Pyrazoloquinoline KRAS inhibitors

Abstract

Disclosed are compounds of formula (I), methods of using the compounds to inhibit KRAS activity, and pharmaceutical compositions comprising such compounds. The compounds are useful for treating, preventing, or ameliorating diseases or disorders associated with KRAS activity, such as cancer. [Formula 1] TIFF2024537824000112.tif69164

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 261,982, filed October 1, 2021, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure provides compounds, as well as compositions and methods of use thereof, that modulate KRAS activity and are useful in the treatment of a variety of diseases, including cancer. [Background technology]

[0003] Ras proteins are part of a family of small GTPases that are activated by growth factors and various extracellular stimuli. The Ras family regulates intracellular signaling pathways involved in cell proliferation, migration, survival and differentiation. Activation of RAS proteins at the cell membrane leads to binding of key effectors and initiation of a cascade of intracellular signaling pathways within the cell, including the RAF and PI3K kinase pathways. While somatic mutations in RAS can result in uncontrolled cell proliferation and malignant transformation, activation of RAS proteins is tightly regulated in normal cells (Simanshu, D. et al. Cell 170.1 (2017): 17-33).

[0004] The Ras family consists of three members: KRAS, NRAS, and HRAS. RAS-mutated cancers account for approximately 25% of human cancers. KRAS is the most frequently mutated isoform, accounting for 85% of all RAS mutations, while NRAS and HRAS are mutated in 12% and 3% of all RAS-mutated cancers, respectively (Simanshu, D. et al. Cell 170.1 (2017): 17-33). KRAS mutations are prevalent among the top three most lethal cancer types: pancreatic (97%), colorectal (44%), and lung (30%) (Cox, AD et al. Nat Rev Drug Discov (2014) 13: 828-51). The majority of RAS mutations occur at amino acid residues 12, 13, and 61. The frequency of certain mutations varies between RAS gene isoforms, with G12 and Q61 mutations predominating in KRAS and NRAS, respectively, while G12, G13 and Q61 mutations are most frequent in HRAS. Furthermore, the spectrum of mutations in RAS isoforms differs between cancer types. For example, KRAS G12D mutations predominate in pancreatic cancer (51%), followed by colorectal adenocarcinoma (45%) and lung cancer (17%), and KRAS G12V mutations are associated with pancreatic cancer (30%), followed by colorectal adenocarcinoma (27%) and lung adenocarcinoma (23%) (Cox, AD et al. Nat Rev Drug Discov (2014) 13:828-51). In contrast, KRAS G12C mutations predominate in non-small cell lung cancer (NSCLC), including 11-16% of lung adenocarcinomas, and 2-5% of pancreatic and colorectal adenocarcinomas (Cox, AD et al. Nat. Rev. Drug Discov. (2014) 13:828-51). Genomic studies across hundreds of cancer cell lines have demonstrated that cancer cells with KRAS mutations are highly dependent on KRAS function for cell proliferation and survival (McDonald, R. et al. Cell 170 (2017):577-592).The role of mutant KRAS as an oncogenic driver is further supported by extensive in vivo experimental evidence showing that mutant KRAS is required for the development and maintenance of primary tumors in animal models (Cox, AD et al. Nat Rev Drug Discov (2014) 13:828-51).

[0005] Taken together, these findings suggest that KRAS mutations play an important role in human cancer, and therefore, the development of inhibitors targeting mutant KRAS may be useful in the clinical treatment of diseases characterized by KRAS mutations. Summary of the Invention

[0006] The present disclosure relates, inter alia, to a compound of formula I [ka] or a pharma- ceutically acceptable salt thereof, wherein the constituent variables are defined herein.

[0007] The present disclosure further provides a pharmaceutical composition comprising a compound of the present disclosure, or a pharma- ceutically acceptable salt thereof, and at least one pharma- ceutically acceptable carrier or excipient.

[0008] The present disclosure further provides a method for inhibiting KRAS activity, comprising administering to an individual a compound of the present disclosure or a pharma- ceutically acceptable salt thereof.The present disclosure also provides the use of the compounds described herein in the manufacture of a medicament for use in therapy.The present disclosure also provides the compounds described herein for use in therapy.

[0009] The present disclosure further provides a method of treating a disease or disorder in a patient, comprising administering to the patient a therapeutically effective amount of a compound of the present disclosure, or a pharma- ceutically acceptable salt thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] compound In some aspects, provided herein is a compound represented by formula I: [ka] or a pharma- ceutically acceptable salt thereof, wherein R 1 is selected from Cl, CH3, CH2F, CHF2, and CF3; Cy 1 is selected from: [ka] R 2 is selected from F and Cl; R 3 is selected from: [ka] and, Cy 2 is selected from: [ka] With the proviso that the compound of formula I is other than the following, or a pharma- ceutically acceptable salt thereof: [Table 1]

[0011] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, R 1 is selected from Cl, CH2F, CHF2, and CF3; Cy 1 is selected from: [ka] R 2 is selected from F and Cl; R 3 is selected from the following: [ka] and, Cy 2 is selected from the following: [ka]

[0012] In another embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, R 1 is Cl, Cy 1 is selected from: [ka] R 2 is F, R 3 is [ka] and, Cy 2 is selected from the following: [ka]

[0013] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, R 1 is selected from Cl, CHF, CHF, and CF. In yet another embodiment, R 1 is selected from CH3, CH2F, CHF2, and CF3. In yet another embodiment, R 1 is selected from Cl, CH3, and CF3.

[0014] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, R 1 is selected from CH2F, CHF2, and CF3. In another embodiment, R 1 is selected from Cl and CH3. In yet another embodiment, R 1 is selected from Cl and CF. In yet another embodiment, R 1 is selected from CH3 and CF3.

[0015] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, R 1 is Cl. In another embodiment, R 1 is CH3. In yet another embodiment, R 1 is CHF. In yet another embodiment, R 1 is CHF2. In one embodiment, R 1 is CF3.

