Inhibitors of mutant RET kinase for use in the treatment of cancer - Patents.com

Abstract

Disclosed herein are methods and pharmaceutical compositions for treating a cancer associated with RET kinase activity in a subject in need of such treatment, where the cancer has developed resistance to a prior treatment and / or the subject has a solvent front mutation in RET kinase.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 256,037, filed October 15, 2021, and U.S. Provisional Patent Application No. 63 / 358,897, filed July 7, 2022, the disclosures of each of which are incorporated by reference in their entireties herein. [Background technology]

[0002] Reconstituted during transfection (RET) kinase is a transmembrane receptor protein kinase that has been implicated in certain cancers. Complex formation of glycosylphosphatidylinositol (GPI)-anchored coreceptors with glial-derived neurotrophic factor (GDNF) family ligands induces dimerization of two RET receptors and triggers transphosphorylation of specific RET tyrosine residues, which in turn activates signaling pathways that regulate cell survival, differentiation, proliferation, migration, and chemotaxis.

[0003] Activating point mutations in RET kinase have been associated with medullary thyroid carcinoma (MTC), as well as RET fusions with non-small cell lung cancer (NSCLC) and papillary thyroid carcinoma (PTC) (Liu, X. et al., Cancer Drug Resist. 2020, 3, 472-481). The oncogenic relevance of RET has prompted the development of RET-selective tyrosine kinase inhibitors (TKIs), such as selpercatinib and pralsetinib. These US Food and Drug Administration (FDA)-approved inhibitors show effective antitumor activity, but these inhibitors tend to be resistant by various RETs.

[0004] Recent X-ray crystallographic studies have provided insight into the structural basis of resistance. In one study, the multikinase inhibitor nintedanib was used to gain understanding of the interaction between TKIs and RET kinase mutations (Terzyan, SSet al. Biol. Chem. 2019, 294(27), 10428-10437). The crystal structures of the RET-nintedanib complex and mutant RET kinase (G810A) revealed that mutations in specific amino acid residues of RET that alter the hydrophobic interactions with nintedanib are closely associated with the development of resistance. In another study, crystallographic structures of RET kinase with selective inhibitors selpercatinib or pralsetinib showed that these two drugs circumvent resistance induced by gatekeeper mutations by adopting a non-conventional binding mode to RET, but both drugs are vulnerable to other mutations such as solvent front mutations of RET kinase (Subbiah, V. et al., Ann. Oncol. 2021, 32(2), 261-268).

[0005] RET G810 solvent-front mutations (G810R, G810S, G810C, and G810V) have been associated with acquired resistance to RET inhibition by selpercatinib (Solomon, BJ et al. Oncol. 2020, 15(4), 541-549). RET solvent-front mutations are also driven by off-target, RET-independent mechanisms such as MET or KRAS amplification in a study investigating specimens from selpercatinib- and pralsetinib-resistant patients (Lin JJ et al., Ann. Oncol. 2020, 31(12), 1725-1733).

[0006] Therefore, there is a need for inhibitors of RET kinase that are active against both gatekeeper and solvent front mutations. Summary of the Invention

[0007] In certain embodiments, described herein is a method of treating a cancer associated with RET kinase activity in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the cancer has developed resistance following prior treatment and / or the subject has a solvent front mutation in the RET kinase. Also provided herein is a method of inhibiting a mutant RET kinase, comprising contacting the mutant RET kinase with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0008] The following embodiments are provided:

[0009] Embodiment 1 is a method of treating a cancer associated with RET kinase activity in a subject in need thereof comprising administering to the subject an effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof, wherein the cancer has developed resistance following prior treatment and / or the subject has a solvent front mutation in RET kinase, and wherein the compound of formula (I) has the structure: [ka] During the ceremony X1 is N or CH; R1 is a C1-C6 alkyl or a C3-C6 cycloalkyl, each of which is optionally substituted by 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H or C1-C6 alkyl; R3 is halo or C1-C6 alkyl; R4 is H or halo; Q is a C1-C6 alkyl or a C3-C6 cycloalkyl, each of which is optionally substituted by 1 to 5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo.

[0010] In embodiment 2, X1 is CH; R1 is a C3-C6 alkyl optionally substituted by 1-3 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H; R3 is a halo; R4 is H; The method of embodiment 1, wherein Q is C1-C3 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 3 substituents independently selected from hydroxy and -O(C1-C3 alkyl).

[0011] In embodiment 3, R1 is isopropyl or tert-butyl; R3 is Cl; The method of embodiment 2, wherein Q is -CH3, -CH2CH2OH, -(CH2)3OCH(CH3)2, or cyclopropyl.

[0012] Embodiment 4 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0013] Embodiment 5 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0014] Embodiment 6 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0015] Embodiment 7 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0016] Embodiment 8 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0017] Embodiment 9 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0018] Embodiment 10 is the method of any one of embodiments 1 to 9, wherein the subject has previously responded to a prior treatment.

[0019] Embodiment 11 is the method of any one of embodiments 1 to 10, wherein the subject is no longer responsive to the previous treatment.

[0020] Embodiment 12 is the method of any one of embodiments 1 to 11, wherein the prior treatment is a kinase inhibitor, immunotherapy, chemotherapy, surgery, or radiation.

[0021] Embodiment 13 is the method of embodiment 12, wherein the prior treatment is a kinase inhibitor.

[0022] Embodiment 14 is the method of embodiment 13, wherein the kinase inhibitor is a selective RET kinase inhibitor or a multikinase inhibitor.

[0023] Embodiment 15 is the method of embodiment 14, wherein the selective RET kinase inhibitor is selpercatinib or pralsetinib.

[0024] Embodiment 16 is the method of embodiment 14, wherein the multikinase inhibitor is nintedanib, vandetanib, cabozantinib, lenvatinib, RXDX-105, sunitinib, sorafenib, alectinib, ponatinib, or regorafenib.

[0025] Embodiment 17 is the method according to any one of embodiments 1 to 16, wherein the cancer is a malignant neoplasm, a malignant tumor, or a solid tumor.

[0026] Embodiment 18 is the method of any one of embodiments 1 to 17, wherein the cancer is leukemia, lung cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, skin cancer, thyroid cancer, salivary gland cancer, endocrine cancer, urothelial cancer, uterine cancer, fallopian tube cancer, gastrointestinal cancer, or esophageal cancer.

[0027] Embodiment 19 is the method of embodiment 18, wherein the cancer is medullary thyroid carcinoma, non-small cell lung carcinoma, lung carcinosarcoma, lung adenocarcinoma, atypical pulmonary carcinoid, multiple endocrine neoplasia type 2, ovarian epithelial carcinoma, uterine carcinosarcoma, fallopian tube carcinoma, chronic myelomonocytic leukemia (CMML), melanoma, basal cell carcinoma, Merkel cell tumor, salivary gland carcinoma, papillary thyroid carcinoma (PTC), anaplastic thyroid carcinoma, meningioma, esophageal adenocarcinoma, gastric adenocarcinoma, ureteral urothelial carcinoma, duodenal adenocarcinoma, or colorectal adenocarcinoma.

[0028] Embodiment 20 is the method of any one of embodiments 1 to 19, wherein the resistance of the cancer is due to a solvent front mutation in RET kinase.

[0029] Embodiment 21 is the method according to any one of embodiments 1 to 20, wherein the solvent front mutation is at G810 in the amino acid sequence of RET kinase.

[0030] Embodiment 22 is the method of any one of embodiments 1 to 21, wherein the solvent front mutation is G810A, G810C, G810R, G810V, or G810S.

[0031] Embodiment 23 is the method of any one of embodiments 1 to 22, wherein the RET kinase further comprises a RET fusion translocation and / or a mutation at the RET gatekeeper residue V804.

[0032] Embodiment 24 is the method of embodiment 23, wherein the RET fusion translocation is KIF5B-RET or CCDC6-RET.

[0033] Embodiment 25 is a method for determining whether the RET gatekeeper residue V804 is a RET V804M 25. The method according to embodiment 23 or 24, wherein

[0034] Embodiment 26 is a method according to any one of embodiments 1 to 25, wherein the solvent front mutation, the RET fusion translocation, and / or the mutation at the RET gatekeeper residue V804 in the RET kinase is identified by a detection method comprising evaluating circulating tumor (cell-free) DNA and / or evaluating a tissue biopsy.

[0035] Embodiment 27 is the method of embodiment 26, wherein the detection method comprises sequencing.

[0036] Embodiment 28 is a method of inhibiting mutant RET kinase, comprising administering to the patient an effective amount of a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof; In the formula, X1 is N or CH; R1 is a C1-C6 alkyl or a C3-C6 cycloalkyl, each of which is optionally substituted by 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H or C1-C6 alkyl; R3 is halo or C1-C6 alkyl; R4 is H or halo; Q is a C1-C6 alkyl or a C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo.

[0037] Embodiment 29 is X1 is CH; R1 is a C3-C6 alkyl optionally substituted by 1-3 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H; R3 is a halo; R4 is H; The method of embodiment 28, wherein Q is C1-C3 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 3 substituents independently selected from hydroxy and -O(C1-C3 alkyl).

[0038] Embodiment 30 is R1 is isopropyl or tert-butyl; R3 is Cl; 30. The method of embodiment 29, wherein Q is -CH3, -CH2CH2OH, -(CH2)3OCH(CH3)2, or cyclopropyl.

[0039] Embodiment 31 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0040] Embodiment 32 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0041] Embodiment 33 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0042] Embodiment 34 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0043] Embodiment 35 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0044] Embodiment 36 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0045] Embodiment 37 is a method according to any one of embodiments 28 to 36, which is an in vitro method.

[0046] Embodiment 38 is a method according to any one of embodiments 28 to 36, which is an in vivo method.

[0047] Embodiment 39 is the method according to any one of embodiments 28 to 38, wherein the mutated RET kinase is due to resistance developed from a prior treatment.

[0048] Embodiment 40 is the method of embodiment 39, wherein the prior treatment is a kinase inhibitor, immunotherapy, chemotherapy, surgery, or radiation.

