thiophen-7-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-ethylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-isopropyl-2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methoxy-6-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(6-ethyl-2-methoxynaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidin-7-amine; 6-(2-methylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 6-(2-isopropylnaphthalen-1-yl)-3-(1H-tetrazol-5-yl)pyrazolo[1,5-a]pyrimidine; 3-(7-amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)- one; 3-(7-amino-6-(5-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(6-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(3-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)- one; 3-(7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)- one; 3-(7-amino-6-(6-methylbenzo[b]thiophen-4-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(5-methylbenzo[b]thiophen-4-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(2-(difluoromethyl)naphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(4-methylbenzo[b]thiophen-3-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol- 5(4H)-one; 3-(7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-1,2,4-oxadiazol-5(4H)-one; 7-amino-6-(2-methoxynaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(3-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(5-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(naphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(6-methylbenzo[b]thiophen-7-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(2-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(5-methylbenzo[b]thiophen-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(7-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid; 7-amino-6-(8-methylnaphthalen-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid, or a pharmaceutically acceptable salt thereof.
20. A pharmaceutical composition comprising a compound according to any one of claims 1- 19, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
21. The pharmaceutical composition of claim 20, further comprising at least one additional pharmaceutical agent.
22. The pharmaceutical composition of claims 20 or 21 for use in the treatment of a disease or disorder that is affected by the inhibition of TET2.
23. A method of inhibiting TET2 comprising administering to the patient in need thereof a compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof.
24. A method of reducing the proliferation of a cell, the method comprising contacting the cell with a compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof, and inhibition TET2.
25. A method of treating cancer comprising administering to the patient in need thereof a compound of any one of claims 1-19, or a pharmaceutically acceptable salt thereof.
26. The method of claim 25, wherein the cancer is selected from non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS).
27. The method according to claim 26, wherein the non-small cell lung cancer (NSCLC) is selected from adenocarcinoma, squamous cell carcinoma, large cell carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, and sarcomatoid carcinoma.
28. A compound according to any one of claims 1-19, or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or disorder that is affected by the inhibition of TET2.
29. Use of a compound according to any one of claims 1-19, or a pharmaceutically acceptable salt thereof for use in the treatment of a disease or disorder that is affected by the inhibition of TET2.
30. The use according to claim 29, wherein the disease or disorder is selected from non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS).
31. A compound according to any one of claims 1-19, or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for treating a disease or disorder that is affected by the inhibition of TET2.
32. The compound of claim 31, wherein the disease or disorder is selected from non-small cell lung cancer (NSCLC), liver cancer, Hepatocellular Carcinoma (HCC), head and neck cancer, esophageal cancer, uterine cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, kidney cancer, melanoma, stomach, castration-resistant prostate cancer (CRPC), gastrointestinal stromal tumor (GIST), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), Diffuse Large B-Cell Lymphoma (DLCBL), Clonal hematopoiesis of indeterminate potential (CHIP), and myelodysplastic syndrome (MDS).
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