[0016] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, Cy 1 Cy 1 -a and Cy 1 In another embodiment, Cy is selected from 1 Cy 1 -a and Cy 1 In yet another embodiment, Cy is selected from 1 Cy 1 -b and Cy 1 -c is selected.

[0017] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, Cy 1 Cy1 In another embodiment, Cy 1 Cy 1 In another embodiment, Cy 1 Cy 1 -c.

[0018] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, R 2 is F. In another embodiment, R 2 is Cl.

[0019] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, R 3 is R 3 In another embodiment, R 3 is R 3 -b.

[0020] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, Cy 2 Cy 2 -a and Cy 2 In another embodiment, Cy is selected from 2 Cy 2 -a and Cy 2 In another embodiment, Cy is selected from 2 Cy 2 -b and Cy 2 In another embodiment, Cy is selected from 2 Cy 2 In yet another embodiment, Cy 2 Cy 2 In yet another embodiment, Cy 2 Cy 2 -c.

[0021] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, Cy 1 Cy 1 -a and Cy 2 Cy 2 -a.

[0022] In one embodiment of Formula I, or a pharma- ceutically acceptable salt thereof, the compound of Formula I is other than: [Table 2]

[0023] In one embodiment, the compound of formula I is 1-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-8-chloro-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-((E)-4-methoxybut-2-enoyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile, 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-((E)-4-methoxybut-2-enoyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, and 8-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, or a pharma- ceutically acceptable salt thereof.

[0024] In another embodiment, the compound of formula I is 1-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-8-chloro-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-((E)-4-methoxybut-2-enoyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile, 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, and 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-((E)-4-methoxybut-2-enoyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, or a pharma- ceutically acceptable salt thereof.

[0025] In yet another embodiment, the compound of formula I is 8-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, or a pharma- ceutically acceptable salt thereof.

[0026] In yet another embodiment, the compound of formula I is 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, or a pharma- ceutically acceptable salt thereof.

[0027] In another embodiment, the compound of the formula herein is a compound of the formula: or a pharma- ceutically acceptable salt thereof.

[0028] It is further understood that certain features of the invention that are described for clarity in the context of separate embodiments can also be provided in combination in a single embodiment (whereas the embodiments are intended to be combined as described in a multiple dependent form). Conversely, various features of the invention that are described for brevity in the context of a single embodiment can also be provided separately or in any suitable subcombination. Thus, it is contemplated that the features described in the embodiments of the compounds of formula I can be combined in any suitable combination.

[0029] In certain places, definitions or embodiments refer to certain rings (e.g., azetidine ring, pyridine ring, etc.). Unless otherwise specified, these rings can be bonded to any ring member, provided that the valence of the atom is not exceeded. For example, an azetidine ring can be bonded at any position of the ring, whereas an azetidin-3-yl ring is bonded at the 3-position.

[0030] The compounds described herein may be asymmetric (e.g., have one or more stereocenters). Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like, can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as separated isomeric forms.

[0031] Resolution of racemic mixtures of compounds can be carried out by any of a number of methods known in the art. One method involves fractional recrystallization using chiral resolving acids that are optically active salt-forming organic acids. Suitable resolving agents for fractional recrystallization methods are optically active acids such as, for example, the D and L forms of various optically active camphorsulfonic acids, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or β-camphorsulfonic acid. Other suitable resolving agents for fractional crystallization methods include stereoisomerically pure forms (e.g., S and R forms, or diastereomerically pure forms) of α-methylbenzylamine, 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

[0032] Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by those skilled in the art.

[0033] In some embodiments, the compounds of the invention have the (R)-configuration. In other embodiments, the compounds have the (S)-configuration. In compounds with more than one chiral center, each of the chiral centers in the compound may be independently (R) or (S), unless otherwise indicated.

[0034] The compounds of the present invention also include tautomeric forms. Tautomeric forms arise from the interchange of a single bond and an adjacent double bond with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position of a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically fixed in one form by appropriate substitution.

[0035] The compounds of the present invention may also include all isotopes of atoms occurring in intermediates or final compounds. Isotopes include atoms with the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the present invention can be replaced or substituted with an isotope of the atom in natural or non-natural abundance. In some embodiments, the compounds include at least one deuterium atom. For example, one or more hydrogen atoms in the compounds of the present disclosure can be replaced or substituted with deuterium. In some embodiments, the compounds include two or more deuterium atoms. In some embodiments, the compounds include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deuterium atoms. Synthetic methods for incorporating isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, NY, Appleton-Century-Crofts, 1971; The Renaissance of H / D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in a variety of studies, such as NMR spectroscopy, metabolic experiments, and / or assays.

[0036] Substitution with heavier isotopes such as deuterium may confer certain therapeutic advantages, such as increased half-life in vivo or reduced dosage requirements, resulting from greater metabolic stability and therefore may be preferred in some circumstances (A. Kerekes et.al. J. Med. Chem. 2011, 54, 201-210; R. Xu et.al. J. Label Compd. Radiopharm. 2015, 58, 308-312).

[0037] The term "compound" as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. The term is also meant to refer to the compounds of the present invention regardless of how they are prepared, for example, synthetically, via a biological process (e.g., metabolic or enzymatic transformation), or via a combination thereof.

[0038] All compounds and their pharma- ceutically acceptable salts may be found together with other substances such as water and solvents (e.g., hydrates and solvates) or may be isolated. When in the solid state, the compounds and their salts described herein may occur in various forms, for example, in the form of a solvate, including a hydrate. The compounds may be in any solid form, such as a polymorph or solvate, and therefore, unless otherwise expressly indicated, references herein to the compounds and their salts should be understood to include any solid form of the compounds.

[0039] In some embodiments, the compound of the present invention or its salt is substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which the compound was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present invention. Substantial separation can include a composition that contains at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compound of the present invention or its salt.