[0049] Embodiment 41 is the method of embodiment 40, wherein the prior treatment is a kinase inhibitor.

[0050] Embodiment 42 is the method of embodiment 41, wherein the kinase inhibitor is a selective RET kinase inhibitor or a multikinase inhibitor.

[0051] Embodiment 43 is the method of embodiment 42, wherein the selective RET kinase inhibitor is selpercatinib or pralsetinib.

[0052] Embodiment 44 is the method of embodiment 42, wherein the multikinase inhibitor is nintedanib, vandetanib, cabozantinib, lenvatinib, RXDX-105, sunitinib, sorafenib, alectinib, ponatinib, or regorafenib.

[0053] Embodiment 45 is the method of any one of embodiments 28 to 44, wherein the mutant RET kinase comprises a solvent front mutation.

[0054] Embodiment 46 is the method of embodiment 45, wherein the solvent front mutation is at G810 in the amino acid sequence of RET kinase.

[0055] Embodiment 47 is the method of embodiment 46, wherein the solvent front mutation is G810A, G810C, G810R, G810V, or G810S.

[0056] Embodiment 48 is the method of any one of embodiments 28 to 47, wherein the RET kinase further comprises a RET fusion translocation and / or a mutation at the RET gatekeeper residue V804.

[0057] Embodiment 49 is the method of embodiment 48, wherein the RET fusion translocation is KIF5B-RET or CCDC6-RET.

[0058] Embodiment 50 is a method for determining whether RET gatekeeper residue V804 is a RET V804M 50. The method of embodiment 48 or 49, wherein [Brief description of the drawings]

[0059] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0060] [Figure 1A] Figure 1 shows the % inhibition curves of Compound A against RET WT and mutant RET kinases.

[0061] [Figure 1B] 4 shows the percent inhibition curves of selpercatinib against RET WT and mutant RET kinases.

[0062] [Figure 1C] 4 shows the % inhibition curves of pralsetinib against RET WT and mutant RET kinases.

[0063] [Figure 1D] 4 shows the percent inhibition curves of staurosporine against RET WT and mutant RET kinases.

[0064] [Diagram 2] Fold change in IC50 values ​​for mutant RET kinase treated with Compound A, selpercatinib, pralsetinib, or staurosporine are shown. Data is normalized to the IC50 value of RET WT.

[0065] [Figure 3A]4 shows the % of viable cells of Ba / F3 cell line stably expressing kinesin family 5B (KIF5B)-RET with wild-type RET domain (Ba / F3 KIF5B-RET) treated with Compound A, pralsetinib, or selpercatinib.

[0066] [Figure 3B] 1 shows the % of viable cells of Ba / F3 cell line stably expressing KIF5B-RET with a G810R mutant RET domain (Ba / F3 KIF5B-RET-G810R) treated with Compound A, pralsetinib, or selpercatinib.

[0067] [Figure 3C] 1 shows the % of viable cells of Ba / F3 cell line stably expressing KIF5B-RET with a G810C mutant RET domain (Ba / F3 KIF5B-RET-G810C) treated with Compound A, pralsetinib, or selpercatinib.

[0068] [Figure 3D] 1 shows the % of viable cells of Ba / F3 cell line stably expressing KIF5B-RET with a G810S mutant RET domain (Ba / F3 KIF5B-RET-G810S) treated with Compound A, pralsetinib, or selpercatinib. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description of the Disclosure definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit any claimed subject matter. To the extent that any material incorporated herein by reference is inconsistent with the explicit content of this disclosure, the explicit content shall prevail. In this application, the use of the singular includes the plural unless expressly stated otherwise. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless the context requires otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0070] References in this specification to "some embodiments," "some embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present invention, but not necessarily in all embodiments.

[0071] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error, such as within 15%, 10%, or 5%.

[0072] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0073] "Solvent front mutation" or "solvent front mutation" refers to one or more amino acid mutations located at the solvent front of a protein (i.e., at positions on the protein exposed to the solvent). In some embodiments, the solvent front mutation causes direct steric hindrance to inhibitor binding and / or destabilizes favorable electrostatic interactions between the inhibitor and its binding site.

[0074] "Alkyl" refers to an unbranched or branched saturated hydrocarbon chain. As used herein, alkyl refers to an alkyl group having 1 to 20 carbon atoms (i.e., C1 to C6). 20 alkyl), 1 to 10 carbon atoms (i.e., C1 to C 10 alkyl), 1 to 6 carbon atoms (i.e., C1 to C6 alkyl), or 1 to 3 carbon atoms (i.e., C1 to C3 alkyl). Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When an alkyl residue having a specific number of carbons is named by a chemical name or identified by a molecular formula, all positional isomers having that number of carbons can be included. Thus, for example, "butyl" includes n-butyl (i.e., -(CH2)3CH3), isobutyl (i.e., -CH2CH(CH3)2), sec-butyl (i.e., -CH(CH3)CH2CH3), and tert-butyl (i.e., -C(CH3)3), and "propyl" includes n-propyl (i.e., -(CH2)2CH3) and isopropyl (i.e., -CH(CH3)2).

[0075] "Cycloalkyl" refers to a saturated or partially unsaturated cyclic alkyl group having a single ring or multiple rings, including fused, bridged, and spiro ring systems. The term "cycloalkyl" includes cycloalkenyl groups (i.e., cyclic groups having at least one double bond). As used herein, cycloalkyl refers to a cyclic group having 3 to 20 ring carbon atoms (i.e., C3 to C4). 20cycloalkyl), 3 to 10 ring carbon atoms (i.e., C 10 Cycloalkyl has from 1 to 6 ring carbon atoms (i.e., C3-C6 cycloalkyl). Cycloalkyl also includes "spirocycloalkyl" when there are two substitution positions on the same carbon atom. Non-limiting examples of cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Additionally, the term cycloalkyl is intended to encompass any non-aromatic ring that may be fused to an aryl ring, regardless of the attachment to the remainder of the molecule.

[0076] "Halogen" or "halo" includes fluoro, chloro, bromo and iodo.

[0077] "Hydroxy" refers to the group --OH.

[0078] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur. Also, the term "optionally substituted" refers to any one or more hydrogen atoms on a specified atom or group may or may not be replaced by a non-hydrogen moiety.

[0079] Any compound or formula described herein is intended to represent unlabeled forms of the compound as well as isotopically labeled forms. Isotopically labeled compounds have the structure shown by the formula given herein, except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine, e.g., 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N,15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I. Various isotopically labeled compounds of the present disclosure, such as 3 H, 13 C and 14 Included in the disclosure are compounds that incorporate a radioactive isotope, such as C. Such isotopically labeled compounds may be useful in detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including metabolic studies, reaction kinetic studies, drug or substrate tissue distribution assays, or in radiation treatment of patients.

[0080] The present disclosure also includes "deuterated analogs" of the compounds described herein in which 1 to n hydrogens attached to carbon atoms are replaced by deuterium, where n is the number of hydrogens in the molecule. Such compounds exhibit increased resistance to metabolism and are therefore useful for increasing the half-life of any compound when administered to a mammal, particularly a human. See, for example, Foster, "Deuterium Isotope Effects in Studies of Drug Metabolism," Trends Pharmacol.Sci.5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example, by using starting materials in which one or more hydrogens have been replaced by deuterium.

[0081] "Pharmaceutically acceptable" refers to compounds, salts, compositions, dosage forms, and other materials that are useful in preparing pharmaceutical compositions suitable for veterinary or human pharmaceutical use.

[0082] The term "pharmaceutically acceptable salt" of a given compound refers to a salt that retains the biological effectiveness and properties of the given compound and is not biologically or otherwise undesirable. "Pharmaceutically acceptable salt" includes, for example, salts with inorganic acids and salts with organic acids. Furthermore, when a compound described herein is obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, when the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, can be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art will recognize various synthetic methodologies that can be used to prepare non-toxic pharmaceutically acceptable addition salts. Pharmaceutically acceptable acid addition salts can be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Similarly, pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, such as alkylamines. Specific examples of suitable amines include, by way of example only, isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

[0083] The compounds disclosed herein, or their pharma- ceutically acceptable salts, may contain asymmetric centers and therefore give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or (D)- or (L)-, for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, e.g., chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from appropriate optically pure precursors, or resolution of the racemates (or racemates of salts or derivatives) using, e.g., chiral high-pressure liquid chromatography (HPLC).

[0084] "Tautomer" refers to alternative forms of a compound that differ in the location of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups that contain ring atoms attached to both the ring -NH- and ring =N moieties, such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole. All tautomeric forms of the compounds described herein are intended to be included.

[0085] "Stereoisomers" refer to compounds composed of the same atoms joined by the same bonds, but with different three-dimensional structures that are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0086] "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.

[0087] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.

[0088] An "effective amount" or dose of a compound or composition refers to the amount of the compound or composition that produces the intended result as desired based on the disclosure herein. Effective amounts can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including LD 50 (the dose lethal to 50% of the population) and ED 50 Examples of therapeutic options include, but are not limited to, determining the dose that is therapeutically effective in 50% of the population.

[0089] A "therapeutically effective amount" or dose of a compound or composition refers to that amount of the compound or composition that results in a reduction or inhibition of symptoms or prolongation of survival in a subject (i.e., a human patient). The result may require multiple administrations of the compound or composition.

[0090] "Treating" or "treatment" of a disease in a subject refers to 1) preventing the disease from occurring in a patient who is predisposed to the disease or who does not yet show symptoms of the disease; 2) inhibiting or arresting the development of the disease; or 3) causing the amelioration or regression of the disease. As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this disclosure, beneficial or desired results include, but are not limited to, one or more of the following: reducing one or more symptoms resulting from a disease or disorder, reducing the extent of a disease or disorder, stabilizing a disease or disorder (e.g., preventing or slowing the worsening of a disease or disorder), delaying the onset or recurrence of a disease or disorder, slowing or slowing the progression of a disease or disorder, improving the condition of a disease or disorder, providing remission (whether partial or total) of a disease or disorder, reducing the dose of one or more other drugs required to treat a disease or disorder, enhancing the effect of another drug used to treat a disease or disorder, slowing the progression of a disease or disorder, increasing the quality of life, and / or prolonging the survival of a subject. "Treatment" also encompasses the reduction of the pathological consequences of a disease or disorder. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0091] As used herein, the terms "subject" and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human, such as a primate, dog, cat, rabbit, or rodent. Neither term requires or is limited to a situation characterized by the supervision (e.g., constant or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician's assistant, janitor, or hospice worker).

