Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

• the present invention relates to compounds that inhibit the KRAS G12C and KRAS G12D proteins; methods of treating diseases or conditions, such as cancer, using the compounds; and pharmaceutical compositions containing the compounds.
• KRAS gene mutations are common in pancreatic cancer, lung adenocarcinoma, colorectal cancer, gall bladder cancer, thyroid cancer, and bile duct cancer. KRAS mutations are also observed in about 13% of patients with NSCLC, and some studies have indicated that KRAS mutations are a negative prognostic factor in patients with NSCLC. Recently, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations have been found to confer resistance to epidermal growth factor receptor (EGFR) targeted therapies in colorectal cancer; accordingly, the mutational status of KRAS can provide important information prior to the prescription of TKI therapy.
• EGFR epidermal growth factor receptor
• pancreatic cancer lung adenocarcinoma, or colorectal cancer, especially those who have been diagnosed to have such cancers characterized by a KRAS mutation, and including those who have progressed after chemotherapy.
• the compounds disclosed herein can be in the form of a pharmaceutically acceptable salt.
• the compounds provided can be formulated into a pharmaceutical composition comprising a compound disclosed herein and a pharmaceutically acceptable excipient.
• KRAS G12C is contacted in a cell, comprising contacting the cell with a compound or composition disclosed herein.
• a method of inhibiting KRAS G12D in a cell comprising contacting the cell with a compound or composition disclosed herein.
• the KRAS G12D mutation arises from a single nucleotide change (c.35G>A) and results in an amino acid substitution of the glycine (G) at position 12 by an aspartic acid (D).
• KRAS G12D is present in 3.99% of AACR GENIE cases, with colorectal adenocarcinoma, pancreatic exocrine neoplasm, non-small cell lung carcinoma, uterine corpus neoplasm, and ovarian neoplasm having the greatest prevalence. Further provided is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a compound or composition disclosed herein. In some embodiments, the cancer is lung cancer, pancreatic cancer, or colorectal cancer.
• One aspect of the present invention provides various compounds, stereoisomers, atropisomers, pharmaceutically acceptable salts, pharmaceutically acceptable salts of the stereoisomers, and pharmaceutically acceptable salts of the atropisomers as described in the embodiments set forth below.
• Another aspect of the present invention provides a pharmaceutical composition that includes the compound of any of the embodiments or the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
• Another aspect of the present invention provides a method of treating cancer.
• Such methods include: administering to a patient in need thereof a therapeutically effective amount of the compound of any of the embodiments or a pharmaceutically acceptable salt thereof.
• the cancer is a hematologic malignancy.
• the cancer is selected from the group consisting of breast cancer, colorectal cancer, skin cancer, melanoma, ovarian cancer, kidney cancer, lung cancer, non-small cell lung cancer, lymphoma, non- Hodgkin's lymphoma, myeloma, multiple myeloma, leukemia, and acute myelogenous leukemia.
• the cancer is multiple myeloma.
• the cancer is acute myelogenous leukemia. In some other such methods, the cancer is non- Hodgkin's lymphoma.
• the method further includes administering to a patient in need thereof a therapeutically effective amount of an additional pharmaceutically active compound.
• the additional pharmaceutically active compound is pembrolizumab.
• the additional pharmaceutically active compound is nivolumab.
• the additional pharmaceutically active compound is trametinib.
• the additional pharmaceutically active compound is RMC-4630. In still other such methods, the additional pharmaceutically active compound is AMG 404.
• alkyl refers to straight chained and branched C1-C8 hydrocarbon groups, including but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethybutyl.
• alkyl group has “m” to “n” carbon atoms.
• alkylene refers to an alkyl group having a substituent.
• An alkyl (e.g., methyl), or alkylene (e.g., -CH2-), group can be substituted with one or more, and typically one to three, of independently selected, for example, halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, -NC, amino, - CO2H, -CO2C1-C6alkyl, -OCO C1-C6alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C5-C10aryl, and C5-C10 heteroaryl.
• haloalkyl specifically refers to an alkyl group
• alkenyl and alkynyl indicate an alkyl group that further includes a double bond or a triple bond, respectively.
• halo refers to fluoro, chloro, bromo, and iodo.
• alkoxy is defined as -OR, wherein R is alkyl.
• amino or “amine” interchangeably refers to a -NR2 group, wherein each R is, e.g., H or a substituent.
• the amino group is further substituted to form an ammonium ion, e.g., NR3+.
• Ammonium moieties are specifically included in the definition of “amino” or “amine.”
• Substituents can be, for example, an alkyl, alkoxy, cycloalkyl, heterocycloalkyl, amide, or carboxylate.
• An R group may be further substituted, for example, with one or more, e.g., one to four, groups selected from halo, cyano, alkenyl, alkynyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, urea, carbonyl, carboxylate, amine, and amide.
• An “amide” or “amido” group interchangeably refers to a group similar to an amine or amino group but further including a C(O), e.g., -C(O)NR 2 .
• Some contemplated amino or amido groups include CH 2 NH 2 , CH(CH 3 )NH 2 , CH(CH 3 ) 2 NH 2 , CH 2 CH 2 NH 2 ,
• aryl refers to a C6-14 monocyclic or polycyclic aromatic group, preferably a C6-10 monocyclic or bicyclic aromatic group, or 00-14 polycyclic aromatic group.
• aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl.
• Aryl also refers to C10-14 bicyclic and tricyclic carbon rings, where one ring is aromatic and the others are saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl).
• an aryl group can be unsubstituted or substituted with one or more, and in particular one to four, groups independently selected from, for example, halo, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, -CF3, -OCF3, -NO2, -CN, -NC,
• cycloalkyl refers to a monocyclic or polycyclic non- aromatic carbocyclic ring, where the polycyclic ring can be fused, bridged, or spiro.
• the carbocyclic ring can have 3 to 10 carbon ring atoms.
• Contemplated carbocyclic rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclononyl.
• heterocycloalkyl means a monocyclic or polycyclic (e.g., bicyclic), saturated or partially unsaturated, ring system containing 3 or more (e.g., 3 to 12, 4 to 10, 4 to 8, or 5 to 7) total atoms, of which one to five (e.g., 1, 2, 3, 4, or 5) of the atoms are independently selected from nitrogen, oxygen, and sulfur.
• heterocycloalkyl groups include azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, dihydropyrrolyl, morpholinyl, thiomorpholinyl, dihydropyridinyl, oxacycloheptyl, dioxacycloheptyl, thiacycloheptyl, and diazacycloheptyl.
• a cycloalkyl or heterocycloalkyl group can be unsubstituted or substituted with one or more, and in particular one to four, groups.
• substituents include halo, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, -OCF3, -NO2, - CN, -NC, -OH, alkoxy, amino, -CO2H, -CO2C1-C6alkyl, -OCOC1-C6alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C5-C10aryl, and C5-C10 heteroaryl.
• heteroaryl refers to a monocyclic or polycyclic ring system (for example, bicyclic) containing one to three aromatic rings and containing one to four (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur in an aromatic ring.
• the heteroaryl group has from 5 to 20, from 5 to 15, from 5 to 10 ring, or from 5 to 7 atoms.
• Heteroaryl also refers to C10-14 bicyclic and tricyclic rings, where one ring is aromatic and the others are saturated, partially unsaturated, or aromatic.
• heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, triazolyl, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofur
• a heteroaryl group can be unsubstituted or substituted with one or more, and in particular one to four or one or two, substituents.
• Contemplated substituents include halo, C1-6alkyl, C2- 6alkenyl, C2-6alkynyl, -OCF3, -NO2, -CN, -NC, -OH, alkoxy, amino, -CO2H, -CO2C1- C6alkyl, -OCOC1-C8alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C5-C10aryl, and C5- C10 heteroaryl.
• Boc refers to the structure o
• trifluoroacetamide refers to the structure
• trityl refers to the structure
• tosyl refers to the structure o
• Troc refers to the structure
• Teoc refers to the structure o
• Alloc refers to the structure
• Fmoc refers to the structure
• the compounds disclosed herein include all pharmaceutically acceptable isotopically- labeled compounds wherein one or more atoms of the compounds disclosed herein are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
• isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N,
• radiolabelled compounds could be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action.
• Certain isotopically-labeled compounds of the disclosure for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies.
• the radioactive isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
• substitution with heavier isotopes such as deuterium, i.e. 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence are preferred in some circumstances.
• Substitution with positron emitting isotopes such as 11C, 18F, 150 and 13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
• PET Positron Emission Topography
• Isotopically-labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Preparations and Examples as set out below using an appropriate isotopically- labeled reagent in place of the non-labeled reagent previously employed.