[0040] The phrase "pharmacologically acceptable" is employed herein to refer to compounds, substances, compositions, and / or dosage forms that are, within the scope of safe medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio.

[0041] The expressions "ambient temperature" and "room temperature" as used herein are understood in the art and generally refer to a temperature that is about the temperature of the room in which the reaction is carried out, e.g., a temperature of about 20° C. to about 30° C., e.g., the reaction temperature.

[0042] The present invention also includes pharma- ceutically acceptable salts of the compounds described herein. The term "pharma- ceutically acceptable salts" refers to derivatives of the disclosed compounds, where the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharma- ceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharma- ceutically acceptable salts of the present invention include non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharma- ceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two, generally with non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol) or acetonitrile (MeCN) being preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17, pp. 171-175, 1997. th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19, and Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). In some embodiments, the compounds described herein include N-oxide forms.

[0043] synthesis The compounds of the present invention, including their salts, may be prepared using known organic synthesis techniques, or may be synthesized according to any of a number of possible synthetic routes, such as those in the following schemes.

[0044] The reaction for preparing the compound of the present invention can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis.A suitable solvent can be substantially non-reactive with the starting material (reactant), intermediate or product at the temperature at which the reaction is carried out, which can range from the freezing temperature of the solvent to the boiling temperature of the solvent.A given reaction can be carried out in one solvent or a mixture of more than one solvent.Depending on the specific reaction step, a suitable solvent for a specific reaction step can be selected by those skilled in the art.

[0045] Preparation of the compounds of the present invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one of ordinary skill in the art. The chemistry of protecting groups can be found, for example, in Kocienski, Protecting Groups, (Thieme, 2007); Robertson, Protecting Group Chemistry, (Oxford University Press, 2000); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 6 th Ed. (Wiley, 2007), Peturssion et al., “Protective Groups in Carbohydrate Chemistry,” J. Chem. Educ., 1997, 74(11), 1297, and Wuts et al., Protective Groups in Organic Synthesis, 4th Ed., (Wiley, 2006).

[0046] The reaction can be monitored according to any suitable method known in the art. For example, product formation can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13C), by spectroscopic means such as infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).

[0047] The following schemes provide general guidance relating to the preparation of compounds of the invention. Those skilled in the art will appreciate that the preparations shown in the schemes can be modified or optimized using general knowledge of organic chemistry to prepare various compounds of the invention. [ka]

[0048] Compounds of formula 1-17 can be prepared via the synthetic route outlined in Scheme 1. Esterification of commercially available starting material 1-1 with H2SO4 in ethanol. Halogenation of compound 1-2 with a suitable reagent such as N-chlorosuccinimide (NCS) results in intermediate 1-3 (Hal is a halide such as F, Cl, Br, or I). Compound 1-5 can be prepared by treating 1-3 with a reagent such as ethyl malonyl chloride (1-4). Intermediate 1-5 can undergo a cyclization reaction (such as sodium ethoxide in ethanol) to produce compounds 1-6, which can be treated with a suitable reagent (e.g., POCl3) to provide compounds 1-7. Condensation of intermediate 1-7 with amine 1-8 (PG is a suitable protecting group such as Boc) can be carried out to produce compound 1-9. Reduction of the ester with a reducing reagent (such as DIBAL), followed by oxidation of the intermediate with an oxidizing reagent (such as Dess-Martin periodinane) gives the aldehyde 1-10. Treatment of intermediate 1-10 with hydroxylamine hydrochloride and pyridine gives compound 1-11. Intermediate 1-11 can undergo a cyclization reaction (e.g., methanesulfonyl chloride, aminopyridine in DCM) to produce compounds 1-12. The R 3 The group is S NThe intermediate 1-13 can be placed via a suitable transformation such as an Ar reaction or a coupling reaction. The intermediate 1-13 can first undergo deprotection of the protecting group PG, followed by functionalization of the resulting amine (e.g., coupling with an acid chloride, e.g., acryloyl chloride), and then provide compound 1-14. The halogen of 1-14 (Hal) can be optionally converted to R via transition metal-mediated coupling or other suitable methods. 2 to give 1-15. The desired product 1-17 can be prepared by a cross-coupling reaction between 1-15 and an adduct of formula 1-16 under standard Suzuki cross-coupling conditions (e.g., in the presence of a palladium catalyst and a suitable base), or under standard Stille cross-coupling conditions (e.g., in the presence of a palladium catalyst), or under standard Negishi cross-coupling conditions (e.g., in the presence of a palladium catalyst), where M is a boronic acid, a boronic ester, or an appropriately substituted metal (e.g., M is B(OR)2, Sn(alkyl)3, or Zn-Hal). The sequence of the above chemical reactions can be rearranged appropriately to suit the preparation of different analogs.

[0049] KRAS protein The Ras family consists of three members: KRAS, NRAS, and HRAS. RAS-mutated cancers account for approximately 25% of human cancers. KRAS is the most frequently mutated isoform in human cancers: 85% of all RAS mutations are found in KRAS, 12% in NRAS, and 3% in HRAS (Simanshu, D. et al. Cell 170.1 (2017): 17-33). KRAS mutations are prevalent among the top three most lethal cancer types: pancreatic cancer (97%), colorectal cancer (44%), and lung cancer (30%) (Cox, AD et al. Nat Rev Drug Discov (2014) 13: 828-51). The majority of RAS mutations occur at amino acid residues / codons 12, 13, and 61, with codon 12 mutations being the most frequent in KRAS. The frequency of specific mutations that vary between RAS genes and G12D mutations is most prevalent in KRAS, whereas Q61R and G12R mutations are most frequent in NRAS and HRAS. Furthermore, the spectrum of mutations in RAS isoforms differs between cancer types. For example, G12D mutations in KRAS are prevalent in pancreatic cancer (51%), followed by colorectal adenocarcinoma (45%) and lung cancer (17%) (Cox, AD et al. Nat Rev Drug Discov (2014) 13:828-51). In contrast, G12C mutations in KRAS predominate in non-small cell lung cancer (NSCLC), which constitutes 11-16% of lung adenocarcinomas (nearly half of mutant KRAS are G12C), as well as in 2-5% of pancreatic and colorectal adenocarcinomas, respectively (Cox, AD et al. Nat. Rev. Drug Discov. (2014) 13:828-51). Genomic studies, using shRNA knockdown of thousands of genes across hundreds of cancer cell lines, have demonstrated that cancer cells exhibiting KRAS mutations are highly dependent on KRAS function for cell proliferation (McDonald, R. et al. Cell 170 (2017):577-592). Taken together, these findings suggest that KRAS mutations play an important role in human cancer, and therefore, the development of inhibitors targeting mutant KRAS may be useful in the clinical treatment of diseases characterized by KRAS mutations.