[0092] As used herein, the term "pharmaceutical composition" or "medicament" refers to a composition suitable for pharmaceutical use in a subject (eg, as a RET kinase inhibitor).

[0093] Although various features of the invention may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein for clarity in the context of separate embodiments, the invention may also be implemented in a single embodiment.

[0094] compound The present disclosure provides a compound having the structure of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, In the formula, X1 is N or CH; R1 is a C1-C6 alkyl or a C3-C6 cycloalkyl, each of which is optionally substituted by 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H or C1-C6 alkyl; R3 is halo or C1-C6 alkyl; R4 is H or halo; Q is C1-C6 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo.

[0095] In some embodiments, X1 is N. In some embodiments, X1 is CH.

[0096] In some embodiments, R1 is a C1-C6 alkyl optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, R1 is a C1-C3 alkyl optionally substituted with 1-5 substituents independently selected from halo, hydroxy and -O(C1-C6 alkyl). In some embodiments, R1 is a C1-C3 alkyl optionally substituted with 1-3 substituents independently selected from halo, hydroxy and -O(C1-C6 alkyl). In some embodiments, R1 is a C1 alkyl (i.e., methyl) optionally substituted with 1-5 substituents independently selected from halo, hydroxy and -O(C1-C6 alkyl). In some embodiments, R1 is a C2 alkyl (i.e., ethyl) optionally substituted with 1-5 substituents independently selected from halo, hydroxy and -O(C1-C6 alkyl). In some embodiments, R1 is a C3 alkyl, such as n-propyl or isopropyl, optionally substituted with 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl). In some embodiments, R1 is a C4 alkyl, such as n-butyl, sec-butyl, or tert-butyl, optionally substituted with 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl). In some embodiments, R1 is a C5 alkyl, such as n-pentyl, sec-pentyl, or iso-pentyl, optionally substituted with 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl). In some embodiments, R1 is a C6 alkyl, such as n-hexyl, optionally substituted with 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl). In some embodiments, R1 is a C1-C6 alkyl optionally substituted by 1-5 substituents independently selected from -O(C1-C3 alkyl), such as hydroxy, methoxy, or ethoxy, and halo (such as F, Cl, or Br).

[0097] In some embodiments, R1 is a C3-C6 cycloalkyl optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, R1 is a C3-C6 cycloalkyl optionally substituted with 1-3 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, R1 is a C3 cycloalkyl (i.e., cyclopropyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, R1 is a C4 cycloalkyl (i.e., cyclobutyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, R1 is a C5 cycloalkyl (i.e., cyclopentyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, R1 is a C6 cycloalkyl (i.e., cyclohexyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, R1 is a C3-C6 cycloalkyl optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C3-C6 alkyl), such as methoxy or ethoxy, and halo (such as F, Cl, or Br).

[0098] In some embodiments, R2 is H. In some embodiments, R2 is a C1-C6 alkyl. In some embodiments, R2 is a C1 alkyl (i.e., methyl). In some embodiments, R2 is a C2 alkyl (i.e., ethyl). In some embodiments, R2 is a C3 alkyl, such as n-propyl or isopropyl. In some embodiments, R2 is a C4 alkyl, such as n-butyl or tert-butyl. In some embodiments, R2 is a C5 alkyl, such as n-pentyl, sec-pentyl, or iso-pentyl. In some embodiments, R2 is a C6 alkyl, such as n-hexyl.

[0099] In some embodiments, R3 is halo. In some embodiments, R3 is F, Cl, Br, or I. In some embodiments, R3 is F. In some embodiments, R3 is Br. In some embodiments, R3 is Cl. In some embodiments, R3 is I. In some embodiments, R3 is a C1-C6 alkyl. In some embodiments, R3 is a C1-C3 alkyl. In some embodiments, R3 is a C1 alkyl (i.e., methyl). In some embodiments, R3 is a C2 alkyl (i.e., ethyl). In some embodiments, R3 is a C3 alkyl, such as n-propyl or isopropyl. In some embodiments, R3 is a C4 alkyl, such as n-butyl, sec-butyl, or tert-butyl. In some embodiments, R3 is a C5 alkyl, such as n-pentyl, sec-pentyl, or iso-pentyl. In some embodiments, R3 is a C6 alkyl, such as n-hexyl.

[0100] In some embodiments, R4 is H. In some embodiments, R4 is halo, such as F, Cl, Br, or I. In some embodiments, R4 is F. In some embodiments, R4 is Cl. In some embodiments, R4 is Br. In some embodiments, R4 is I.

[0101] In some embodiments, Q is a C1-C6 alkyl optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C1-C6 alkyl optionally substituted with 1-3 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C1-C3 alkyl optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C1 alkyl (i.e., methyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C2 alkyl (i.e., ethyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C3 alkyl, such as n-propyl or isopropyl, optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl), and halo. In some embodiments, Q is a C4 alkyl, such as n-butyl, sec-butyl, or tert-butyl, optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl), and halo. In some embodiments, Q is a C5 alkyl, such as n-pentyl, sec-pentyl, or iso-pentyl, optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl), and halo. In some embodiments, Q is a C6 alkyl, such as n-hexyl, optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl), and halo. In some embodiments, Q is a C1-C6 alkyl optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C3 alkyl), and halo (e.g., F, Cl, or Br).

[0102] In some embodiments, Q is a C3-C6 cycloalkyl optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C3-C6 cycloalkyl optionally substituted with 1-3 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C3 cycloalkyl (i.e., cyclopropyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C4 cycloalkyl (i.e., cyclobutyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C5 cycloalkyl (i.e., cyclopentyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo. In some embodiments, Q is a C6 cycloalkyl (i.e., cyclohexyl) optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C1-C6 alkyl), and halo. In some embodiments, Q is a C3-C6 cycloalkyl optionally substituted with 1-5 substituents independently selected from hydroxy, -O(C3-C6 alkyl), such as methoxy or ethoxy, and halo (such as F, Cl, or Br).

[0103] In some embodiments, X1 is CH; R1 is C3-C6 alkyl optionally substituted with 1-3 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H; R3 is halo; R4 is H; and Q is C1-C3 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1-3 substituents independently selected from hydroxy and -O(C1-C3 alkyl).

[0104] In some embodiments, R1 is isopropyl or tert-butyl; R3 is Cl; and Q is -CH3, -CH2CH2OH, -(CH2)3OCH(CH3)2, or cyclopropyl.

[0105] In some embodiments, the compound of Formula (I) or a pharma- ceutically acceptable salt thereof is any one of the compounds provided in Table 1 or a pharma- ceutically acceptable salt thereof. [Table 1] TIFF2024539641000018.tif85147

[0106] In other embodiments, the compound of Formula (I) or a pharma- ceutically acceptable salt thereof is any one of the compounds provided in Table 2, or a pharma- ceutically acceptable salt thereof. [Table 2] TIFF2024539641000020.tif255137TIFF2024539641000021.tif223141TIFF2024539641 000022.tif255139TIFF2024539641000023.tif218141TIFF2024539641000024.tif70141

[0107] All compounds of formula (I) described herein, or any variation thereof, that exist in free base or acid form can be converted to their pharma- ceutically acceptable salts by treatment with an appropriate inorganic or organic base or acid by methods known to those skilled in the art. Salts of the compounds of the present disclosure can be converted to their free base or acid form by standard techniques.

[0108] Compounds of formula (I) or pharma- ceutically acceptable salts thereof can be prepared according to the procedures described in PCT Publication No. WO 2017 / 178844, the disclosure of which is incorporated herein by reference.

[0109] In some embodiments, the compounds of formula (I) or pharma- ceutically acceptable salts thereof exhibit similar potency against both wild-type RET kinase and RET kinase mutants, e.g., solvent front RET kinase mutants (e.g., G810 mutant RET kinase). In some embodiments, potency is measured by the IC 50 value, for example, as determined by the methods described in Examples 1 and 2. In some embodiments, a compound of formula (I) or a pharma- ceutically acceptable salt thereof promotes a decrease in cell viability relative to a cell line expressing both wild-type RET kinase and a RET kinase mutant, such as a cell line having a G810 mutant RET kinase. In some embodiments, cell viability or cell viability is determined using a luminescence assay as described in Example 2.

[0110] Pharmaceutical Compositions In another aspect, a pharmaceutical composition of a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided herein. Thus, the present disclosure includes a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition according to the present disclosure may be in a form suitable for oral, buccal, sublingual, parenteral (subcutaneous, intramuscular, intravenous, or intrathecal), nasal, topical, vaginal, rectal, intracerebral, intradermal, intravitreal, intraosseous injection, intraperitoneal, or inhalation administration. The pharmaceutical composition of the present disclosure comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, or excipient.

[0111] The compounds described herein can be used in preparing pharmaceutical compositions by combining the compounds as active ingredients with pharma- ceutically acceptable excipients. Some examples of materials which may function as pharma- ceutically acceptable excipients include sugars such as lactose, glucose and sucrose; starches such as corn starch, potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; surfactants such as polysorbate 80 (i.e., Tween 80); powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil, glycols, e.g., propylene glycol; polyols such as glycerin, sorbitol, mannitol, polyethylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffers such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic, compatible substances used in pharmaceutical formulations. Pharmaceutical formulations can be prepared by known pharmaceutical methods. Suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21, incorporated herein by reference. st ed.(2005).

[0112] Wetting agents, emulsifying agents and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0113] Examples of pharma- ceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0114] The pharmaceutical compositions may be conveniently provided in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the compound that produces a therapeutic effect. Generally, this amount ranges from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0115] In certain embodiments, the pharmaceutical composition of the present disclosure comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, such as bile acids, and polymeric carriers, such as polyesters and polyanhydrides; and a compound of formula (I) or a pharma- ceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition renders the compound of formula (I) or a pharma- ceutically acceptable salt thereof orally bioavailable.