• Isotopically-labeled compounds as disclosed herein can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples and schemes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.
• Certain of the compounds as disclosed herein may exist as stereoisomers (i.e., isomers that differ only in the spatial arrangement of atoms) including optical isomers and conformational isomers (or conformers).
• the compounds disclosed herein include all stereoisomers, both as pure individual stereoisomer preparations and enriched preparations of each, and both the racemic mixtures of such stereoisomers as well as the individual diastereomers and enantiomers that may be separated according to methods that are known to those skilled in the art. Additionally, the compounds disclosed herein include all tautomeric forms of the compounds.
• Certain of the compounds disclosed herein may exist as atropisomers, which are conformational stereoisomers that occur when rotation about a single bond in the molecule is prevented, or greatly slowed, as a result of steric interactions with other parts of the molecule.
• the compounds disclosed herein include all atropisomers, both as pure individual atropisomer preparations, enriched preparations of each, or a non-specific mixture of each. Where the rotational barrier about the single bond is high enough, and interconversion between conformations is slow enough, separation and isolation of the isomeric species may be permitted.
• groups such as, but not limited to, the following RIO groups, may exhibit restricted rotation.
• the present invention comprises a compound having a structure of formula (I) wherein
• R 1 is H, halo, or -CH 3 ;
• R 2 is H, halo, or -CH 3 ;
• b is optionally a single or a double bond;
• ring A is a monocyclic 4-7 membered ring or a bicyclic, bridged, fused, or spiro 6-11 membered ring;
• L is a bond or NR4
• R 5 is H, halo, an -C 1-6 alkyl, -C2-6alkynyl, -C 1-6 alkylene-O-C 1-4 alkyl, -C 1-
• R 5a is selected from H, -C 1-6 alkyl, -C 2-6 alkynyl, -C 1-6 alkylene-O-C 1-4 alkyl, -C 1- 6 alkylene-OH, -C 1-6 haloalkyl, -C 1-6 alkyleneamine, -C 0-6 alkylene-amide, -C(O)OH, -C(O)OC 1- 4alkyl, -C 0-6 alkylene-O-C 6-14 aryl, -C 0-3 alkylene-C(O)C 1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C 0-3 alkylene-C 3-14 cycloalkyl, -C 0-3 alkylene-C 2 - 14 heterocycloalkyl, -C 0-3 alkylene-C 6-14 aryl, or -C 0-3 alkylene-C 2-14 heteroaryl;
• R 5b is selected from H, -C 1-6 alkyl, -C 2-6 alkynyl, -C 1-6 alkylene-O-C 1-4 alkyl, -C 1- 6 alkylene-OH, -C 1-6 haloalkyl, -C 1-6 alkyleneamine, -C 0-6 alkylene-amide, -C(O)OH, -C(O)OC 1- 4alkyl, -C 0-6 alkylene-O-C 6-14 aryl, -C 0-3 alkylene-C(O)C 1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C 0-3 alkylene-C 3-14 cycloalkyl, -C 0-3 alkylene-C 2 - 14 heterocycloalkyl, -C 0-3 alkylene-C 6-14 aryl, or -C 0-3 alkylene-C 2-14 heteroaryl; or
• R 6 is H, halo, -C 1-6 alkyl, -C 2-6 alkynyl, -C 1-6 alkylene-O-C 1-4 alkyl, -C 1-6 alkylene- OH, -C 1-6 haloalkyl, -C 1-6 alkyleneamine, -C 0-6 alkylene-amide, -C(O)OH, -C(O)OC 1-4 alkyl, -C 0-6 alkylene-O-C 6-14 aryl, -C 0-3 alkylene-C(O)C 1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C 0-3 alkylene-C 3-14 cycloalkyl, -C 0-3 alkylene-C 2-14 heterocycloalkyl, -C 0-3 alkylene-C 6- 14 aryl, or -C 0-3 alkylene-C 2-14 heteroaryl;
• R 5a and R 6a together with the atoms to which they are attached, may form a 3-6 membered ring that optionally includes one or two heteroatoms selected from O, S or N; or
• R 5a and R 6a are absent when b is a double bond
• R 6a is H, or -C 1-6 alkyl
• R 6b is H, -C 1-6 alkyl, -C 2-6 alkynyl, -C 1-6 alkylene-O-C 1-4 alkyl, -C 1-6 alkylene-OH, - C 1-6 haloalkyl, -C 1-6 alkyleneamine, -C 0-6 alkylene-amide, -C(O)OH, -C(O)OC 1-4 alkyl, -C 1-6 alkylene-O-aryl, -C 0-3 alkylene-C(O)C 1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C 0-3 alkylene-C 3-14 cycloalkyl, -C 0-3 alkylene-C 2-14 heterocycloalkyl, -C 0-3 alkylene-C 6- 14 aryl, -C 0-3 alkylene-C 2-14 heteroaryl, or cyano; or R 6a and R
• R 7 is H or C 1-8 alkyl
• R 8 is H, OH, NR a R b ; wherein R a and R b are each independently H, halo, -C 1-6 alkyl, -C 2-6 alkynyl; wherein the ring A or the -C 1-6 alkyl, -C 2-6 alkynyl, -C 1-6 alkylene-O-C 1-4 alkyl, -C 1- 6 alkylene-OH, -C 1-6 haloalkyl, -C 1-6 alkyleneamine, -C 0-6 alkylene-amide, -C(O)OC 1- 4alkyl, -C 1-6 alkylene-O-aryl, -C 0-3 alkylene-C(O)C 1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -C 0-3 alkylene-C 3-14 cycloalkyl, -C 0-3 alkylene-C 2 - 1 4
• the present invention comprises the compound of Embodiment 1, having the structure:
• R 5a is selected from H, -C 1- 6 alkyl, -C 1-6 alkylene-OH, -C(O)OH, -C(O)OC 1-4 alkyl, and -C 0-3 alkylene-C 2-14 heterocycloalkyl, unless b is a double bond.
• R 6b is H, -C 1-6 alkyl, C 1- 6 alkylene-OH, -C(O)OH, -C(O)OC 1-4 alkyl, or -C 0-3 alkylene-C 2-14 heterocycloalkyl, unless R 3 has the following structure
• R 3 has the following structure and unless b is a double bond.
• the compound of embodiment 43 wherein R 5a and R 6a , together with the atoms to which they are attached, may form a 3-6 membered ring that optionally includes one or two heteroatoms selected from O, S or N.
• R 5b is selected from H, -C 1-6 alkyl, -C 1- 6 alkylene-OH, -C(O)OH, -C(O)OC 1-4 alkyl, or -C 0-3 alkylene-C 2-14 heterocycloalkyl.
• the compound of embodiment 56 wherein R 5a and R 6a , together with the atoms to which they are attached, may form a 3-6 membered ring that optionally includes one or two heteroatoms selected from O, S or N.
• the compound of embodiment 59, wherein R 5a and R 6a , together with the atoms to which they are attached, may form a 3-6 membered ring that optionally includes one or two heteroatoms selected from O, S or N.
• the compound of embodiment 62 wherein R 5a and R 6a , together with the atoms to which they are attached, may form a 3-6 membered ring that optionally includes one or two heteroatoms selected from O, S or N.
• Embodiment 84 is a diagrammatic representation of Embodiment 84.
• a pharmaceutical composition comprising the compound of any one embodiments 1- 83 and a pharmaceutically acceptable excipient.
• kits for treating cancer comprising a compound of any one of embodiments 1- 84 and an additional pharmaceutical active compound.
• kits of embodiment 85 wherein the cancer is a solid tumor.
• kits of embodiment 85, wherein the cancer is a KRAS G12C mutated cancer.
• kits of embodiment 85 wherein the cancer is a KRAS G12D mutated cancer.
• a method of inhibiting KRAS G12C in a cell comprising contacting the cell with the compound of any one embodiments 1-91.
• a method of inhibiting KRAS G12D in a cell comprising contacting the cell with the compound of any one embodiments 1-91.
• a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the compound of any one embodiments 1-84.
• Embodiment 107 is a diagrammatic representation of Embodiment 107.
• Embodiment 109 is a diagrammatic representation of Embodiment 109.
• a compound according to any one of embodiments 1-84 in the preparation of a medicament for treating cancer is a compound according to any one of embodiments 1-84 in the preparation of a medicament for treating cancer.
• a compound of any one of claims 1-83 or the pharmaceutical composition of claim 84 for use as a medicament is a compound of any one of claims 1-83 or the pharmaceutical composition of claim 84 for use as a medicament.
• Contemplated halogenating agents include, but are not limited to, chlorine, bromine, N-chlorosuccinimide, and N-bromosuccinimide, optionally in the presence of a catalyst, e.g., iron or aluminum.