[0050] How to use Cancer types involving KRAS with G12C, G12V, and G12D mutations include, but are not limited to, carcinomas (e.g., pancreatic carcinoma, colorectal carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical skin carcinoma, thyroid carcinoma), hematopoietic malignancies (e.g., myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), chronic and juvenile myelomonocytic leukemia (CMML and JMML), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), and multiple myeloma (MM)), and other neoplasms (e.g., glioblastoma and sarcoma). In addition, KRAS mutations have been found in acquired resistance to anti-EGFR therapy (Knickelbein, K. et al. Genes & Cancer, (2015): 4-12). KRAS mutations have been found in immunological and inflammatory disorders (Fernandez-Medarde, A. et al. Genes & Cancer, (2011):344-358), such as Ras-associated lymphoproliferative disorder (RALD) or juvenile myelomonocytic leukemia (JMML), caused by somatic mutations in KRAS or NRAS.

[0051] The compound of the present disclosure can inhibit the activity of KRAS protein.For example, the compound of the present disclosure can be used to inhibit the activity of KRAS in cells or individuals or patients that require enzyme inhibition by administering to cells, individuals or patients an inhibitory amount of one or more compounds of the present disclosure.

[0052] As KRAS inhibitors, the compounds of the present disclosure are useful for treating various diseases associated with abnormal expression or activity of KRAS. Compounds that inhibit KRAS are useful for providing a means of preventing growth or inducing apoptosis in tumors by inhibiting angiogenesis. It is therefore expected that the compounds of the present disclosure will prove useful for treating or preventing proliferative disorders such as cancer. In particular, tumors that have activating mutants of receptor tyrosine kinases or have upregulation of receptor tyrosine kinases may be particularly sensitive to inhibitors.

[0053] Provided herein in an aspect is a method of inhibiting KRAS activity, comprising contacting a compound of the present disclosure with KRAS. In one embodiment, the contacting comprises administering the compound to a patient.

[0054] Provided herein, in one aspect, is a method of inhibiting a KRAS protein having a G12C mutation, the method comprising contacting KRAS with a compound of the present disclosure.

[0055] Provided herein in certain aspects is a method of inhibiting a KRAS protein having a G12D mutation, said method comprising contacting KRAS with a compound of the present disclosure.

[0056] Provided herein in one aspect is a method of inhibiting a KRAS protein having a G12V mutation, the method comprising contacting KRAS with a compound of the present disclosure.

[0057] Provided herein, in another aspect, is a method for treating a disease or disorder associated with inhibition of KRAS interaction, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of any of the formulas disclosed herein, or a pharma- ceutically acceptable salt thereof.

[0058] In yet another aspect, there is provided herein a method for treating a disease or disorder associated with inhibiting a KRAS protein having a G12C mutation, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of any of the formulas disclosed herein, or a pharma- ceutically acceptable salt thereof.

[0059] Also provided herein, in yet another aspect, is a method of treating cancer in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of a compound disclosed herein, wherein the cancer is characterized by interaction with a KRAS protein having a G12C mutation.

[0060] In yet another aspect, there is provided herein a method for treating a disease or disorder associated with inhibiting a KRAS protein having a G12D mutation, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of any of the formulas disclosed herein, or a pharma- ceutically acceptable salt thereof.

[0061] Provided herein, in yet another aspect, is a method of treating cancer in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of a compound disclosed herein, wherein the cancer is characterized by interaction with a KRAS protein having a G12D mutation.

[0062] In yet another aspect, there is provided herein a method for treating a disease or disorder associated with inhibiting a KRAS protein having a G12V mutation, comprising administering to a patient in need of treatment a therapeutically effective amount of a compound of any of the formulas disclosed herein, or a pharma- ceutically acceptable salt thereof.

[0063] Provided herein, in yet another aspect, is a method of treating cancer in a patient in need of such treatment comprising administering to the patient a therapeutically effective amount of a compound disclosed herein, wherein the cancer is characterized by interaction with a KRAS protein having a G12V mutation.

[0064] Provided herein in yet another aspect is a method of treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of any one of the compounds disclosed herein, or a pharma- ceutically acceptable salt thereof.

[0065] Provided herein in certain aspects is a method of treating a disease or disorder associated with inhibition of KRAS interaction or a mutant thereof in a patient in need thereof, the method comprising administering to the patient a compound disclosed herein or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound disclosed herein or a pharma- ceutically acceptable salt thereof, in combination with another therapy or treatment described herein.

[0066] In one embodiment, the disease or disorder is an immunological or inflammatory disorder. In another embodiment, the immunological or inflammatory disorder is a Ras-associated lymphoproliferative disorder caused by somatic mutations in KRAS and juvenile myelomonocytic leukemia.

[0067] In one embodiment, the cancer is selected from hematological cancer, sarcoma, lung cancer, gastrointestinal cancer, genitourinary tract cancer, liver cancer, bone cancer, nervous system cancer, gynecological cancer, and skin cancer.