[0116] Pharmaceutical compositions of the present disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or pastilles (with an inert base, such as gelatin and glycerin or sucrose and acacia), and / or mouthwashes, each containing a predetermined amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof as an active ingredient. A compound of formula (I) or a pharma- ceutically acceptable salt thereof may also be administered as a bolus, electuary, or paste.

[0117] In solid dosage forms of the present disclosure for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate and / or any of the following: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; wetting agents, such as glycerol. disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; solution retarders such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol, glycerol monostearate and non-ionic surfactants; absorbents such as kaolin, bentonite clay; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0118] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants or dispersants. Molded tablets may be produced in a suitable machine by moistening a mixture of powdered compounds with an inert liquid diluent.

[0119] Tablets and other solid dosage forms of the pharmaceutical composition of the present disclosure, such as dragees, capsules, pills and granules, can be optionally divided or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres in various proportions to provide the desired release profile. They can also be formulated for rapid release, for example, lyophilized. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents, and can be compositions that release the active ingredient only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0120] The liquid dosage form for oral administration of the compound of formula (I) or its pharma- ceutically acceptable salt includes pharma-ceutically acceptable emulsion, microemulsion, solution, suspension, syrup and elixir.In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.

[0121] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0122] Suspensions may contain, in addition to the active compounds, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0123] Pharmaceutical compositions of the disclosure for rectal or vaginal administration can be prepared by mixing one or more compounds of the disclosure with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and can be presented as a suppository that is solid at room temperature but liquid at body temperature and thus will melt in the rectal or vaginal cavity and release the active compound.

[0124] Dosage forms for topical or transdermal administration of the compounds of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound (i.e., the compound of formula (I) or a pharma- ceutically acceptable salt thereof) may be mixed under sterile conditions with a pharma- ceutically acceptable carrier and any preservatives, buffers or propellants which may be required.

[0125] The ointments, pastes, creams and gels may contain, in addition to a compound of formula (I) or a pharma- ceutically acceptable salt thereof, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

[0126] Powders and sprays can contain, in addition to the compound of formula (I) or a pharma- ceutically acceptable salt thereof, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these substances.Sprays can further contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0127] Pharmaceutical compositions of the disclosure suitable for parenteral administration comprise one or more compounds of formula (I) or pharma- ceutically acceptable salts thereof in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which can be reconstituted into sterile injectable solutions or dispersions immediately prior to use and which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0128] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0129] The pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents. Prevention of microbial action on the target compound can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenylsorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. Furthermore, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0130] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of the drug.This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility.The absorption rate of the drug then depends on its dissolution rate, which in turn depends on the crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0131] Injectable depot forms are prepared by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0132] Treatment method The compounds of formula (I) or pharma- ceutically acceptable salts thereof, and pharmaceutical compositions comprising the compounds of formula (I) or pharma- ceutically acceptable salts thereof, may be used in the methods of administration and treatment provided herein. The compounds and pharmaceutical compositions may also be used in in vitro methods, such as in vitro methods in which the compounds or pharmaceutical compositions are administered to cells for screening purposes and / or to perform quality control assays.

[0133] In one aspect, provided herein is a method of treating a cancer associated with RET kinase activity in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof, wherein the cancer has developed resistance following prior treatment and / or the subject has a solvent front mutation in RET kinase.

[0134] In another aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof for treating a subject having a cancer associated with RET kinase activity, where the cancer has developed resistance following prior treatment and / or the subject has a solvent front mutation in RET kinase.

[0135] In a further aspect, provided herein is a method of inhibiting a mutant RET kinase, comprising contacting the mutant RET kinase with an effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof. In some embodiments, the method is an in vitro method. In other embodiments, the method is an in vivo method.

[0136] Also provided herein is a compound of Formula (I) or a pharma- ceutically acceptable salt thereof, for use in the methods disclosed herein for treating a cancer associated with RET kinase activity in a subject in need thereof.

[0137] In a further aspect, provided herein is the use of a compound of formula (I) or a pharma- ceutically acceptable salt thereof for the manufacture of a medicament for the methods disclosed herein of treating a cancer associated with RET kinase activity in a subject in need thereof.

[0138] Pretreatment In some embodiments, the cancer has developed resistance following prior treatment. In some embodiments, the prior treatment is a kinase inhibitor, immunotherapy, chemotherapy, surgery, or radiation, hi some embodiments, the prior treatment is a combination of two or more of a kinase inhibitor, immunotherapy, chemotherapy, surgery, and radiation.

[0139] In some embodiments, the prior treatment is a kinase inhibitor, hi some embodiments, the kinase inhibitor is a selective RET kinase inhibitor or a multikinase inhibitor.

[0140] In some embodiments, the kinase inhibitor is a selective RET kinase inhibitor. In some embodiments, the selective RET kinase inhibitor is selpercatinib or pralsetinib. In some embodiments, the selective RET kinase inhibitor is selpercatinib. In some embodiments, the selective RET kinase inhibitor is pralsetinib.

[0141] In some embodiments, the kinase inhibitor is a multikinase inhibitor. In some embodiments, the multikinase inhibitor is nintedanib, vandetanib, cabozantinib, lenvatinib, RXDX-105, sunitinib, sorafenib, alectinib, ponatinib, or regorafenib. In some embodiments, the multikinase inhibitor is nintedanib. In some embodiments, the multikinase inhibitor is vandetanib. In some embodiments, the multikinase inhibitor is cabozantinib. In some embodiments, the multikinase inhibitor is lenvatinib. In some embodiments, the multikinase inhibitor is RXDX-105. In some embodiments, the multikinase inhibitor is sunitinib. In some embodiments, the multikinase inhibitor is sorafenib. In some embodiments, the multikinase inhibitor is alectinib. In some embodiments, the multikinase inhibitor is ponatinib. In some embodiments, the multikinase inhibitor is regorafenib.

[0142] In some embodiments, the prior treatment is immunotherapy. As used herein, immunotherapy refers to a type of treatment that utilizes a subject's immune system to fight a disease, such as cancer. Immunotherapy often involves stimulating the subject's immune system or supplementing laboratory-derived immune system mimicking components to find and attack cancer cells. In some variations, immunotherapy includes anti-PD-1 / PD-L1 antibodies and anti-CTLA-4 antibodies. In some embodiments, the prior treatment is chemotherapy. As used herein, chemotherapy refers to cancer treatments that include medicines or drugs to kill cancer cells. In some variations, chemotherapy includes doxorubicin, cisplatin, and 5-fluorouracil. In some embodiments, the prior treatment is surgery. As used herein, surgery refers to the removal of tumors and nearby tissues during surgery. Surgery is a localized treatment that affects only a portion of the body. In some variations, surgery includes open surgery and minimally invasive surgery. In some embodiments, the prior treatment is radiation. As used herein, radiation refers to the treatment of cancer with high doses of radiation to kill cancer cells and shrink tumors. In some variations, radiation includes x-rays (ie, from an external beam source) as well as brachytherapy, where radiation is placed inside the subject's body.

[0143] In some embodiments, the prior treatment is a first-line treatment. As used herein, first-line treatment refers to the first treatment given for a disease. First-line treatment is often part of a standard course of treatment, such as chemotherapy and radiation therapy after surgery. When used alone, first-line treatment is the one that is accepted as the best treatment by the treating physician. In some embodiments, the prior treatment is a second-line treatment. As used herein, second-line treatment refers to treatment given when the first treatment (first-line treatment) does not work or stops working. In some embodiments, the prior treatment is a third-line treatment. As used herein, third-line treatment refers to treatment given when both the first treatment (first-line treatment) and the subsequent treatment (second-line treatment) do not work or stop working. In some embodiments, the first-line, second-line, or third-line treatment is a kinase inhibitor, immunotherapy, chemotherapy, surgery, or radiation as described herein. In some embodiments, the prior treatment includes one or more of the prior treatments listed herein. In other embodiments, the prior treatment is limited to a single prior treatment as described herein.

[0144] Type of Cancer In some embodiments, the cancer has developed resistance following a prior treatment. In some embodiments, the cancer is a malignant neoplasm, a malignant tumor, or a solid tumor. In some embodiments, the cancer is a malignant neoplasm. In some embodiments, the cancer is a malignant tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the original cancer (e.g., tumor) is not resistant to the prior treatment, but has metastasized to a cancer that is resistant to the prior treatment.

[0145] In some embodiments, the cancer is leukemia, lung cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, skin cancer, thyroid cancer, salivary gland cancer, endocrine cancer, urothelial cancer, uterine cancer, fallopian tube cancer, gastrointestinal cancer, or esophageal cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is salivary gland cancer. In some embodiments, the cancer is endocrine cancer. In some embodiments, the cancer is urothelial cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is esophageal cancer.

[0146] In some embodiments, the cancer is medullary thyroid carcinoma, non-small cell lung carcinoma, lung carcinosarcoma, lung adenocarcinoma, atypical lung carcinoid, multiple endocrine neoplasia type 2, ovarian epithelial carcinoma, uterine carcinosarcoma, fallopian tube carcinoma, ovarian epithelial carcinoma, chronic myelomonocytic leukemia (CMML), melanoma, basal cell carcinoma, Merkel cell tumor, salivary gland carcinoma, papillary thyroid carcinoma (PTC), anaplastic thyroid carcinoma, meningioma, esophageal adenocarcinoma, gastric adenocarcinoma, ureteral urothelial carcinoma, duodenal adenocarcinoma, or colorectal adenocarcinoma. In some embodiments, the cancer is medullary thyroid carcinoma. In some embodiments, the cancer is non-small cell lung carcinoma. In some embodiments, the cancer is lung carcinosarcoma. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the cancer is atypical lung carcinoid. In some embodiments, the cancer is multiple endocrine neoplasia type 2. In some embodiments, the cancer is uterine carcinosarcoma. In some embodiments, the cancer is fallopian tube carcinoma. In some embodiments, the cancer is ovarian epithelial carcinoma. In some embodiments, the cancer is chronic myelomonocytic leukemia (CMML). In some embodiments, the cancer is melanoma. In some embodiments, the cancer is basal cell carcinoma. In some embodiments, the cancer is Merkel cell carcinoma. In some embodiments, the cancer is salivary gland carcinoma. In some embodiments, the cancer is papillary thyroid carcinoma (PTC). In some embodiments, the cancer is anaplastic thyroid carcinoma. In some embodiments, the cancer is meningioma. In some embodiments, the cancer is esophageal adenocarcinoma. In some embodiments, the cancer is gastric adenocarcinoma. In some embodiments, the cancer is ureteral urothelial carcinoma. In some embodiments, the cancer is duodenal adenocarcinoma. In some embodiments, the cancer is colorectal adenocarcinoma.