• a catalyst e.g., iron or aluminum.
• the ordinarily skilled synthetic chemist will readily understand that other halogenating agents and catalysts can be used.
• Contemplated amidating agents include, but are not limited to, N, N’- diisopropylcarbodiimide, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide, benzotriazo1-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate, O-(benzotriazo1-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate, thionyl chloride, isobutyl chloroformate, diethyl cyanophosphonate, carbonyl diimidazole, and polyphosphonic anhydride.
• the ordinarily skilled synthetic chemist will readily understand that other amidating agents can be used.
• Contemplated sulfurizing agents include, but are not limited to, sulfur, phosphorus pentasulfide, and Lawesson’s reagent. The ordinarily skilled synthetic chemist will readily understand that other sulfurizing agents can be used.
• Contemplated oxidants include, but are not limited to, hydrogen peroxide, iodobenzene diacetate, t-butyl hydroperoxide, N-bromosuccinimide, and ammonium peroxodisulfate. The ordinarily skilled synthetic chemist will readily understand that other oxidants can be used.
• Contemplated metalating agents include, but are not limited to, bis(pinacolato)diboron, magnesium, zinc, hexamethyldistannane, and n-butyllithium. The ordinarily skilled synthetic chemist will readily understand that other metalating agents and catalysts can be used.
• Contemplated activating agents include, but are not limited to, sodium nitrite and t- butyl nitrite. The ordinarily skilled synthetic chemist will readily understand that other activating agents can be used.
• Contemplated cross-coupling reactions include, but are not limited to, Suzuki coupling, Negishi coupling, Hiyama coupling, Kumada coupling, and Stille coupling.
• Suzuki coupling Negishi coupling
• Hiyama coupling Hiyama coupling
• Kumada coupling Kumada coupling
• Stille coupling The ordinarily skilled chemist will readily understand that couplings as shown in the following Methods can be performed under a number of conditions.
• compositions for injection, administration, and routes of administration
• compositions that includes a compound as disclosed herein, together with a pharmaceutically acceptable excipient, such as, for example, a diluent or carrier.
• a pharmaceutically acceptable excipient such as, for example, a diluent or carrier.
• Compounds and pharmaceutical compositions suitable for use in the present invention include those wherein the compound can be administered in an effective amount to achieve its intended purpose. Administration of the compound described in more detail below.
• Suitable pharmaceutical formulations can be determined by the skilled artisan depending on the route of administration and the desired dosage. See, e.g ., Remington’s Pharmaceutical Sciences, 1435-712 (18th ed., Mack Publishing Co, Easton, Pennsylvania, 1990). Formulations may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the administered agents.
• a suitable dose may be calculated according to body weight, body surface areas or organ size. Further refinement of the calculations necessary to determine the appropriate treatment dose is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein as well as the pharmacokinetic data obtainable through animal or human clinical trials.
• phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
• pharmaceutically acceptable excipients includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such excipients for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the therapeutic compositions, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
• the formulation may comprise corn syrup solids, high-oleic safflower oil, coconut oil, soy oil, L-leucine, calcium phosphate tribasic, L-tyrosine, L-proline, L-lysine acetate, DATEM (an emulsifier), L-glutamine, L-valine, potassium phosphate dibasic, L-isoleucine, L-arginine, L-alanine, glycine, L-asparagine monohydrate, L-serine, potassium citrate, L-threonine, sodium citrate, magnesium chloride, L- histidine, L-methionine, ascorbic acid, calcium carbonate, L-glutamic acid, L-cystine dihydrochloride, L-tryptophan, L-aspartic acid, choline chloride, taurine, m-inositol, ferrous sulfate, ascorbyl palmitate, zinc sulfate, L
• DATEM
• the compound can be present in a pharmaceutical composition as a pharmaceutically acceptable salt.
• pharmaceutically acceptable salts include, for example base addition salts and acid addition salts.
• Pharmaceutically acceptable base addition salts may be formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
• Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
• metals used as cations are sodium, potassium, magnesium, ammonium, calcium, or ferric, and the like.
• suitable amines include isopropylamine, trimethylamine, histidine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.
• Pharmaceutically acceptable acid addition salts include inorganic or organic acid salts.
• suitable acid salts include the hydrochlorides, formates, acetates, citrates, salicylates, nitrates, phosphates.
• Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include, for example, formic, acetic, citric, oxalic, tartaric, or mandelic acids, hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, trifluoroacetic acid (TFA), propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucur
• compositions containing the compounds disclosed herein can be manufactured in a conventional manner, e.g., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.
• suitable compositions can be formulated readily by combining a compound disclosed herein with pharmaceutically acceptable excipients such as carriers well known in the art. Such excipients and carriers enable the present compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• compositions for oral use can be obtained by adding a compound as disclosed herein with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients include, for example, fillers and cellulose preparations. If desired, disintegrating agents can be added.
• Pharmaceutically acceptable ingredients are well known for the various types of formulation and may be for example binders (e.g., natural or synthetic polymers), lubricants, surfactants, sweetening and flavoring agents, coating materials, preservatives, dyes, thickeners, adjuvants, antimicrobial agents, antioxidants and carriers for the various formulation types.
• the composition typically is in the form of a solid (e.g., tablet, capsule, pill, powder, or troche) or a liquid formulation (e.g., aqueous suspension, solution, elixir, or syrup).
• a solid e.g., tablet, capsule, pill, powder, or troche
• a liquid formulation e.g., aqueous suspension, solution, elixir, or syrup.
• the composition can additionally contain a functional solid and/or solid carrier, such as a gelatin or an adjuvant.
• the tablet, capsule, and powder can contain about 1 to about 95% compound, and preferably from about 15 to about 90% compound.
• a functional liquid and/or a liquid carrier such as water, petroleum, or oils of animal or plant origin can be added.
• the liquid form of the composition can further contain physiological saline solution, sugar alcohol solutions, dextrose or other saccharide solutions, or glycols.
• the composition can contain about 0.5 to about 90% by weight of a compound disclosed herein, and preferably about 1 to about 50% of a compound disclosed herein.
• the liquid carrier is non-aqueous or substantially non-aqueous.
• the composition may be supplied as a rapidly-dissolving solid formulation for dissolution or suspension immediately prior to administration.
• composition When a therapeutically effective amount of a compound disclosed herein is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution.
• parenterally acceptable aqueous solution having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
• a preferred composition for intravenous, cutaneous, or subcutaneous injection typically contains, in addition to a compound disclosed herein, an isotonic vehicle.
• compositions may be prepared for administration as solutions of free base or pharmacologically acceptable salts in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
• Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can optionally contain a preservative to prevent the growth of microorganisms.
• injectable compositions can include sterile aqueous solutions, suspensions, or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions, suspensions, or dispersions. In all embodiments the form must be sterile and must be fluid to the extent that easy syringability exists.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the carrier is non-aqueous or substantially non-aqueous.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size of the compound in the embodiment of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• Slow release or sustained release formulations may also be prepared in order to achieve a controlled release of the active compound in contact with the body fluids in the GI tract, and to provide a substantially constant and effective level of the active compound in the blood plasma.
• release can be controlled by one or more of dissolution, diffusion, and ion- exchange.
• the slow release approach may enhance absorption via saturable or limiting pathways within the GI tract.
• the compound may be embedded for this purpose in a polymer matrix of a biological degradable polymer, a water-soluble polymer or a mixture of both, and optionally suitable surfactants. Embedding can mean in this context the incorporation of micro-particles in a matrix of polymers. Controlled release formulations are also obtained through encapsulation of dispersed micro-particles or emulsified micro-droplets via known dispersion or emulsion coating technologies.
• compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant.
• the dosage unit can be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• compositions disclosed herein can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).
• Formulations for injection can be presented in unit dosage form (e.g., in ampules or in multidose containers), with an added preservative.
• the compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the compounds in water-soluble form.
• suspensions of the compounds can be prepared as appropriate oily injection suspensions.
• Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters.
• Aqueous injection suspensions can contain substances which increase the viscosity of the suspension.
• the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
• a present composition can be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
• Compounds disclosed herein also can be formulated in rectal compositions, such as suppositories or retention enemas (e.g., containing conventional suppository bases).
• the compounds also can be formulated as a depot preparation.
• Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• a compound disclosed herein can be administered orally, buccally, or sublingually in the form of tablets containing excipients, such as starch or lactose, or in capsules or ovules, either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents.
• excipients such as starch or lactose
• capsules or ovules either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents.
• Such liquid preparations can be prepared with pharmaceutically acceptable additives, such as suspending agents.