[0068] In another embodiment, the cancer is selected from carcinoma, hematological cancer, sarcoma, and glioblastoma.

[0069] In yet another embodiment, the hematological cancer is selected from myeloproliferative neoplasms, myelodysplastic syndromes, chronic and juvenile myelomonocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, and multiple myeloma.

[0070] In yet another embodiment, the carcinoma is selected from pancreatic carcinoma, colon carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical carcinoma, skin carcinoma, and thyroid carcinoma.

[0071] In another embodiment, the lung cancer is selected from non-small cell lung cancer (NSCLC), small cell lung carcinoma, bronchogenic carcinoma, squamous cell bronchogenic carcinoma, undifferentiated small cell bronchogenic carcinoma, undifferentiated large cell bronchogenic carcinoma, adenocarcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchial carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular carcinoma and non-pavicellular carcinoma, bronchial adenoma, and pleuropulmonary blastoma.

[0072] In yet another embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In yet another embodiment, the lung cancer is adenocarcinoma.

[0073] In one embodiment, the gastrointestinal cancer is selected from esophageal squamous cell carcinoma, esophageal adenocarcinoma, esophageal leiomyosarcoma, esophageal lymphoma, gastric carcinoma, gastric lymphoma, gastric leiomyosarcoma, exocrine pancreatic carcinoma, pancreatic ductal adenocarcinoma, pancreatic insulinoma, pancreatic glucagonoma, pancreatic gastrinoma, pancreatic carcinoid tumor, pancreatic vipoma, small intestinal adenocarcinoma, small intestinal lymphoma, small intestinal carcinoid tumor, Kaposi's sarcoma, small intestinal leiomyoma, small intestinal hemangioma, small intestinal lipoma, small intestinal neurofibroma, small intestinal fibroma, colon adenocarcinoma, colon ductal adenoma, colonic villous adenoma, colonic hamartoma, colonic leiomyoma, colon carcinoma, gallbladder carcinoma, and anal carcinoma.

[0074] In one embodiment, the gastrointestinal cancer is colorectal cancer.

[0075] In another embodiment, the cancer is a carcinoma. In yet another embodiment, the carcinoma is selected from pancreatic cancer, colon carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical skin carcinoma, and thyroid carcinoma.

[0076] In yet another embodiment, the cancer is a hematopoietic malignancy. In one embodiment, the hematopoietic malignancy is selected from multiple myeloma, acute myeloid leukemia, and myeloproliferative neoplasm.

[0077] In another embodiment, the cancer is a neoplasm. In yet another embodiment, the neoplasm is a glioblastoma or a sarcoma.

[0078] In certain embodiments, the disclosure provides a method of treating a KRAS-mediated disorder in a patient in need of such treatment, the method comprising administering to said patient a compound according to the invention or a pharma- ceutically acceptable composition thereof.

[0079] In some embodiments, diseases and indications treatable using compounds of the present disclosure include, but are not limited to, hematological cancers, sarcomas, lung cancer, gastrointestinal cancer, genitourinary cancer, liver cancer, bone cancer, nervous system cancer, gynecological cancer, and skin cancer.

[0080] Exemplary hematological cancers include lymphomas and leukemias, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, non-Hodgkin's lymphoma (including relapsed or refractory NHL and relapsed follicular), Hodgkin's lymphoma, myeloproliferative disorders (e.g. These include primary myelofibrosis (PMF), polycythemia vera (PV), essential thrombocytosis (ET), 8p11 myeloproliferative syndrome, myelodysplastic syndromes (MDS), T-cell acute lymphoblastic lymphoma (T-ALL), multiple myeloma, cutaneous T-cell lymphoma, adult T-cell lymphoma, Waldenstrom's macroglobulinemia, hairy cell lymphoma, marginal zone lymphoma, chronic myelogenous lymphoma, and Burkitt's lymphoma.

[0081] Exemplary sarcomas include chondrosarcoma, Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdomyosarcoma, fibroma, lipoma, hamartoma, lymphosarcoma, leiomyosarcoma, and teratoma.

[0082] Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer, bronchial carcinoma (squamous cell, small undifferentiated cell, large undifferentiated cell, adenocarcinoma), alveolar (bronchial) carcinoma, bronchial adenoma, chondromatous hamartoma, mesothelioma, pavicellular and non-small cell carcinoma, bronchial adenoma, and pleuropulmonary blastoma.

[0083] Exemplary gastrointestinal cancers include cancer of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), cancer of the stomach (carcinoma, lymphoma, leiomyosarcoma), cancer of the pancreas (exocrine pancreatic carcinoma, ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, vipoma), cancer of the small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), cancer of the colon (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon carcinoma, gallbladder carcinoma, and anal carcinoma.

[0084] Exemplary genitourinary tract cancers include cancer of the kidney (adenocarcinoma, Wilms' tumor [nephroblastoma], renal cell carcinoma), cancer of the bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), cancer of the prostate (adenocarcinoma, sarcoma), cancer of the testes (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, lipoma), and urothelial carcinoma.

[0085] Exemplary liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

[0086] Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (osteochondral exostosis), benign cartilage tumor, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, and giant cell tumor.

[0087] Exemplary nervous system cancers include cancer of the skull (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), cancer of the meninges (meningioma, meningeal sarcoma, gliomatosis), cancer of the brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma, glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, neuroectodermal tumors), and cancer of the spinal cord (neurofibroma, meningioma, glioma, sarcoma), neuroblastoma and Lhermitte-Dacros disease and pineal tumor.

[0088] Exemplary gynecologic cancers include cancer of the breast (ductal carcinoma, lobular carcinoma, breast sarcoma, triple-negative breast cancer, HER2-positive breast cancer, inflammatory breast cancer, papillary carcinoma), cancer of the uterus (endometrial carcinoma), cancer of the cervix (cervical carcinoma, preneoplastic cervical dysplasia), cancer of the ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granular-squamous cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), cancer of the vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), cancer of the vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma)), and cancer of the fallopian tubes (carcinoma).