[0147] In some embodiments, the cancer shows evidence of activation and / or oncogenic RET alteration. In some embodiments, the cancer is associated with abnormal RET kinase activity. Abnormal RET kinase activity refers to a level of RET kinase activity that is significantly different (i.e., higher or lower) than the level of RET kinase activity in a subject that does not have a cancer associated with RET kinase activity or in a healthy subject (e.g., a subject that does not have cancer).

[0148] RET kinase mutations In some embodiments, the resistance caused by the cancer is due to a solvent front mutation in the RET kinase. In some embodiments, the solvent front mutation is at G810 in the amino acid sequence of the RET kinase. In some embodiments, the solvent front mutation is G810A, G810C, G810R, G810V, or G810S. In some embodiments, the solvent front mutation is G810A. In some embodiments, the solvent front mutation is G810C. In some embodiments, the solvent front mutation is G810R. In some embodiments, the solvent front mutation is G810V. In some embodiments, the solvent front mutation is G810S.

[0149] In some embodiments, the amino acid sequence of the RET kinase comprises the amino acid sequence of a wild-type RET kinase (RET WT). In some embodiments, the amino acid sequence of the RET kinase comprises the amino acid sequence of the cytoplasmic domain of RET WT. In some embodiments, the cytoplasmic domain of RET WT comprises amino acids 658-1114 of Accession No. NP_066124.1. In some embodiments, the amino acid sequence of the cytoplasmic domain of RET kinase comprises the amino acid sequence of SEQ ID NO: 1:HCYHKFAHKPPISSAEMTFRRPAQAFPVSYSSSGARRPSLDSMENQVSVDAFKILEDPKWEFPRKNLVLGKTLGEGEFGKVVKATAFHLKGRAGYTTVAVKMLKENASPSELRDLLSEFNVLKQVNHPHVIKLYGACSQDGPLLLIVEYAKYGSLRGFLRESRKVGPGYLGSGGSRNSSSLDHPDERALTMGDLISFAWQISQGMQYLAEMKL It contains the sequence of VHRDLAARNILVAEGRKMKISDFGLSRDVYEEDSYVKRSQGRIPVKWMAIESLFDHIYTTQSDVWSFGVLLWEIVTLGGNPYPGIPPERLFNLLKTGHRMERPDNCSEEMYRLMLQCWKQEPDKRPVFADISKDLEKMMVKRRDYLDLAASTPSDSLIYDDGLSEEETPLVDCNNAPLPRALPSTWIENKLYGMSDPNWPGESPVPLTRADGTNTGFPRYPNDSVYANWMLSPSAAKLMDTFDS (sequence number 1).

[0150] In some embodiments, the amino acid sequence of the RET kinase comprises at least about 80% sequence identity, e.g., at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RET kinase comprises about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RET kinase comprises the sequence of SEQ ID NO:1.

[0151] In some embodiments, the amino acid sequence of the RET kinase comprises the amino acid sequence of full-length RET WT. In some embodiments, full-length RET WT comprises amino acids 1-1114 of Accession No. NP_066124.1. In some embodiments, the amino acid sequence of the full-length RET kinase comprises the sequence of SEQ ID NO:2.

[0152] In some embodiments, the amino acid sequence of the RET kinase comprises at least about 80% sequence identity, e.g., at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the RET kinase comprises about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence of the RET kinase comprises the sequence of SEQ ID NO:2.

[0153] In some embodiments, the solvent front mutations are identified by a detection method that includes evaluating circulating tumor DNA and / or evaluating a tissue biopsy. Circulating tumor DNA is cell-free DNA that is released into the bloodstream as dead cancer cells are destroyed. In some embodiments, the circulating tumor DNA includes 200 or fewer nucleotides. In some embodiments, the solvent front mutations are identified by a detection method that includes evaluating circulating tumor DNA and evaluating a tissue biopsy. In some embodiments, the solvent front mutations are identified by a detection method that includes evaluating circulating tumor DNA or evaluating a tissue biopsy. In some embodiments, the solvent front mutations are identified by a detection method that includes evaluating circulating tumor DNA. In some embodiments, the solvent front mutations are identified by a detection method that includes evaluating a tissue biopsy.

[0154] In some embodiments, the solvent front mutations are identified by detection methods that include sequencing.

[0155] In some embodiments, the original cancer (eg, tumor) does not have a solvent front mutation, but has metastasized to a cancer that does have a solvent front mutation.

[0156] In some embodiments, the RET kinase further comprises a RET fusion translocation and / or a mutation at the RET gatekeeper residue V804. In some embodiments, the RET kinase further comprises a RET fusion translocation and a mutation at the RET gatekeeper residue V804. In some embodiments, the RET kinase further comprises a RET fusion translocation or a mutation at the RET gatekeeper residue V804. In some embodiments, the RET kinase further comprises a RET fusion translocation. In some embodiments, the RET fusion translocation is KIF5B-RET or CCDC6-RET. In some embodiments, the RET fusion translocation is KIF5B-RET. In some embodiments, the RET fusion translocation is CCDC6-RET. In some embodiments, the RET kinase further comprises a mutation at the RET gatekeeper residue V804. In some embodiments, the RET gatekeeper residue V804 is a RET V804M It is.

[0157] In some embodiments, the RET fusion translocation is identified by a detection method comprising evaluating circulating tumor DNA and / or evaluating a tissue biopsy. In some embodiments, the RET fusion translocation is identified by a detection method comprising evaluating circulating tumor DNA and evaluating a tissue biopsy. In some embodiments, the RET fusion translocation is identified by a detection method comprising evaluating circulating tumor (cell-free) DNA or evaluating a tissue biopsy. In some embodiments, the RET fusion translocation is identified by a detection method comprising evaluating circulating tumor DNA. In some embodiments, the RET fusion translocation is identified by a detection method comprising evaluating a tissue biopsy. In some embodiments, the RET fusion translocation is identified by a detection method comprising sequencing.

[0158] In some embodiments, the mutation at RET gatekeeper residue V804 in RET kinase is identified by a detection method comprising evaluating circulating tumor DNA and / or evaluating a tissue biopsy. In some embodiments, the mutation at RET gatekeeper residue V804 in RET kinase is identified by a detection method comprising evaluating circulating tumor DNA and evaluating a tissue biopsy. In some embodiments, the mutation at RET gatekeeper residue V804 in RET kinase is identified by a detection method comprising evaluating circulating tumor DNA or evaluating a tissue biopsy. In some embodiments, the mutation at RET gatekeeper residue V804 in RET kinase is identified by a detection method comprising evaluating circulating tumor DNA. In some embodiments, the mutation at RET gatekeeper residue V804 in RET kinase is identified by a detection method comprising evaluating a tissue biopsy. In some embodiments, the RET gatekeeper residue V804 in RET kinase is identified by a detection method comprising sequencing.

[0159] Dosage and Administration The compounds of formula (I) or pharma- ceutically acceptable salts thereof, or pharmaceutical compositions of formula (I) or pharma- ceutically acceptable salts thereof described herein may be administered to a subject by any suitable route of administration.

[0160] In some embodiments, the compound of formula (I) or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of formula (I) or a pharma- ceutically acceptable salt thereof described herein is administered to a subject by oral, buccal, sublingual, parenteral (subcutaneous, intramuscular, intravenous, or intrathecal), nasal, topical, vaginal, rectal, intracerebral, intradermal, intravitreal, intraosseous injection, intraperitoneal, or inhalation administration. In some embodiments, the compound of formula (I) or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of formula (I) or a pharma- ceutically acceptable salt thereof described herein is administered to a subject by oral administration. In some embodiments, the compound of formula (I) or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of formula (I) or a pharma- ceutically acceptable salt thereof described herein is administered to a subject by intravenous, subcutaneous, or intramuscular administration. In some embodiments, the compound of formula (I) or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of formula (I) or a pharma- ceutically acceptable salt thereof, described herein, is administered to the subject by intravenous administration. In some embodiments, the compound of formula (I) or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of formula (I) or a pharma- ceutically acceptable salt thereof, described herein, is administered to the subject by subcutaneous administration. In some embodiments, the compound of formula (I) or the composition described herein is administered to the subject by intramuscular administration.

[0161] Regardless of the route of administration selected, the compounds of formula (I) or a pharma- ceutically acceptable salt thereof, or pharmaceutical compositions of formula (I) or a pharma- ceutically acceptable salt thereof, are formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0162] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied for a particular patient, composition and mode of administration so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response without being toxic to the patient.

[0163] The selected dose level will depend on a variety of factors, including the activity of the particular compound of the present disclosure being used, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound being used, the age, sex, weight, condition, general health and previous medical history of the subject being treated, and similar factors well known in the medical arts. Daily, weekly or monthly doses (or other time intervals) can be used.

[0164] A physician or veterinarian having ordinary skill in the art can determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start the dosage of the compound of formula (I) or a pharma- ceutically acceptable salt thereof used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and then gradually increase the dosage until the desired effect is achieved.