• a compound also can be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily.
• the compound is best used in the form of a sterile aqueous solution which can contain other substances, for example, salts, or sugar alcohols, such as mannitol, or glucose, to make the solution isotonic with blood.
• a sterile aqueous solution which can contain other substances, for example, salts, or sugar alcohols, such as mannitol, or glucose, to make the solution isotonic with blood.
• a compound disclosed herein is administered as a suitably acceptable formulation in accordance with normal veterinary practice.
• the veterinarian can readily determine the dosing regimen and route of administration that is most appropriate for a particular animal.
• kits for use in the therapeutic intervention of the disease comprising a packaged set of medicaments that include the compound disclosed herein as well as buffers and other components for preparing deliverable forms of said medicaments, and/or devices for delivering such medicaments, and/or any agents that are used in combination therapy with the compound disclosed herein, and/or instructions for the treatment of the disease packaged with the medicaments.
• the instructions may be fixed in any tangible medium, such as printed paper, or a computer readable magnetic or optical medium, or instructions to reference a remote computer data source such as a world wide web page accessible via the internet.
• a “therapeutically effective amount” means an amount effective to treat or to prevent development of, or to alleviate the existing symptoms of, the subject being treated.
• a “therapeutically effective dose” refers to that amount of the compound that results in achieving the desired effect.
• a therapeutically effective amount of a compound disclosed herein decreases KRAS activity by at least 5%, compared to control, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.
• the amount of compound administered can be dependent on the subject being treated, on the subject's age, health, sex, and weight, the kind of concurrent treatment (if any), severity of the affliction, the nature of the effect desired, the manner and frequency of treatment, and the judgment of the prescribing physician.
• the frequency of dosing also can be dependent on pharmacodynamic effects on arterial oxygen pressures.
• the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This typically involves adjustment of a standard dose (e.g., reduction of the dose if the patient has a low body weight).
• typical dosages of the compounds of the present invention can be about 0.05 mg/kg/day to about 50 mg/kg/day, for example at least 0.05 mg/kg, at least 0.08 mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, or at least 0.5 mg/kg, and preferably 50 mg/kg or less, 40 mg/kg or less, 30 mg/kg or less, 20 mg/kg or less, or 10 mg/kg or less, which can be about 2.5 mg/day (0.5 mg/kg x 5kg) to about 5000 mg/day (50mg/kg x 100kg), for example.
• dosages of the compounds can be about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.05 mg/kg/day to about 10 mg/kg/day, about 0.05 mg/kg/day to about 5 mg/kg/day, about 0.05 mg/kg/day to about 3 mg/kg/day, about 0.07 mg/kg/day to about 3 mg/kg/day, about 0.09 mg/kg/day to about 3 mg/kg/day, about 0.05 mg/kg/day to about 0.1 mg/kg/day, about 0.1 mg/kg/day to about 1 mg/kg/day, about 1 mg/kg/day to about 10 mg/kg/day, about 1 mg/kg/day to about 5 mg/kg/day, about 1 mg/kg/day to about 3 mg/kg/day, about 3 mg/day to about 1000 mg/day, about 5 mg/day to about 500 mg/day, about 10 mg/day to about 200 mg/day, about 3 mg/day to about 100 mg/day, or about 100 mg/day to about 250 mg/day
• the present disclosure provides a method of inhibiting RAS-mediated cell signaling comprising contacting a cell with an effective amount of one or more compounds disclosed herein. Inhibition of RAS-mediated signal transduction can be assessed and demonstrated by a wide variety of ways known in the art.
• Non-limiting examples include a showing of (a) a decrease in GTPase activity of RAS; (b) a decrease in GTP binding affinity or an increase in GDP binding affinity; (c) an increase in K off of GTP or a decrease in K off of GDP; (d) a decrease in the levels of signaling transduction molecules downstream in the RAS pathway, such as a decrease in pMEK, pERK, or pAKT levels; and/or (e) a decrease in binding of RAS complex to downstream signaling molecules including but not limited to Raf. Kits and commercially available assays can be utilized for determining one or more of the above.
• the disclosure also provides methods of using the compounds or pharmaceutical compositions of the present disclosure to treat disease conditions, including but not limited to conditions implicated by G12C KRAS, G12D KRAS, HRAS or NRAS mutation (e.g., cancer).
• a method for treatment of cancer comprising administering an effective amount of any of the foregoing pharmaceutical compositions comprising a compound as disclosed herein to a subject in need thereof.
• the cancer is mediated by a KRAS, HRAS or NRAS G12C or G12D mutation.
• the cancer is pancreatic cancer, colorectal cancer or lung cancer.
• the cancer is gall bladder cancer, thyroid cancer, and bile duct cancer.
• the disclosure provides method of treating a disorder in a subject in need thereof, wherein the said method comprises determining if the subject has a KRAS, HRAS or NRAS G12C or G12D mutation and if the subject is determined to have the KRAS, HRAS or NRAS G12C or G12D mutation, then administering to the subject a therapeutically effective dose of at least one compound as disclosed herein or a pharmaceutically acceptable salt thereof.
• the disclosed compounds inhibit anchorage-independent cell growth and therefore have the potential to inhibit tumor metastasis.
• the disclosure provides a method for inhibiting tumor metastasis, the method comprising administering an effective amount a compound disclosed herein.
• KRAS, HRAS or NRAS G12C or G12D mutations have also been identified in hematological malignancies (e.g., cancers that affect blood, bone marrow and/or lymph nodes). Accordingly, certain embodiments are directed to administration of a disclosed compounds (e.g., in the form of a pharmaceutical composition) to a patient in need of treatment of a hematological malignancy.
• hematological malignancies include, but are not limited to leukemias and lymphomas.
• the presently disclosed compounds can be used for treatment of diseases such as Acute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML), Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Chronic myelogenous leukemia (CML), Acute monocytic leukemia (AMoL) and/ or other leukemias.
• ALL Acute lymphoblastic leukemia
• AML Acute myelogenous leukemia
• CLL Chronic lymphocytic leukemia
• SLL small lymphocytic lymphoma
• CML Chronic myelogenous leukemia
• Acute monocytic leukemia Acute monocytic leukemia
• the compounds are useful for treatment of lymphomas such as all subtypes of Hodgkins lymphoma or non-Hodgkins lymphoma.
• the compounds are useful for treatment of plasma cell malignancies such as multiple myeloma, mant
• Determining whether a tumor or cancer comprises a G12C KRAS, HRAS or NRAS mutation or a G12D KRAS, HRAS or NRAS mutation can be undertaken by assessing the nucleotide sequence encoding the KRAS, HRAS or NRAS protein, by assessing the amino acid sequence of the KRAS, HRAS or NRAS protein, or by assessing the characteristics of a putative KRAS, HRAS or NRAS mutant protein.
• the sequence of wild-type human KRAS, HRAS or NRAS is known in the art, (e.g. Accession No. NP203524).
• PCR-RFLP polymerase chain reaction-restriction fragment length polymorphism
• PCR-SSCP polymerase chain reaction-single strand conformation polymorphism
• MASA mutant allele-specific PCR amplification
• samples are evaluated for G12C or G12D KRAS, HRAS or NRAS mutations by real-time PCR.
• real-time PCR fluorescent probes specific for the KRAS, HRAS or NRAS G12C or G12D mutation are used. When a mutation is present, the probe binds and fluorescence is detected.
• the KRAS, HRAS or NRAS G12C or G12D mutation is identified using a direct sequencing method of specific regions (e.g., exon 2 and/or exon 3) in the KRAS, HRAS or NRAS gene. This technique will identify all possible mutations in the region sequenced.
• Methods for detecting a mutation in a KRAS, HRAS or NRAS protein are known by those of skill in the art. These methods include, but are not limited to, detection of a KRAS, HRAS or NRAS mutant using a binding agent (e.g., an antibody) specific for the mutant protein, protein electrophoresis and Western blotting, and direct peptide sequencing.
• a binding agent e.g., an antibody
• Methods for determining whether a tumor or cancer comprises a G12C or G12D KRAS, HRAS or NRAS mutation can use a variety of samples.
• the sample is taken from a subject having a tumor or cancer.
• the sample is a fresh tumor/cancer sample.
• the sample is a frozen tumor/cancer sample.
• the sample is a formalin-fixed paraffin-embedded sample. In some embodiments, the sample is a circulating tumor cell (CTC) sample. In some embodiments, the sample is processed to a cell lysate. In some embodiments, the sample is processed to DNA or RNA.
• CTC circulating tumor cell
• the disclosure also relates to a method of treating a hyperproliferative disorder in a mammal that comprises administering to said mammal a therapeutically effective amount of a compound as disclosed herein, or a pharmaceutically acceptable salt thereof.