[0089] Exemplary skin cancers include melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, Merkel cell skin cancer, lenticular dysplasia nevus, lipoma, hemangioma, dermatofibroma, and keloids.

[0090] Exemplary head and neck cancers include glioblastoma, melanoma, rhabdomyosarcoma, lymphosarcoma, osteosarcoma, squamous cell carcinoma, adenocarcinoma, oral cancer, laryngeal cancer, nasopharyngeal cancer, nasal and paranasal sinus cancer, thyroid and parathyroid cancer, eye tumors, lip and oral cavity tumors, and squamous cell head and neck cancer.

[0091] Compounds of the present disclosure may also be useful in inhibiting tumor metastasis.

[0092] In addition to oncogenic neoplasms, the compounds of the present invention are useful for treating skeletal and chondrocyte disorders, including, but not limited to, achondroplasia, hypochondroplasia, dwarfism, thanatophoric dysplasia (TD) (clinical forms TDI and TDII), Apert syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrate syndrome, Pfeiffer syndrome, and craniosynostosis syndrome. In some embodiments, the present disclosure provides a method of treating a patient suffering from a skeletal and chondrocyte disorder.

[0093] In some embodiments, the compounds described herein can be used to treat Alzheimer's disease, HIV, or tuberculosis.

[0094] As used herein, the term "8p11 myeloproliferative syndrome" is meant to refer to myeloid / lymphoid neoplasms associated with eosinophilia and FGFR1 abnormalities.

[0095] As used herein, the term "cell" is meant to refer to an in vitro, ex vivo, or in vivo cell. In some embodiments, an ex vivo cell may be part of a tissue sample excised from an organism, such as a mammal. In some embodiments, an in vitro cell may be a cell in cell culture. In some embodiments, an in vivo cell is a cell that lives in an organism, such as a mammal.

[0096] As used herein, the term "contacting" refers to bringing together the indicated moieties in an in vitro system or in an in vivo system. For example, "contacting" KRAS with a compound described herein includes administering a compound described herein to an individual or patient, such as a human, having KRAS, as well as, for example, introducing a compound described herein into a sample, including a cell preparation or purified preparation containing KRAS.

[0097] As used herein, the terms "individual," "subject," or "patient," used interchangeably, refer to any animal, including a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, and most preferably a human.

[0098] As used herein, the phrase "therapeutically effective amount" refers to an amount of an active compound or pharmaceutical agent, such as an amount of any of the solid forms or salts thereof disclosed herein, that elicits the biological or medicinal response in a tissue, system, animal, individual, or human that is desired by a researcher, veterinarian, physician, or other clinician. The appropriate "effective" amount in any individual case may be determined using techniques known to those of ordinary skill in the art.

[0099] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of safe medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0100] As used herein, the phrase "pharmaceutically acceptable carrier or excipient" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients or carriers are generally safe, non-toxic, and not biologically or otherwise undesirable, and include excipients or carriers that are acceptable for veterinary use as well as human pharmaceutical use. In one embodiment, each component is "pharmaceutically acceptable" as defined herein. For example, Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6th ed., Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed., Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed., Gibson Ed.; CRC Press LLC: Boca Raton, Fla., 2009.

[0101] As used herein, the term "treating" or "treatment" refers to inhibiting a disease, e.g., inhibiting a disease, condition, or disorder (i.e., halting further development of the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder, or ameliorating a disease; e.g., ameliorating a disease, condition, or disorder (i.e., reversing the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder, e.g., reducing the severity of the disease.

[0102] As used herein, the terms "prevent," "preventing," or "prevention" include the prevention of at least one symptom associated with or caused by the condition, disease or disorder being prevented.

[0103] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment (wherein the embodiments are intended to be combined as described in a multiple dependent fashion). Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0104] Combination therapy I. Cancer Therapy Cancer cell growth and survival can be affected by the dysfunction of multiple signal transduction pathways.Therefore, to treat such conditions, it is useful to combine different enzyme / protein / receptor inhibitors that show different preferences in the targets that regulate their activity.Targeting more than one signal transduction pathway (or more than one biomolecule involved in a given signal transduction pathway) may reduce the possibility of drug resistance occurring in cell populations and / or reduce the toxicity of treatment.

[0105] In addition to one or more additional pharmaceutical agents, such as chemotherapeutic agents, anti-inflammatory agents, steroids, immunosuppressants, cancer immunotherapeutic agents, metabolic enzyme inhibitors, chemokine receptor inhibitors, and phosphatase inhibitors, targeted therapies, such as Bcr-Abl, Flt-3, EGFR, HER2, JAK, c-MET, VEGFR, PDGFR, c-Kit, IGF-1R, RAF, FAK, and CDK4 / 6 kinase inhibitors, such as those described in WO2006 / 056399, may be used in combination with the compounds of the present disclosure to treat CDK2-related diseases, disorders, or conditions. Other agents, such as therapeutic antibodies, may be used in combination with the compounds of the present disclosure to treat CDK2-related diseases, disorders, or conditions. One or more additional pharmaceutical agents may be administered to a patient simultaneously or sequentially.

[0106] In some embodiments, a CDK2 inhibitor is administered or used in combination with a BCL2 inhibitor or a CDK4 / 6 inhibitor.