[0165] In general, a suitable daily dose of a compound of the present disclosure is that amount of the compound that is the lowest dose effective to produce a therapeutic effect (e.g., inhibit mutant RET kinase). Such an effective dose will generally depend on the factors described above. In general, the dose of a compound of formula (I) or a pharma- ceutically acceptable salt thereof to a subject, when used for the indicated effect, will range from about 0.0001 to about 100 mg per kg of body weight per day. Preferably, the daily dosage will range from about 0.001 to about 50 mg of compound per kg of body weight, and even more preferably from about 0.01 to about 10 mg of compound per kg of body weight.

[0166] If desired, the effective daily dose of the active compound can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.

[0167] When the compounds of formula (I) or pharma- ceutically acceptable salts thereof are administered to humans and animals as pharmaceuticals, they can be given as such or as a pharmaceutical composition containing, for example, about 0.1% to 99.5% (more preferably, about 0.5% to 90%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0168] The compound of formula (I) or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of formula (I) or a pharma- ceutically acceptable salt thereof, can be administered once, twice, three times, or four times a day, using any suitable mode described above. Administration or treatment with the compound may also continue for several days. For example, treatment generally continues for at least about 7 days, about 14 days, or about 28 days during one cycle of treatment. Treatment cycles are well known and frequently alternate with rest periods of about 1 to 28 days, generally about 7 days or about 14 days, between cycles. In certain embodiments, treatment cycles may be continuous.

[0169] When administered orally, the total daily dose for a human subject can be from about 1 to about 1,000 mg / day, from about 1,000 to about 2000 mg / day, from about 10 to about 500 mg / day, from about 50 to about 300 mg / day, from about 75 to about 200 mg / day, or from about 100 to about 150 mg / day.

[0170] The daily dose may also be described as the total amount of the compound described herein administered per dose or per day. The daily dose of the compound may be about 1 mg to about 4000 mg, about 2000 to about 4000 mg / day, about 1 to about 2000 mg / day, about 1 to about 1,000 mg / day, about 10 to about 500 mg / day, about 20 to about 500 mg / day, about 50 to about 300 mg / day, about 75 to about 200 mg / day, or about 15 to about 150 mg / day.

[0171] In certain embodiments, the method includes administering to a subject an initial daily dose of about 1 to about 800 mg of a compound of formula (I) or a pharma- ceutically acceptable salt thereof, and increasing the dose in increments until clinical efficacy is achieved. Increments of about 5, 10, 25, 50, or 100 mg can be used to increase the dose. The dosage can be increased daily, every other day, twice weekly, or once weekly.

[0172] The compound of formula (I), or a pharma- ceutically acceptable salt thereof, or a composition described herein may be administered one or more times, for example, two, three, four, five or more times. In some embodiments, the duration of treatment is up to about 36 months, for example, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 27 months, about 30 months, about 33 months, or about 36 months. In some embodiments, the treatment period is extended further. In some embodiments, the treatment period is the entire life of the subject.

[0173] In some embodiments, a compound of formula (I), or a pharma- ceutically acceptable salt thereof, or a composition described herein is administered to a subject in need thereof about once a day, about once every two days, about once every three days, about once every four days, about once every five days, about once every six days, about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every two months, about once every three months, about once every six months, about once every nine months, or about once a year.

[0174] subject In some embodiments, a compound of formula (I) or a salt thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof, is administered to a subject having a cancer associated with RET kinase activity, the cancer having developed resistance after prior treatment, and / or the subject has a solvent front mutation in RET kinase. In some embodiments, the subject has a cancer associated with RET kinase activity, the cancer having developed resistance after prior treatment, and the subject has a solvent front mutation in RET kinase. In some embodiments, the subject has a cancer associated with RET kinase activity, the cancer having developed resistance after prior treatment, or the subject has a solvent front mutation in RET kinase. In some embodiments, the subject has a cancer associated with RET kinase activity, the cancer having developed resistance after prior treatment, or the subject has a solvent front mutation in RET kinase. In some embodiments, the subject has a cancer associated with RET kinase activity, the cancer having developed resistance after prior treatment. In some embodiments, the subject has a cancer associated with RET kinase activity, where RET kinase has a solvent front mutation.

[0175] In some embodiments, the subject has previously responded to a prior treatment. In some embodiments, the subject is no longer responding to a prior treatment.

[0176] In some embodiments, the prior treatment is a kinase inhibitor, immunotherapy, chemotherapy, surgery, or radiation. In some embodiments, the prior treatment is a kinase inhibitor. In some embodiments, the kinase inhibitor is a selective RET kinase inhibitor or a multikinase inhibitor. In some embodiments, the kinase inhibitor is a selective RET kinase inhibitor. In some embodiments, the selective RET kinase inhibitor is selpercatinib or pralsetinib. In some embodiments, the kinase inhibitor is a multikinase inhibitor. In some embodiments, the multikinase inhibitor is nintedanib, vandetanib, cabozantinib, lenvatinib, RXDX-105, sunitinib, sorafenib, alectinib, ponatinib, or regorafenib. In some embodiments, the multikinase inhibitor is nintedanib. In some embodiments, the prior treatment is an immunotherapy. In some embodiments, the prior treatment is chemotherapy. In some embodiments, the prior treatment is surgery. In some embodiments, the prior treatment is radiation.

[0177] In some embodiments, a compound of formula (I) or a salt thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof, is administered to a subject having a solvent front mutation in RET kinase. In some embodiments, the solvent front mutation is at G810 in the amino acid sequence of RET kinase. In some embodiments, the solvent front mutation is G810A, G810C, G810R, G810V, or G810S. In some embodiments, the amino acid sequence of RET kinase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the RET kinase further comprises a RET fusion translocation and / or a mutation in the RET gatekeeper residue V804. In some embodiments, the RET fusion translocation is KIF5B-RET or CCDC6-RET. In some embodiments, the RET gatekeeper residue V804 is at the RET fusion translocation. V804M It is.

[0178] In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate, dog, cat, rabbit, or rodent. In some embodiments, the subject is a primate. In some embodiments, the subject is a human. In some embodiments, the human is about 1 to about 90 years old, e.g., about 10 to about 80 years old, about 20 to about 70 years old, or about 30 to about 60 years old. In some embodiments, the human is at least about 21, 30, 40, 50, 60, 65, 70, 75, 80, or 85 years old, or any of about 21, 30, 40, 50, 60, 65, 70, 75, 80, or 85 years old. In some embodiments, the subject is a child. In some embodiments, the human is less than or about any of 21, 18, 15, 10, 5, 4, 3, 2, or 1 years old. In some embodiments, the subject is female. In some embodiments, the subject is male.

[0179] Kit / manufactured product In certain embodiments, the present specification discloses kits and articles of manufacture for use with one or more of the methods and compositions described herein. Such kits include carriers, packages, or containers that are compartmentalized to receive one or more containers, such as vials, tubes, and the like, each of the containers (several) includes one of the separate elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from various materials, such as glass or plastic.

[0180] The kit typically includes a label listing the contents and / or instructions for use, as well as a package insert with instructions for use. A set of instructions is also typically included.

[0181] In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when letters, numbers, or other symbols forming the label are attached, molded, or etched into the container itself, and the label is associated with the container when the label is present in a receptacle or carrier that also holds the container, for example, as a package insert. In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates instructions for the use of the contents, such as in the methods described herein.

[0182] In certain embodiments, the pharmaceutical compositions are provided in a pack or dispenser device containing one or more unit dosage forms containing the compound provided herein. The pack comprises, for example, metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser also includes a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the approval by the government agency of the form of the drug for administration to humans or animals. Such notice is, for example, a label approved by the U.S. Food and Drug Administration for a drug, or an approved product insert. In one embodiment, compositions containing the compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in a suitable container, and labeled for the treatment of an indicated condition.

[0183] Exemplary embodiments The present disclosure is further illustrated by the following embodiments, the features of each embodiment may be combined with any of the other embodiments where appropriate and practical.

[0184] Embodiment P1 is a method of treating a cancer associated with RET kinase activity in a subject in need thereof comprising administering to the subject an effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof, wherein the cancer has developed resistance following prior treatment and / or the subject has a solvent front mutation in RET kinase, and wherein the compound of formula (I) has the following structure: [ka] During the ceremony X1 is N or CH; R1 is a C1-C6 alkyl or a C3-C6 cycloalkyl, each of which is optionally substituted by 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H or C1-C6 alkyl; R3 is halo or C1-C6 alkyl; R4 is H or halo; Q is C1-C6 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo.

[0185] Embodiment P2 is X1 is CH; R1 is a C3-C6 alkyl optionally substituted by 1-3 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H; R3 is a halo; R4 is H; The method of embodiment P1, wherein Q is C1-C3 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 3 substituents independently selected from hydroxy and -O(C1-C3 alkyl).

[0186] Embodiment P3 is R1 is isopropyl or tert-butyl; R3 is Cl; The method of embodiment 2, wherein Q is -CH3, -CH2CH2OH, -(CH2)3OCH(CH3)2, or cyclopropyl.

[0187] Embodiment P4 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0188] Embodiment P5 is the compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0189] Embodiment P6 is the compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0190] Embodiment P7 is a compound of formula (I) [ka] or a pharma- ceutically acceptable salt thereof.

[0191] Embodiment P8 is the embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0192] Embodiment P9 is the embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0193] Embodiment P10 is the method of any one of embodiments P1 to P9, wherein the subject has previously responded to a prior treatment.

[0194] Embodiment P11 is the method of any one of embodiments P1 to P10, wherein the subject has become non-responsive to a prior treatment.

[0195] Embodiment P12 is the method of any one of embodiments P1 to P11, wherein the prior treatment is a kinase inhibitor, immunotherapy, chemotherapy, surgery, or radiation.

[0196] Embodiment P13 is the method of embodiment P12, wherein the prior treatment is a kinase inhibitor.

[0197] Embodiment P14 is the method of embodiment P13, wherein the kinase inhibitor is a selective RET kinase inhibitor or a multikinase inhibitor.

[0198] Embodiment P15 is the method of embodiment P14, wherein the selective RET kinase inhibitor is selpercatinib or pralsetinib.

[0199] Embodiment P16 is the method of embodiment P14, wherein the multikinase inhibitor is nintedanib, vandetanib, cabozantinib, lenvatinib, RXDX-105, sunitinib, sorafenib, alectinib, ponatinib, or regorafenib.