• said method relates to the treatment of a subject who suffers from a cancer such as a solid tumor cancer, acute myeloid leukemia, cancer in adolescents, adrenocortical carcinoma childhood, AIDS-related cancers (e.g.
• Lymphoma and Kaposi's Sarcoma anal cancer, appendix cancer, astrocytomas, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, atypical teratoid, embryonal tumors, germ cell tumor, primary lymphoma, cervical cancer, childhood cancers, chordoma, cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myleoproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS), embryonal tumors, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma,
• said method relates to the treatment of a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin (e. g., psoriasis), restenosis, or prostate (e. g., benign prostatic hypertrophy (BPH)).
• a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin (e. g., psoriasis), restenosis, or prostate (e. g., benign prostatic hypertrophy (BPH)).
• the methods for treatment are directed to treating lung cancers, the methods comprise administering an effective amount of any of the above described compound (or a pharmaceutical composition comprising the same) to a subject in need thereof.
• the lung cancer is a non- small cell lung carcinoma (NSCLC), for example adenocarcinoma, squamous-cell lung carcinoma or large-cell lung carcinoma.
• NSCLC non- small cell lung carcinoma
• the lung cancer is a small cell lung carcinoma.
• Other lung cancers treatable with the disclosed compounds include, but are not limited to, glandular tumors, carcinoid tumors and undifferentiated carcinomas.
• the disclosure further provides methods of modulating a G12C and or a G12D Mutant
• KRAS, HRAS or NRAS protein activity by contacting the protein with an effective amount of a compound of the disclosure. Modulation can be inhibiting or activating protein activity.
• the disclosure provides methods of inhibiting protein activity by contacting the G12C and/or G12D Mutant KRAS, HRAS or NRAS protein with an effective amount of a compound of the disclosure in solution.
• the disclosure provides methods of inhibiting the G12C or G12D Mutant KRAS, HRAS or NRAS protein activity by contacting a cell, tissue, or organ that expresses the protein of interest.
• the disclosure provides methods of inhibiting protein activity in subject including but not limited to rodents and mammal (e.g., human) by administering into the subject an effective amount of a compound of the disclosure.
• the percentage modulation exceeds 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
• the percentage of inhibiting exceeds 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
• the disclosure provides methods of inhibiting KRAS, HRAS or NRAS G12C or G12D activity in a cell by contacting said cell with an amount of a compound of the disclosure sufficient to inhibit the activity of KRAS, HRAS or NRAS G12C in said cell. In some embodiments, the disclosure provides methods of inhibiting KRAS, HRAS or NRAS G12C activity in a tissue by contacting said tissue with an amount of a compound of the disclosure sufficient to inhibit the activity of KRAS, HRAS or NRAS G12C in said tissue.
• the disclosure provides methods of inhibiting KRAS, HRAS or NRAS G12C activity in an organism by contacting said organism with an amount of a compound of the disclosure sufficient to inhibit the activity of KRAS, HRAS or NRAS G12C in said organism. In some embodiments, the disclosure provides methods of inhibiting KRAS, HRAS or NRAS G12C activity in an animal by contacting said animal with an amount of a compound of the disclosure sufficient to inhibit the activity of KRAS, HRAS or NRAS G12C in said animal.
• the disclosure provides methods of inhibiting KRAS, HRAS or NRAS G12C activity in a mammal by contacting said mammal with an amount of a compound of the disclosure sufficient to inhibit the activity of KRAS, HRAS or NRAS G12C in said mammal. In some embodiments, the disclosure provides methods of inhibiting KRAS, HRAS or NRAS
• G12C activity in a human by contacting said human with an amount of a compound of the disclosure sufficient to inhibit the activity of KRAS, HRAS or NRAS G12C or G12D in said human.
• the present disclosure provides methods of treating a disease mediated by KRAS,
• HRAS or NRAS G12C activity in a subject in need of such treatment provides methods of treating a disease mediated by KRAS, HRAS or NRAS G12D activity in a subject in need of such treatment.
• the present disclosure also provides methods for combination therapies in which an agent known to modulate other pathways, or other components of the same pathway, or even overlapping sets of target enzymes are used in combination with a compound of the present disclosure, or a pharmaceutically acceptable salt thereof.
• such therapy includes but is not limited to the combination of one or more compounds of the disclosure with chemotherapeutic agents, therapeutic antibodies, and radiation treatment, to provide a synergistic or additive therapeutic effect.
• the method comprises administering to the patient in need thereof a therapeutically effective amount of an additional pharmaceutically active compound.
• the method comprises administering the compound of the present invention before the administration of at least one additional pharmaceutically active compound.
• the method comprises administering the compound of the present invention concurrently with the administration of at least one additional pharmaceutically active compound.
• the method comprises administering the compound of the present invention after the administration of at least one additional pharmaceutically active compound.
• chemotherapeutics are presently known in the art and can be used in combination with the compounds of the disclosure.
• the chemotherapeutic is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti- hormones, angiogenesis inhibitors, and anti androgens.
• Non-limiting examples are chemotherapeutic agents, cytotoxic agents, and non peptide small molecules such as Gleevec® (Imatinib Mesylate), Kyprolis® (carfilzomib), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib), venetoclax, and Adriamycin as well as a host of chemotherapeutic agents.
• Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide
• alkyl sulfonates such as busulfan, improsulfan and piposulfan
• aziridines such as benzodopa, carboquone, meturedopa, and uredopa
• ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine
• nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard
• nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine
• antibiotics such as aclacino
• chemotherapeutic cell conditioners include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, (NolvadexTM), raloxifene, aromatase inhibiting 4(5)- imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine
• the compounds or pharmaceutical composition of the present disclosure can be used in combination with commonly prescribed anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N-Allylamino-17- demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide, Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenic herbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy),
• Indolocarb azole Irofulven, Laniquidar, Larotaxel, Lenalidomide, Lucanthone, Lurtotecan, Mafosfamide, Mitozolomide, Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone, Proteasome inhibitor, Rebeccamycin, Resiquimod, Rubitecan, SN-38, Salinosporamide A, Sapacitabine, Stanford V, Swainsonine, Talaporfm, Tariquidar, Tegafur- uracil, Temodar, Tesetaxel, Triplatin tetranitrate, Tris(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimezan, Vinflunine, ZD6126 or Zosuquidar.
• This disclosure further relates to a method for using the compounds or pharmaceutical compositions provided herein, in combination with radiation therapy for inhibiting abnormal cell growth or treating the hyperproliferative disorder in the mammal.
• Techniques for administering radiation therapy are known in the art, and these techniques can be used in the combination therapy described herein.
• the administration of the compound of the disclosure in this combination therapy can be determined as described herein.
• Radioactive isotopes e.g. At-211, 1-131, 1-125, Y-90, Re-186, Re-188, Sm- 153, Bi-212, P-32, and radioactive isotopes of Lu.
• Suitable radiation sources for use as a cell conditioner of the present disclosure include both solids and liquids.
• the radiation source can be a radionuclide, such as 1-125, 1-131, Yb-169, Ir-192 as a solid source, 1-125 as a solid source, or other radionuclides that emit photons, beta particles, gamma radiation, or other therapeutic rays.
• the radioactive material can also be a fluid made from any solution of radionuclide(s), e.g., a solution of 1-125 or 1-131, or a radioactive fluid can be produced using a slurry of a suitable fluid containing small particles of solid radionuclides, such as Au-198, Y-90.
• the radionuclide(s) can be embodied in a gel or radioactive micro spheres.
• the compounds or pharmaceutical compositions of the disclosure can be used in combination with an amount of one or more substances selected from anti- angiogenesis agents, signal transduction inhibitors, antiproliferative agents, glycolysis inhibitors, or autophagy inhibitors.
• Anti-angiogenesis agents such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloprotienase 9) inhibitors, and COX-11 (cyclooxygenase 11) inhibitors, can be used in conjunction with a compound of the disclosure and pharmaceutical compositions described herein.
• Anti-angiogenesis agents include, for example, rapamycin, temsirolimus (CCI- 779), everolimus (RADOOl), sorafenib, sunitinib, and bevacizumab.
• Examples of useful COX-II inhibitors include alecoxib, valdecoxib, and rofecoxib.
• MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-
• MMP-2 and/or AMP-9 are those that selectively inhibit MMP-2 and/or AMP-9 relative to the other matrix- metalloproteinases (i. e., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP- 7, MMP- 8, MMP-10, MMP-11, MMP-12, andMMP-13).
• MMP inhibitors useful in the disclosure are AG-3340, RO 32-3555, and RS 13-0830.