[0107] Compounds as disclosed herein may be used in combination with one or more other enzyme / protein / receptor inhibitor therapies to treat diseases such as cancer and other diseases or disorders described herein. Examples of diseases and indications treatable with combination therapy include those described herein. Examples of cancer include solid tumors and non-solid tumors such as liquid tumors, blood cancers. Examples of infectious diseases include viral infections, bacterial infections, fungal infections or parasitic infections. For example, compounds of the present disclosure may be combined with one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, BCL2, CDK4 / 6, TGF-βR, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IDH2, IGF-1R, IR-R, PDGFαR, PDGFβR, PI3K (alpha, beta, gamma, delta, and multiple or selective), CSF1R, KIT, FLK-II, KDR / FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, PARP, Ron, Sea, TRKA, TRKB, TRKC, TAM kinases (Axl, Mer, Tyro3), FLT3, VEGFR / Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK, and B-Raf. In some embodiments, the compounds of the present disclosure may be combined with one or more of the following inhibitors to treat cancer or an infectious disease: Non-limiting examples of inhibitors that may be combined with the compounds of the present disclosure for the treatment of cancer and infectious diseases include FGFR inhibitors (FGFR1, FGFR2, FGFR3, or FGFR4, e.g., pemigatinib (INCB54828), INCB62079), EGFR inhibitors (also known as ErB-1 or HER-1, e.g., erlotinib, gefitinib, vandetanib, osimertinib, cetuximab, necitumumab, or panitumumab), VEGFR inhibitors or pathway blockers (e.g., bevacizumab, pazopanib, sunitinib, sorafenib, axitinib,regorafenib, ponatinib, cabozantinib, vandetanib, ramucirumab, lenvatinib, ziv-aflibercept), PARP inhibitors (e.g., olaparib, rucaparib, veliparib, or niraparib), JAK inhibitors (JAK1 and / or JAK2, e.g., ruxolitinib or baricitinib; or JAK1, e.g., itacitinib (INCB39110), INCB052793, or INCB052794), 54707), IDO inhibitors (e.g., epacadostat, NLG919, or BMS-986205, MK7162), LSD1 inhibitors (e.g., GSK2979552, INCB59872 and INCB60003) TDO inhibitors, PI3K-delta inhibitors (e.g., palsaclisib (INCB50465) or INCB50797), PI3K-gamma inhibitors such as PI3K-gamma selective inhibitors, Pim inhibitors (e.g., INCB53914), CSF1R inhibitors, TAM receptor tyrosine kinases (Tyro-3, Axl, and Mer, e.g., INCB081776), adenosine receptor antagonists (e.g., A2a / A2b receptor antagonists), HPK1 inhibitors, chemokine receptor inhibitors (e.g., CCR2 or CCR5 inhibitors), SHP1 / 2 phosphatase inhibitors, histone deacetylase inhibitors (HDACs) such as HDAC8 inhibitors, angiogenesis inhibitors, interleukin receptor inhibitors, bromo and extra-terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors, e.g., INCB54329 and INCB57643), c-MET inhibitors (e.g., capmatinib), anti-CD19 antibodies (e.g., tafasitamab), ALK2 inhibitors (e.g., INCB00928), or combinations thereof.

[0108] In some embodiments, the compounds or salts described herein are administered with a PI3Kδ inhibitor. In some embodiments, the compounds or salts described herein are administered with a JAK inhibitor. In some embodiments, the compounds or salts described herein are administered with a JAK1 or JAK2 inhibitor (e.g., baricitinib or ruxolitinib). In some embodiments, the compounds or salts described herein are administered with a JAK1 inhibitor. In some embodiments, the compounds or salts described herein are administered with a JAK1 inhibitor that is more selective than JAK2.

[0109] Examples of antibodies for use in combination therapy include, but are not limited to, trastuzumab (e.g., anti-HER2), ranibizumab (e.g., anti-VEGF-A), bevacizumab (AVASTIN™, e.g., anti-VEGF), panitumumab (e.g., anti-EGFR), cetuximab (e.g., anti-EGFR), Rituxan (e.g., anti-CD20), and antibodies against c-MET.

[0110] One or more of the following agents may be used in combination with the compounds of the present disclosure, which are presented as a non-limiting list: cytostatic agents, cisplatin, doxorubicin, taxotere, taxol, etoposide, irinotecan, camptosar, topotecan, paclitaxel, docetaxel, epothilone, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH66336, R115777, L778,123, BMS214662, IRESSA™ (gefitinib), TARCEVA™ (erlotinib), antibodies against EGFR, intron, ara-C, adriamycin, cytoxan, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipou Broman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, oxaliplatin, leucovirin, ELOXATIN™ (oxaliplatin), pentostatine, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin, mitomycin C, L-asparaginase, teniposide 17. alpha.-Ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, goserelin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, navelbene, anastrozole, letrazole, capecitabine, reloxafine, droloxafine, hexamethylmelamine, avastin, HERCEPTIN ( VELCADE™ (bortezomib), ZEVALIN™ (ibritumomab tiuxetan), TRISENOX™ (arsenic trioxide), XELODA™ (capecitabine), vinorelbine, porfimer, ERBITUX™ (cetuximab), thiotepa, altretamine, melphalan, trastuzumab, lerozole, Fulvestrant, exemestane, ifosfomide, rituximab, C225 (cetuximab), campath (alemtuzumab), clofarabine, cladribine, aphidicolon, rituxan, sunitinib, dasatinib, tezacitabine, Sml1, fludarabine, pentostatin, triapin, didox, trimidox, amidox, 3-AP, and MDL-101,731.

[0111] The compounds of the present disclosure may also be used in combination with other methods of treating cancer, for example, by chemotherapy, radiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, or surgery. Examples of immunotherapy include cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2), CRS-207 immunotherapy, cancer vaccines, monoclonal antibodies, bispecific or multispecific antibodies, antibody-drug conjugates, adoptive T cell transfer, Toll receptor agonists, RIG-I agonists, oncolytic virus therapy, and immunomodulatory small molecules, including thalidomide or JAK1 / 2 inhibitors, PI3Kδ inhibitors, etc. The compounds may be administered in combination with one or more anti-cancer drugs, for example, chemotherapeutic agents, etc. Examples of chemotherapy drugs include abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, baricitinib, bleomycin, bortezomib, intravenous busulfan, oral busulfan, calcitinib, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, and exonuclease. Lizumab, epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alpha 2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, mechlorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone,Nandrolone phenpropionate, nelarabine, nofetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, and zoledronate.