[0200] Embodiment P17 is the method of any one of embodiments P1 to P16, wherein the cancer is a malignant neoplasm, a malignant tumor, or a solid tumor.

[0201] Embodiment P18 is a method according to any one of embodiments P1 to P17, wherein the cancer is leukemia, lung cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, skin cancer, thyroid cancer, salivary gland cancer, endocrine cancer, urothelial cancer, uterine cancer, fallopian tube cancer, gastrointestinal cancer, or esophageal cancer.

[0202] Embodiment P19 is the method of embodiment P18, wherein the cancer is medullary thyroid carcinoma, non-small cell lung carcinoma, lung carcinosarcoma, lung adenocarcinoma, atypical pulmonary carcinoid, multiple endocrine neoplasia type 2, ovarian epithelial carcinoma, uterine carcinosarcoma, fallopian tube carcinoma, chronic myelomonocytic leukemia (CMML), melanoma, basal cell carcinoma, Merkel cell tumor, salivary gland carcinoma, papillary thyroid carcinoma (PTC), anaplastic thyroid carcinoma, meningioma, esophageal adenocarcinoma, gastric adenocarcinoma, ureteral urothelial carcinoma, duodenal adenocarcinoma, or colorectal adenocarcinoma.

[0203] Embodiment P20 is the method of any one of embodiments P1 to P19, wherein the resistance of the cancer is due to a solvent front mutation in RET kinase.

[0204] Embodiment P21 is the method of any one of embodiments P1 to P20, wherein the solvent front mutation is at G810 in the amino acid sequence of RET kinase.

[0205] Embodiment P22 is the method of any one of embodiments P1 to P21, wherein the solvent front mutation is G810A, G810C, G810R, G810V, or G810S.

[0206] Embodiment P23 is the method of any one of embodiments P1 to P22, wherein the RET kinase further comprises a RET fusion translocation and / or a mutation at the RET gatekeeper residue V804.

[0207] Embodiment P24 is the method of embodiment P23, wherein the RET fusion translocation is KIF5B-RET or CCDC6-RET.

[0208] Embodiment P25 is an embodiment in which the RET gatekeeper residue V804 is V804MThe method according to embodiment P23 or P24, wherein

[0209] Embodiment P26 is the method of any one of embodiments P1 to P25, wherein the solvent front mutation, the RET fusion translocation, and / or the mutation at the RET gatekeeper residue V804 in the RET kinase is identified by a detection method comprising evaluating circulating tumor (cell-free) DNA and / or evaluating a tissue biopsy.

[0210] Embodiment P27 is the method of embodiment P26, wherein the detection method comprises sequencing.

[0211] Embodiment P28 is a pharmaceutical composition for treating a subject having a cancer associated with RET kinase activity, comprising a therapeutically effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof, wherein the cancer has developed resistance following prior treatment and / or the subject has a solvent front mutation in RET kinase; [ka] During the ceremony, X1 is N or CH; R1 is a C1-C6 alkyl or a C3-C6 cycloalkyl, each of which is optionally substituted by 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H or C1-C6 alkyl; R3 is halo or C1-C6 alkyl; R4 is H or halo; Q is C1-C6 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo.

[0212] Embodiment P29 is X1 is CH; R1 is a C3-C6 alkyl optionally substituted by 1-3 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H; R3 is a halo; R4 is H; The pharmaceutical composition according to embodiment P28, wherein Q is C1-C3 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 3 substituents independently selected from hydroxy and -O(C1-C3 alkyl).

[0213] Embodiment P30 is R1 is isopropyl or tert-butyl; R3 is Cl; The pharmaceutical composition of embodiment P29, wherein Q is -CH3, -CH2CH2OH, -(CH2)3OCH(CH3)2, or cyclopropyl.

[0214] Embodiment P31 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0215] Embodiment P32 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0216] Embodiment P33 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0217] Embodiment P34 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0218] Embodiment P35 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0219] Embodiment P36 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0220] Embodiment P37 is a pharmaceutical composition according to any one of embodiments P28 to P36, wherein the subject has previously responded to a prior treatment.

[0221] Embodiment P38 is a pharmaceutical composition according to any one of embodiments P28 to P37, wherein the subject has become non-responsive to prior treatment.

[0222] Embodiment P39 is a pharmaceutical composition according to any one of embodiments P28 to P38, wherein the prior treatment is a kinase inhibitor, immunotherapy, chemotherapy, surgery, or radiation.

[0223] Embodiment P40 is the pharmaceutical composition of embodiment P39, wherein the prior treatment is a kinase inhibitor.

[0224] Embodiment P41 is the pharmaceutical composition according to embodiment P40, wherein the kinase inhibitor is a selective RET kinase inhibitor or a multikinase inhibitor.

[0225] Embodiment P42 is the pharmaceutical composition of embodiment P41, wherein the selective RET kinase inhibitor is selpercatinib or pralsetinib.

[0226] Embodiment P43 is the pharmaceutical composition of embodiment P41, wherein the multikinase inhibitor is nintedanib, vandetanib, cabozantinib, lenvatinib, RXDX-105, sunitinib, sorafenib, alectinib, ponatinib, or regorafenib.

[0227] Embodiment P44 is a pharmaceutical composition according to any one of embodiments P28 to P43, wherein the cancer is a malignant neoplasm, a malignant tumor, or a solid tumor.

[0228] Embodiment P45 is a pharmaceutical composition according to any one of embodiments P28 to P44, wherein the cancer is leukemia, lung cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, skin cancer, thyroid cancer, salivary gland cancer, endocrine cancer, urothelial cancer, uterine cancer, fallopian tube cancer, gastrointestinal cancer, or esophageal cancer.

[0229] Embodiment P46 is the pharmaceutical composition of embodiment P45, wherein the cancer is medullary thyroid carcinoma, non-small cell lung carcinoma, lung carcinosarcoma, lung adenocarcinoma, atypical pulmonary carcinoid, multiple endocrine neoplasia type 2, ovarian epithelial carcinoma, uterine carcinosarcoma, fallopian tube carcinoma, chronic myelomonocytic leukemia (CMML), melanoma, basal cell carcinoma, Merkel cell tumor, salivary gland carcinoma, papillary thyroid carcinoma (PTC), anaplastic thyroid carcinoma, meningioma, esophageal adenocarcinoma, gastric adenocarcinoma, ureteral urothelial carcinoma, duodenal adenocarcinoma, or colorectal adenocarcinoma.

[0230] Embodiment P47 is a pharmaceutical composition according to any one of embodiments P28 to P46, wherein the resistance of the cancer is due to a solvent front mutation in RET kinase.

[0231] Embodiment P48 is a pharmaceutical composition according to any one of embodiments P28 to P47, wherein the solvent front mutation is at G810 in the amino acid sequence of RET kinase.

[0232] Embodiment P49 is a pharmaceutical composition according to any one of embodiments P28 to P48, wherein the solvent front mutation is G810A, G810C, G810R, G810V, or G810S.

[0233] Embodiment P50 is a pharmaceutical composition according to any one of embodiments P28 to P49, wherein the RET kinase further comprises a RET fusion translocation and / or a mutation in the RET gatekeeper residue V804.

[0234] Embodiment 51 is a pharmaceutical composition according to embodiment P50, wherein the RET fusion translocation is KIF5B-RET or CCDC6-RET.

[0235] Embodiment P52 is an embodiment in which the RET gatekeeper residue V804 is V804M The pharmaceutical composition according to embodiment P50 or P51,

[0236] Embodiment P53 is a pharmaceutical composition according to any one of embodiments P28 to P52, wherein the solvent front mutation, the RET fusion translocation, and / or the mutation at the RET gatekeeper residue V804 in the RET kinase is identified by a detection method comprising evaluating circulating tumor DNA and / or evaluating a tissue biopsy.

[0237] Embodiment P54 is the pharmaceutical composition according to embodiment P53, wherein the detection method comprises sequencing.

[0238]

[0033] Embodiment P55 is a method of inhibiting mutant RET kinase comprising administering to the patient an effective amount of a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof; X1 is N or CH; R1 is a C1-C6 alkyl or a C3-C6 cycloalkyl, each of which is optionally substituted by 1-5 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H or C1-C6 alkyl; R3 is halo or C1-C6 alkyl; R4 is H or halo; Q is C1-C6 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 5 substituents independently selected from hydroxy, -O(C1-C6 alkyl) and halo.

[0239] Embodiment P56 is X1 is CH; R1 is a C3-C6 alkyl optionally substituted by 1-3 substituents independently selected from halo, hydroxy, and -O(C1-C6 alkyl); R2 is H; R3 is a halo; R4 is H; The method of embodiment P55, wherein Q is C1-C3 alkyl or C3-C6 cycloalkyl, each of which is optionally substituted with 1 to 3 substituents independently selected from hydroxy and -O(C1-C3 alkyl).

[0240] Embodiment P57 is R1 is isopropyl or tert-butyl; R3 is Cl; The method of embodiment P56, wherein Q is -CH3, -CH2CH2OH, -(CH2)3OCH(CH3)2, or cyclopropyl.

[0241] Embodiment P58 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0242] Embodiment P59 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0243] Embodiment P60 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0244] Embodiment P61 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0245] Embodiment P62 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0246] Embodiment P63 is an embodiment in which the compound of formula (I) is [ka] or a pharma- ceutically acceptable salt thereof.

[0247] Embodiment P64 is the method of any one of embodiments P55 to P63, wherein the method is an in vitro method.

[0248] Embodiment P65 is the method of any one of embodiments P55 to P63, wherein the method is an in vivo method.

[0249] Embodiment P66 is the method of any one of embodiments P55 to P65, wherein the mutated RET kinase is due to resistance developed from a prior treatment.

[0250] Embodiment P67 is the method of embodiment P66, wherein the prior treatment is a kinase inhibitor, immunotherapy, chemotherapy, surgery, or radiation.

[0251] Embodiment P68 is the method of embodiment P67, wherein the prior treatment is a kinase inhibitor.