• the present compounds may also be used in co-therapies with other anti-neoplastic agents, such as acemannan, aclarubicin, aldesleukin, alemtuzumab, alitretinoin, altretamine, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, ANCER, ancestim, ARGLABIN, arsenic trioxide, BAM 002 (Novelos), bexarotene, bicalutamide, broxuridine, capecitabine, celmoleukin, cetrorelix, cladribine, clotrimazole, cytarabine ocfosfate, DA 3030 (Dong- A), daclizumab, denileukin diftitox, deslorelin, dexrazoxane, dilazep, docetaxel, docosanol
• the compounds of the invention may further be used with VEGFR inhibitors.
• Other compounds described in the following patents and patent applications can be used in combination therapy: US 6,258,812, US 2003/0105091, WO 01/37820, US 6,235,764, WO 01/32651, US 6,630,500, US 6,515,004, US 6,713,485, US 5,521,184, US 5,770,599, US 5,747,498, WO 02/68406, WO 02/66470, WO 02/55501, WO 04/05279, WO 04/07481, WO 04/07458, WO 04/09784, WO 02/59110, WO 99/45009, WO 00/59509, WO 99/61422, US 5,990,141, WO 00/12089, and WO 00/02871.
• the combination comprises a composition of the present invention in combination with at least one anti-angiogenic agent.
• Agents are inclusive of, but not limited to, in vitro synthetically prepared chemical compositions, antibodies, antigen binding regions, radionuclides, and combinations and conjugates thereof.
• An agent can be an agonist, antagonist, allosteric modulator, toxin or, more generally, may act to inhibit or stimulate its target (e.g., receptor or enzyme activation or inhibition), and thereby promote cell death or arrest cell growth.
• anti-angiogenic agents include ERBITUXTM (IMC-C225), KDR (kinase domain receptor) inhibitory agents (e.g., antibodies and antigen binding regions that specifically bind to the kinase domain receptor), anti-VEGF agents (e.g., antibodies or antigen binding regions that specifically bind VEGF, or soluble VEGF receptors or a ligand binding region thereof) such as AVASTINTM or VEGF-TRAPTM, and anti-VEGF receptor agents (e.g., antibodies or antigen binding regions that specifically bind thereto), EGFR inhibitory agents (e.g., antibodies or antigen binding regions that specifically bind thereto) such as Vectibix (panitumumab), IRES S ATM (gefitinib), TARCEVATM (erlotinib), anti-Angl and anti-Ang2 agents (e.g., antibodies or antigen binding regions specifically binding thereto or to their receptors, e.g., Tie2/Tek
• compositions of the present invention can also include one or more agents (e.g., antibodies, antigen binding regions, or soluble receptors) that specifically bind and inhibit the activity of growth factors, such as antagonists of hepatocyte growth factor (HGF, also known as Scatter Factor), and antibodies or antigen binding regions that specifically bind its receptor "c-met".
• agents e.g., antibodies, antigen binding regions, or soluble receptors
• HGF hepatocyte growth factor
• c-met antibodies or antigen binding regions that specifically bind its receptor "c-met”.
• anti-angiogenic agents include Campath, IL-8, B-FGF, Tek antagonists (Ceretti et ah, U.S. Publication No. 2003/0162712; U.S. Patent No. 6,413,932), anti-TWEAK agents (e.g., specifically binding antibodies or antigen binding regions, or soluble TWEAK receptor antagonists; see, Wiley, U.S. Patent No. 6,727,225), ADAM distintegrin domain to antagonize the binding of integrin to its ligands (Fanslow et ak, U.S. Publication No.
• anti-eph receptor and/or anti-ephrin antibodies or antigen binding regions specifically binding anti-eph receptor and/or anti-ephrin antibodies or antigen binding regions
• anti-PDGF-BB antagonists e.g., specifically binding antibodies or antigen binding regions
• PDGFR kinase inhibitory agents e.g., antibodies or antigen binding regions that specifically bind thereto.
• Additional anti-angiogenic/anti-tumor agents include: SD-7784 (Pfizer, USA); cilengitide. (Merck KGaA, Germany, EPO 770622); pegaptanib octasodium, (Gilead Sciences, USA); Alphastatin, (BioActa, UK); M-PGA, (Celgene, USA, US 5712291); ilomastat, (Arriva,
• ZD 6126 (Angiogene Pharmaceuticals, UK); PPI 2458, (Praecis, USA); AZD 9935, (AstraZeneca, UK); AZD 2171, (AstraZeneca, UK); vatalanib (pINN), (Novartis, Switzerland and Schering AG, Germany); tissue factor pathway inhibitors, (EntreMed, USA); pegaptanib (Pinn), (Gilead Sciences, USA); xanthorrhizol, (Yonsei University, South Korea); vaccine, gene- based, VEGF-2, (Scripps Clinic and Research Foundation, USA); SPV5.2, (Supratek, Canada); SDX 103, (University of California at San Diego, USA); PX 478, (ProlX, USA);
• METASTATIN (EntreMed, USA); troponin I, (Harvard University, USA); SU 6668, (SUGEN,
• GFB 116 (South Florida University, USA and Yale University, USA); CS 706, (Sankyo, Japan); combretastatin A4 prodrug, (Arizona State University, USA); chondroitinase AC, (IBEX, Canada); BAY RES 2690, (Bayer, Germany); AGM 1470, (Harvard University, USA, Takeda, Japan, and TAP, USA); AG 13925, (Agouron, USA); Tetrathiomolybdate, (University of Michigan, USA); GCS 100, (Wayne State University, USA) CV 247, (Ivy Medical, UK); CKD 732, (Chong Kun Dang, South Korea); MAb, vascular endothelium growth factor, (Xenova,
• irsogladine (INN), (Nippon Shinyaku, Japan); RG 13577, (Aventis, France); WX 360, (Wilex, Germany); squalamine (pINN), (Genaera, USA); RPI 4610, (Sirna, USA); cancer therapy, (Marinova, Australia); heparanase inhibitors, (InSight, Israel); KL 3106, (Kolon, South Korea); Honokiol, (Emory University, USA); ZK CDK, (Schering AG, Germany); ZK Angio, (Schering AG, Germany); ZK 229561, (Novartis, Switzerland, and Schering AG, Germany); XMP 300, (XOMA, USA); VGA 1102, (Taisho, Japan); VEGF receptor modulators, (Pharmacopeia, USA); VE-cadherin-2 antagonists , (ImClone Systems, USA); Vasostatin, (National Institutes of Health
• Autophagy inhibitors include, but are not limited to chloroquine, 3- methyladenine, hydroxychloroquine (PlaquenilTM), bafilomycin Al, 5-amino-4- imidazole carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1, analogues of cAMP, and drugs which elevate cAMP levels such as adenosine, LY204002, N6-mercaptopurine riboside, and vinblastine.
• antisense or siRNA that inhibits expression of proteins including but not limited to ATG5 (which are implicated in autophagy), may also be used.
• Additional pharmaceutically active compounds/agents that can be used in the treatment of cancers and that can be used in combination with one or more compound of the present invention include: epoetin alfa; darbepoetin alfa; panitumumab; pegfilgrastim; palifermin; filgrastim; denosumab; ancestim; AMG 102; AMG 386; AMG 479; AMG 655; AMG 745; AMG 951; and AMG 706, or a pharmaceutically acceptable salt thereof.
• a composition provided herein is conjointly administered with a chemotherapeutic agent.
• chemotherapeutic agents may include, natural products such as vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), mitomycin, enzymes (e.g., L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine), antiplatelet agents, antiproliferative/antimitotic alkylating agents such
• chemotherapeutic agents may include mechlorethamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, sorafenib, or any analog or derivative variant of the foregoing.
• the compounds of the present invention may also be used in combination with radiation therapy, hormone therapy, surgery and immunotherapy, which therapies are well known to those skilled in the art.
• a pharmaceutical composition provided herein is conjointly administered with a steroid.
• Suitable steroids may include, but are not limited to, 21- acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate,
• the compounds of the present invention may also be used in combination with an additional pharmaceutically active compound that disrupts or inhibits RAS-RAF-ERK or PI3K- AKT-TOR signaling pathways.
• the additional pharmaceutically active compound is a PD-1 and PD-L1 antagonist.
• the compounds or pharmaceutical compositions of the disclosure can also be used in combination with an amount of one or more substances selected from EGFR inhibitors, MEK inhibitors, PI3K inhibitors, ART inhibitors, TOR inhibitors, Mcl-1 inhibitors, BCL-2 inhibitors, SHP2 inhibitors, such as (RMC-4630), proteasome inhibitors, and immune therapies, including monoclonal antibodies, immunomodulatory imides (IMiDs), anti-PD-1, anti-PDL-1, anti-CTLA4, anti-LAGl, and anti- 0X40 agents, GITR agonists, CAR-T cells, and BiTEs.
• IMDs immunomodulatory imides
• EGFR inhibitors include, but are not limited to, small molecule antagonists, antibody inhibitors, or specific antisense nucleotide or siRNA.
• Useful antibody inhibitors of EGFR include cetuximab (Erbitux), panitumumab (Vectibix), zalutumumab, nimotuzumab, and matuzumab.
• Small molecule antagonists of EGFR include gefitinib, erlotinib (Tarceva), and most recently, lapatinib (TykerB). See e.g., Yan L, et. al., Pharmacogenetics and Pharmacogenomics In Oncology Therapeutic Antibody Development, BioTechniques 2005;
• Non-limiting examples of small molecule EGFR inhibitors include any of the EGFR inhibitors described in the following patent publications, and all pharmaceutically acceptable salts and solvates of said EGFR inhibitors: European Patent Application EP 520722, published Dec. 30, 1992; European Patent Application EP 566226, published Oct. 20, 1993; PCT International Publication WO 96/33980, published Oct. 31, 1996; U.S. Pat. No. 5,747,498, issued May 5, 1998; PCT International Publication WO 96/30347, published Oct. 3, 1996; European Patent Application EP 787772, published Aug. 6, 1997; PCT International Publication WO 97/30034, published Aug. 21, 1997; PCT International Publication WO 97/30044, published Aug.
• Antibody -based EGFR inhibitors include any anti-EGFR antibody or antibody fragment that can partially or completely block EGFR activation by its natural ligand.
• Non- limiting examples of antibody-based EGFR inhibitors include those described in Modjtahedi, FL, et al., 1993, Br. J. Cancer 67:247-253; Teramoto, T., et al., 1996, Cancer 77:639-645; Goldstein et al., 1995, Clin. Cancer Res. 1:1311-1318; Huang, S. M., et al., 1999, Cancer Res.
• the EGFR inhibitor can be monoclonal antibody Mab E7.6.3 (Yang, 1999 supra), or Mab C225 (ATCC Accession No. HB-8508), or an antibody or antibody fragment having the binding specificity thereof.
• MEK inhibitors include, but are not limited to, trametinib (Mekinist ® ), Cl- 1040, AZD6244, PD318088, PD98059, PD334581, RDEA119, ARRY-142886, ARRY-438162, and PD-325901.
• PI3K inhibitors include, but are not limited to, wortmannin, 17-hydroxy wortmannin analogs described in WO 06/044453, 4-[2-(1H-Indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1- yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941 and described in PCT Publication Nos.
• LY294002 (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one available from Axon Medchem), PI 103 hydrochloride (3-[4-(4-morpholinylpyrido-[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride available from Axon Medchem), PIK 75 (N'-[(lE)-(6-bromoimidazo[1,2- a]pyridin-3-yl)methylene]-N,2-dimethyl-5-nitrobenzenesulfono-hydrazide hydrochloride available from Axon Medchem), PIK 90 (N-(7,8-dimethoxy-2,3-dihydro-imidazo[1,2- c]quinazolin-5-yl)-nicotinamide available from Axon Medchem), GDC-0941 bismesylate (2- (1-1)
• PI3K inhibitors include demethoxyviridin, perifosine, CALIOI, PX-866, BEZ235, SF1126, INK1117, IPI-145, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TGlOO-115, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136.
• AKT inhibitors include, but are not limited to, Akt-1-1 (inhibits Aktl) (Barnett et al. (2005) Biochem. J, 385 (Pt.
• perifosine e.g., interferes with Akt membrane localization; Dasmahapatra et al. (2004) Clin. Cancer Res. 10(15), 5242-52, 2004); phosphatidylinositol ether lipid analogues (e.g., Gills and Dennis (2004) Expert. Opin. Investig. Drugs 13, 787-97); and triciribine (TCN or API-2 or NCI identifier: NSC 154020; Yang et al. (2004) Cancer Res. 64, 4394-9).
• TOR inhibitors include, but are not limited to, inhibitors include AP-23573, CCI-779, everolimus, RAD-001, rapamycin, temsirolimus, ATP-competitive TORC1/TORC2 inhibitors, including PI- 103, PP242, PP30 and Torin 1.
• Other TOR inhibitors in FKBP12 enhancer include rapamycins and derivatives thereof, including: CCI-779 (temsirolimus), RADOOl (Everolimus; WO 9409010) and AP23573; rapalogs, e.g. as disclosed in WO 98/02441 and WO 01/14387, e.g.
• AP23573, AP23464, or AP23841 40-(2-hydroxyethyl)rapamycin, 40-[3- hydroxy(hydroxymethyl)methylpropanoate]-rapamycin (also called CC1779), 40-epi- (tetrazolyt)-rapamycin (also called ABT578), 32-deoxorapamycin, 16-pentynyloxy-32(S)- dihydrorapanycin, and other derivatives disclosed in WO 05005434; derivatives disclosed in U.S. Pat. No. 5,258,389, WO 94/090101, WO 92/05179, U.S. Pat. No. 5,118,677, U.S. Pat. No.
• MC1-1 inhibitors include, but are not limited to, AMG-176, MIK665, and S63845.
• the myeloid cell leukemia-1 (MCL-1) protein is one of the key anti-apoptotic members of the B-cell lymphoma-2 (BCL-2) protein family. Over-expression of MCL-1 has been closely related to tumor progression as well as to resistance, not only to traditional chemotherapies but also to targeted therapeutics including BCL-2 inhibitors such as ABT-263.
• SHP inhibitors include, but are not limited to, SHP099.
• Proteasome inhibitors include, but are not limited to, Kyprolis ® (carfilzomib), Velcade ® (bortezomib), and oprozomib.
• Immune therapies include, but are not limited to, anti -PD- 1 agents, anti-PDL-1 agents, anti-CTLA-4 agents, anti-LAGl agents, and anti-OX40 agents.
• Monoclonal antibodies include, but are not limited to, Darzalex ® (daratumumab), Herceptin ® (trastuzumab), Avastin ® (bevacizumab), Rituxan ® (rituximab), Lucentis ® (ranibizumab), and Eylea ® (aflibercept).
• Immunomodulatory imide drugs are a class of immunomodulatory drugs (drugs that adjust immune responses) containing an imide group.
• the IMiD class includes thalidomide and its analogues (lenalidomide, pomalidomide, and apremilast).
• WO 2006/121168 Al each of which are expressly incorporated by reference herein, include: pembrolizumab (Keytruda ® ), nivolumab (Opdivo ® ), YervoyTM (ipilimumab) or Tremelimumab (to CTLA-4), galiximab (to B7.1), BMS-936558 (to PD-1), MK-3475 (to PD-1), AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A (to B7-H1), MEDI-570 (to ICOS), AMG 404, AMG557 (to B7H2), MGA271 (to B7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF- 05082566 (to CD137), CDX-1127 (to CD27), anti-OX40 (Providence Health Services), huMAbOX40L (to OX40
• Immune therapies also include genetically engineered T-cells (e.g., CAR-T cells) and bispecific antibodies (e.g., BiTEs).
• T-cells e.g., CAR-T cells
• bispecific antibodies e.g., BiTEs
• GITR agonists include, but are not limited to, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as, a GITR fusion protein described in U.S.
• the one or more compounds of the disclosure will be co-administered with other agents as described above.
• the compounds described herein are administered with the second agent simultaneously or separately.
• This administration in combination can include simultaneous administration of the two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, a compound described herein and any of the agents described above can be formulated together in the same dosage form and administered simultaneously. Alternatively, a compound of the disclosure and any of the agents described above can be simultaneously administered, wherein both the agents are present in separate formulations. In another alternative, a compound of the present disclosure can be administered just followed by and any of the agents described above, or vice versa. In some embodiments of the separate administration protocol, a compound of the disclosure and any of the agents described above are administered a few minutes apart, or a few hours apart, or a few days apart.
• kits comprises two separate pharmaceutical compositions: a compound of the present invention, and a second pharmaceutical compound.
• the kit comprises a container for containing the separate compositions such as a divided bottle or a divided foil packet. Additional examples of containers include syringes, boxes, and bags. In some embodiments, the kit comprises directions for the use of the separate components.
• kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, [0257] or when titration of the individual components of the combination is desired by the prescribing health care professional.
• Step 1 2-Isopropyl-4-methylpyridin-3-amine (Intermediate R).
• THF 4- mL
• 2-propylzinc bromide 0.5 M solution in THF, 5.40 mL, 2.7 mmol, Sigma-Aldrich, St.