[0252] Embodiment P69 is the method of embodiment P68, wherein the kinase inhibitor is a selective RET kinase inhibitor or a multikinase inhibitor.

[0253] Embodiment P70 is the method of embodiment P69, wherein the selective RET kinase inhibitor is selpercatinib or pralsetinib.

[0254] Embodiment P71 is the method of embodiment P69, wherein the multikinase inhibitor is nintedanib, vandetanib, cabozantinib, lenvatinib, RXDX-105, sunitinib, sorafenib, alectinib, ponatinib, or regorafenib.

[0255] Embodiment P72 is the method of any one of embodiments P55 to P71, wherein the mutant RET kinase comprises a solvent front mutation.

[0256] Embodiment P73. The method of embodiment P72, wherein the solvent front mutation is at G810 in the amino acid sequence of RET kinase.

[0257] Embodiment P74 is the method of embodiment P73, wherein the solvent front mutation is G810A, G810C, G810R, G810V, or G810S.

[0258] Embodiment P75 is the method of any one of embodiments P55 to P74, wherein the RET kinase further comprises a RET fusion translocation and / or a mutation at RET gatekeeper residue V804.

[0259] Embodiment 76 is the method of embodiment P75, wherein the RET fusion translocation is KIF5B-RET or CCDC6-RET.

[0260] Embodiment P77 is an embodiment in which the RET gatekeeper residue V804 is V804M The method of embodiment P75 or P76, wherein EXAMPLES

[0261] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0262] The following abbreviations may be relevant to this application: Abbreviation ATP: Adenosine triphosphate CSK: C-terminal Src kinase DMSO: Dimethyl sulfoxide DTT: Dithiothreitol FBS: fetal bovine serum. h: time MSA: Mobility shift assay PBS: Phosphate-buffered saline TKI: Tyrosine kinase inhibitor WT: wild type

[0263] Example 1. In vitro RET kinase enzyme assay. material. Wild-type human RET kinase (RET WT; cytoplasmic domain (amino acids 658-1114 of accession no. NP_066124.1) was expressed as an N-terminal GST fusion protein (79 kDa) in a baculovirus expression system and purified using glutathione sepharose chromatography.

[0264] Human RET kinase G810 mutants (cytoplasmic domain, amino acids 658-1114 of accession number NP_066124.1, mutants [G810C], [G810R] or [G810S]) were expressed as N-terminally DYKDDDK-tagged (SEQ ID NO: 3) biotinylated proteins (56 kDa) in a baculovirus expression system and activated with ATP. The proteins were purified using DYKDDDK-tag (SEQ ID NO: 3) antibody agarose and activated with ATP. The activated proteins were purified using gel filtration.

[0265] A 2x kinase solution (containing 2mM RET kinase) was prepared using assay buffer containing 20mM HEPES pH 7.5, 0.01% Triton X-100, and 1mM DTT.

[0266] A 4x substrate / ATP / metal solution was prepared using kit buffer containing 20 mM HEPES pH 7.5, Triton X-100, and 5 mM DTT such that the final 1x substrate / ATP / metal solution was 1 / 1000 / 5000 μM. The substrate was a synthetic peptide CSKtide peptide conjugated to a fluorophore and the metal was MgCl2.

[0267] Compound 2-(4-amino-1-tert-butyl-pyrazolo[3,4-d]pyrimidin-3-yl)-3-chloro-N-methyl-1H-indole-6-carboxamide (referred to herein as "Compound A") was prepared according to Example 4 of PCT Publication No. WO 2017 / 178844. Compounds selpercatinib, pralsetinib, and staurosporine, all of which are TKIs, were obtained from commercial sources. Solutions of Compound A, selpercatinib, pralsetinib, and staurosporine were each prepared in DMSO at concentrations 100-fold higher than the reaction conditions and further diluted 25-fold with assay buffer for 4× compound solutions. Final 1× concentrations of Compound A, selpercatinib, and pralsetinib were 10 μM to 0.3 nM. Final 1× concentrations of staurosporine were 0.1 μM to 0.003 nM (RET WT) or 10 μM to 0.3 nM (RET G810C, G810R, G810S).

[0268] procedure. 5 μL of 4× compound solution, 5 μL of 4× substrate / ATP / metal solution, and 10 μL of 2× kinase solution were mixed in each well of a 384-well plate. After 1 h incubation at room temperature, 70 μL of Termination Buffer (QuickScout Screening Assist MSA; Carna Biosciences) was added to each well. Tyrosine kinase activity was measured using an off-chip mobility shift assay (MSA) by applying the reaction mixture to a LabChip™ system (Perkin Elmer) and quantifying product (P) and substrate (S) peptide peaks. Kinase reactions were evaluated based on the product ratio (P / (P+S)) calculated from the peak intensities.

[0269] The percent inhibition of each test solution was calculated by setting the reaction control (complete reaction mixture for each kinase without TKI) readout as 0% inhibition and the background (reaction mixture without enzyme) readout as 100% inhibition. IC 50Values ​​were calculated from concentration versus % inhibition curves by fitting to a four-parameter logistic curve.

[0270] result. Activity data (IC) of test compounds against wild-type RET kinase (RET WT) and mutant RET kinases 50 ) are shown in Table 3 below. [Table 3]

[0271] The inhibition curves of each test compound against RET WT and mutant RET kinase are shown in Figures 1A to 1D.

[0272] IC for each compound tested against mutant RET kinase, normalized to RET WT 50 The fold change in values ​​is shown in FIG.

[0273] Overall, the data demonstrate that compound A exhibits consistent efficacy against both the RET WT and G810 mutant RET kinases tested. In contrast, two clinically approved TKIs, selpercatinib and pralsetinib, as well as the widely used agent staurosporine, all showed significantly reduced inhibitory activity against mutant RET kinase, with IC 50 The values ​​increased approximately 10-100 fold.

[0274] Example 2. In vitro cell viability assay. material. The following reagents were obtained commercially from the indicated suppliers: RPMI 1640 medium (Hyclone), FBS (Gibco), PBS (Solarbio), DMSO (Sigma), and CellTiter-Glo® Luminescent Cell Viability Assay (Vazyme). The CellTiter-Glo® Luminescent Cell Viability Assay is a homogenous method for determining the number of viable cells in culture based on quantification of the amount of ATP present, which is directly proportional to the number of viable metabolically active cells. The homogenous "add-mix-measure" format results in cell lysis and the generation of a luminescent signal that is proportional to the amount of ATP present.

[0275] The Ba / F3 mouse pro-B cell line transduced with the KIF5B-RET fusion gene was used in this study. The KIF5B-RET fusion gene, a result of gene rearrangement, is expressed in a subset of cancers, including non-small cell lung cancer, and consists of the 5' portion of the KIF5B gene fused in frame to the 3' portion of the RET gene (encoding the RET kinase domain). Ba / F3 cells stably expressing KIF5B-RET fusion proteins in which the RET domain was either wild type or had a mutation at G810 (G810R, G810C, G810S) were used for these experiments. Ba / F3 KIF5B-RET (wild-type RET kinase, cell line number KC-1041), Ba / F3 KIF5B-RET-G810R (G810R mutant RET kinase, cell line number KC-1623), Ba / F3 KIF5B-RET-G810C (G810C mutant RET kinase, cell line number KC-1623), and Ba / F3 KIF5B-RET-G810S (G810S mutant RET kinase, cell line number KC-1556) were cultured in RPMI 1640 medium supplemented with 10% FBS.

[0276] Compound A, described in Example 1, and two clinically approved TKIs, pralsetinib and selpercatinib, were used in this study.

[0277] Procedure (cytotoxicity and IC 50 ). Cells were harvested in logarithmic growth phase and counted using a hemocytometer. Cell viability was greater than 90% by trypan blue assay. Cell concentration was adjusted with culture medium and cell suspension was added to two 96-well plates to obtain a final cell density of 3000 cells / well. Plates were incubated at 37°C and 5% CO2 in a humidified incubator.

[0278] Stock solutions of compound A, pralsetinib, and selpercatinib were prepared in DMSO. Cells were incubated with nine concentrations of each compound (3.16-fold serial dilutions) ranging from 10 μM to 1 nM. The final concentration of DMSO in the culture medium was 0.1% [v / v].

[0279] The plates were cultured for 3 days. On the third day, CellTiter-Glo® reagent was added to each well and the contents of each well were mixed on an orbital shaker for 5 minutes to induce cell lysis. The cell plates were incubated at room temperature for 20 minutes to stabilize the luminescence signal, and luminescence was recorded using a multimode microplate reader.

[0280] Using GraphPad Prism 7.0, IC 50 Values ​​were calculated. Graph curves were fitted using a nonlinear regression model with a sigmoidal dose-response.

[0281] The cell viability was determined according to formula (1). Cell viability (%)=(Lum 試験化合物 -Lum 培地対照 ) / (Lum ビヒクル対照 -Lum 培地対照 )(Formula 1) Lum = Luminescence Test Compound = Test Compound (targeting RET kinase)

[0282] result. IC of test compounds against Ba / F3 KIF5B-RET cell lines harboring wild-type RET or mutant RET kinase domains 50 The data is shown in Table 4 below. [Table 4]

[0283] The cell viability curves of each of the test compounds against the Ba / F3 KIF5B-RET cell line harboring wild-type RET or a mutant RET kinase domain are shown in Figures 3A-3D.

[0284] The data demonstrate that compound A is highly potent against Ba / F3 cells expressing KIF5B-RET with the wild-type RET domain as well as the G810 RET mutant, with IC that was largely unperturbed by the presence of the G810 mutation. 50 In contrast, selpercatinib and pralsetinib had IC 50 values, indicating increased cell viability in mutant RET kinase cell lines treated with the two approved compounds. Notably, selpercatinib IC 50 The IC value increased 8-44 fold, and pralsetinib IC 50 The values ​​increased by 3 to 44 fold. These results are consistent with the results of the in vitro RET kinase enzyme assay in Example 1.

[0285] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in carrying out the present invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims

[Claim 1] The novel products, methods and processes substantially as herein described.

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