• Step 2 2,5,6-Trichloro-/V-((2-isopropyl-4-methylpyridin-3- yl)carbamoyl)nicotinamide.
• 2,5,6-trichloronicotinamide (Intermediate P, 3.10 g, 13.8 mmol) in THF (46 mL) was added oxalyl chloride (2 M solution in DCM, 7.4 mL, 14.7 mmol) slowly via syringe.
• oxalyl chloride (2 M solution in DCM, 7.4 mL, 14.7 mmol
• Triethylamine (6.0 mL, 42.6 mmol) was added followed by a solution of 2-isopropyl-4-methylpyridin-3-amine (Intermediate R, 2.12 g, 14.1 mmol) via cannula.
• the resulting slurry was warmed to room temperature and stirred for 1 h, then was partitioned between water (120 mL) and EtOAc (175 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was suspended in 9:1 heptane/EtOAc and filtered.
• Step 3 6,7-Dichloro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidine- 2,4(1H,3H)-dione.
• 2, 5, 6-tri chi oro-A-((2-isopropyl -4-methyl pyridin- 3-yl)carbamoyl)nicotinamide (4.71 g, 11.7 mmol) in THF (55 mL) was added KHMDS (1 M solution in THF, 23.5 mL, 23.5 mmol) slowly via syringe.
• Step 4 4,6,7-Trichloro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one.
• acetonitrile 45 mL
• Step 5 (ri)-tert-Butyl-4-(6,7-dichloro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2- dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine- 1 -carboxylate.
• Step 6 (S)-tert-Butyl-4-(6-chloro-7-(2-fluorophenyl)-1-(2-i sopropyl -4-methyl pyridin- 3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate.
• aqueous phase was acidified by addition of concentrated hydrochloric acid to pH 3 and extracted four times with DCM.
• the combined organic phase was dried over magnesium sulphate and concentrated under reduced pressure to give ethyl (27?,3ri)-3-(hydroxymethyl)oxirane-2-carboxylate (413.8 mg, 2.83 mmol, 59.2 % yield) as a yellow oil.
• the resulting yellow paste was dissolved in N, N-dimethylformamide (12.00 mL), and potassium iodide (23.3 mg, 0.14 mmol, Fisher Scientific UK) was added.
• the yellow solution was cooled to 0 °C before the dropwise addition of piperidine (0.83 mL, 8.42 mmol, Spectrum Chemicals & Laboratory Products).
• the resulting suspension solution was slowly warmed to room temperature and stirred for 24 h.
• the resulting reaction mixture was diluted with saturated aqueous NaHCCb (10 mL) and extracted with DCM (20 mL x 2).
• reaction mixture was diluted with water ( 10 mL) and extracted with DCM ( 3 x 10 mL). The organic extract was washed with dried over MgSCri. The solution was filtered, concentrated in vacuo and purified by silica gel chromatography (eluent: 0-10% MeOH/DCM) to give 6-chloro- 7-(2-fluorophenyl)-4-((S)-4-((2R ,3S)-3 -(hydroxymethyl )oxirane-2-carbonyl)-2-methylpiperazin- l-yl)-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (8.1 mg, 0.01 mmol, 7.9 % yield, 99% purity) as a light-yellow solid.
• Coupled Nucleotide Exchange Assay Purified GDP -bound KRAS protein (aa 1-169), containing either G12C or G12D, as well as C118A amino acid substitutions and an A -terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl 2 , and 0.01% Triton X-100) with a compound dose-response titration for either 5 min or 20 hours (see Table 15). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min.
• assay buffer 25 mM HEPES pH 7.4, 10 mM MgCl 2 , and 0.01% Triton X-100
• MIA PaCa-2 (ATCC® CRL-1420TM) cells were cultured in RPMI 1640 Medium (ThermoFisher Scientific 11875093) containing 10% fetal bovine serum (ThermoFisher Scientific 16000044) and lx penicillin-streptomycin-glutamine (ThermoFisher Scientific 10378016). Sixteen hours prior to compound treatment, MIA PaCa-2 cells were seeded in 96-well cell culture plates at a density of 25,000 cells/well and incubated at 37°C, 5% CO2.
• a compound dose-response titration was diluted in growth media, added to appropriate wells of a cell culture plate, and then incubated at 37°C, 5% CO2 for 2 or 4 hours (see Table 15). Following compound treatment, cells were washed with ice-cold Dulbecco's phosphate-buffered saline, no Ca 2+ or Mg 2+ (ThermoFisher Scientific 14190144), and then lysed in RIPA buffer (50 mM Tris-HC1 pH 7.5, 1% Igepal, 0.5% sodium deoxycholate, 150 mM NaC1, and 0.5% sodium dodecyl sulfate) containing protease inhibitors (Roche 4693132001) and phosphatase inhibitors (Roche 4906837001).
• RIPA buffer 50 mM Tris-HC1 pH 7.5, 1% Igepal, 0.5% sodium deoxycholate, 150 mM NaC1, and 0.5% sodium dodecyl sul
• Phosphorylation of ERK1/2 in compound-treated lysates was assayed using Phospho-ERKl/2 Whole Cell Lysate kits (Meso Scale Discovery K151DWD) according to the manufacturer’s protocol. Assay plates were read on a Meso Scale Discovery Sector Imager 6000, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
• KRAS G12D 25 mM HEPES pH 7.4, 20 mM MgCl2, 10 mM NaC1, 3% DMSO, 0.0005% CHAPS with 100 mM GDP made fresh and added to the assay buffer.
• the final concentration of KRAS G12D was 2 uM.
• Assay incubation times were 20 h and the inhibitor concentration was 100 mM.
• Greiner polypropylene clear V-bottom plates were used for the mass spectrometry assays. Typically, the reaction was initiated by addition of KRAS G12D to a well containing inhibitor followed by Rapidfire MS analysis. Relative % Bound values at the various incubation times were used to estimate a kobs value at 100 uM inhibitor.
• RapidFire Sample Liquid handling System The RapidFire liquid handling unit used was the RF 360 model. Solvents were delivered by Agilent 1260 Infinity pumps. The instrument control software was the Agilent RapidFire High Throughput MS-system software
• the sample load/wash time was 6000 ms at a flow rate of 1.25 mL/min (H2O, 0.1% v/v formic acid, 0.05% v/v trifluoroacetic acid); elution time was 7000 ms (acetonitrile, 0.1% v/v formic acid, 0.05% v/v trifluoroacetic acid); re-equilibration time was 700 ms at a flow rate of 1.25 mL/min (H2O, 0.1% v/v formic acid, 0.05% v/v trifluoroacetic acid).
• Mass Spectrometry Mass spectrometric data was acquired on an Agilent 6530 quadrupole time-of-flight (ToF) MS system, operated with a dual Agilent Jet Spray (AJS) ion source, in positive ionization mode.
• the instrument parameters were as follows: gas temperature 350 °C; drying gas 5 L/min; nebulizer 60 psi; sheath gas 350 °C; sheath gas flow 11 L/min; capillary 3.5 kV (0.037 ⁇ A); nozzle 1.75 kV; fragmentor 200 V; skimmer 75 V; octapole RF 800 V peak-to-peak. Data was acquired at the rate of 5 spectra/s. The mass scale was calibrated using the Agilent positive ion tune mix, over the m/z range 300-3200.
• the instrument control software was Agilent MassHunter Workstation Data Acquisition (vB.05.01).
• Mass Spectrometry Data Processing and Informatics All data processing was performed using a HP Z620, 64-bit, 12-processor, Windows 7 PC, using a combination of Agilent MassHunter Qualitative Analysis (B.05.00) Agilent RapidFire control software (v4.0.12333.14070) and the Agilent DA Reprocessor Offline Utilities B.05.00, Build 162.
• the SPE-MS data acquired from the RapidFire-MS system is typically a single SPE-MS data file composed of multiple injection events. For effective charge state extraction, SPE-peak integration and subsequent MS-spectral deconvolution, the single SPE-MS data file is split into single injection events.
• the specific input files for the Pipeline Pilot script are the Excel report/result file directory; the compound molecular weight file (Amgen registry compound number and expected covalent modification) and the Injection Sequence File (RFTIME file; not shown), which is produced during the RapidFire-MS acquisition and contains the injection sequence, injection position and the plate bar code (which corresponds with the 384-well plate map dispensed by the Amgen Sample Bank).
• Additional information that is manually input to the Pipeline Pilot routine via a user interface are the protein’s molecular weight (average molecular weight in Da) and the molecular weight variance, typically 0.01% ( ⁇ 2 Da for a 19.5 kDa protein).

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