[0112] Additional examples of chemotherapeutic agents include proteasome inhibitors (eg, bortezomib), thalidomide, REVLIMID, and DNA damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine, and the like.

[0113] Exemplary steroids include corticosteroids, such as dexamethasone or prednisone.

[0114] Exemplary Bcr-Abl inhibitors include imatinib mesylate (GLEEVAC™), nilotinib, dasatinib, bosutinib, and ponatinib, and pharmaceutically acceptable salts. Other exemplary suitable Bcr-Abl inhibitors include the genera and species of compounds disclosed in U.S. Patent No. 5,521,184, WO04 / 005281, and U.S. Patent No. 60 / 578,491, and pharmaceutically acceptable salts thereof.

[0115] Exemplary suitable Flt-3 inhibitors include midostaurin, lestaurtinib, linifanib, sunitinib, sunitinib, maleate, sorafenib, quizartinib, crenolanib, pacritinib, tanzutinib, PLX3397 and ASP2215, and pharmaceutically acceptable salts thereof. Other exemplary suitable Flt-3 inhibitors include compounds as disclosed in WO03 / 037347, WO03 / 099771, and WO04 / 046120, and pharmaceutically acceptable salts thereof.

[0116] Exemplary and suitable RAF inhibitors include dabrafenib, sorafenib, and vemurafenib, and pharmaceutically acceptable salts thereof. Other exemplary and suitable RAF inhibitors include compounds as disclosed in WO00 / 09495 and WO05 / 028444, and pharmaceutically acceptable salts thereof.

[0117] Exemplary suitable FAK inhibitors include VS-4718, VS-5095, VS-6062, VS-6063, BI853520, and GSK2256098, and pharma- ceutically acceptable salts thereof. Other exemplary suitable FAK inhibitors include those compounds disclosed in WO04 / 080980, WO04 / 056786, WO03 / 024967, WO01 / 064655, WO00 / 053595, and WO01 / 014402, and pharma- ceutically acceptable salts thereof.

[0118] Exemplary and suitable CDK4 / 6 inhibitors include palbociclib, ribociclib, trilaciclib, relociclib, and abemaciclib, and pharmaceutically acceptable salts thereof. Other exemplary and suitable CDK4 / 6 inhibitors include compounds as disclosed in WO09 / 085185, WO12 / 129344, WO11 / 101409, WO03 / 062236, WO10 / 075074, and WO12 / 061156, and pharmaceutically acceptable salts thereof.

[0119] In some embodiments, compounds of the present disclosure may be used in combination with one or more other kinase inhibitors, including imatinib, particularly to treat patients who are resistant to imatinib or other kinase inhibitors.

[0120] In some embodiments, the compounds of the present disclosure may be used in combination with chemotherapeutic agents in the treatment of cancer, and may improve treatment response compared to the response of the chemotherapeutic agent alone, without exacerbating its toxic effects. In some embodiments, the compounds of the present disclosure may be used in combination with chemotherapeutic agents provided herein. For example, additional pharmaceutical agents used in the treatment of multiple myeloma may include, but are not limited to, melphalan, melphalan and prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Further additional agents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3, RAF, and FAK kinase inhibitors. In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of alkylating agents include cyclophosphamide (CY), melphalan (MEL), and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM).Additive or synergistic effects are desirable outcomes when CDK2 inhibitors of the present disclosure are combined with additional agents.

[0121] The agents may be combined with the compound in a single or sequential dosage form, or the agents may be administered simultaneously or sequentially as separate dosage forms.

[0122] The compounds of the present disclosure can be used in combination with one or more other inhibitors or one or more therapies for the treatment of infectious diseases, including viral, bacterial, fungal or parasitic infections.

[0123] In some embodiments, a corticosteroid such as dexamethasone is administered to a patient in combination with a compound of the present disclosure, and the dexamethasone is administered intermittently rather than continuously.

[0124] A compound of formula (I) or any of the formulae described herein, a compound recited in any of the claims and described herein, or a salt thereof, can be combined with another immunogenic agent, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines. Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MARTI and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

[0125] Compounds of formula (I) or any of the formulas described herein, compounds recited in any of the claims and described herein, or salts thereof, can be used in combination with vaccination protocols for the treatment of cancer. In some embodiments, tumor cells are transduced to express GM-CSF. In some embodiments, tumor vaccines include proteins derived from viruses implicated in human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). In some embodiments, compounds of the present disclosure can be used in combination with tumor-specific antigens, such as heat shock proteins isolated from the tumor tissue itself. In some embodiments, compounds of formula (I) or any of the formulas described herein, compounds recited in any of the claims and described herein, or salts thereof can be combined with dendritic cell immunity to activate a strong anti-tumor response.

[0126] The compounds of the present disclosure can be used in combination with bispecific macrocyclic peptides that target tumor cells to Fe alpha or Fe gamma receptor expressing effector cells. The compounds of the present disclosure can also be combined with macrocyclic peptides that activate host immune responsiveness.

[0127] In some further embodiments, the combination of the disclosed compounds and other therapeutic agents may be administered to a patient before, during, and / or after bone marrow or stem cell transplantation. The disclosed compounds can be used in combination with bone marrow transplantation to treat various tumors of hematopoietic origin.

[0128] The compounds of formula (I) or any of the formulas described herein, the compounds cited in any of the claims and described herein, or salts thereof, can be used in combination with vaccines to stimulate immune responses against pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include those for which there is currently no effective vaccine, or for which conventional vaccines are not completely effective. These include, but are not limited to, HIV, hepatitis (types A, B, and C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, and Pseudomonas Aeruginosa.

[0129] Viruses causing infectious diseases treatable by the methods of the present disclosure include, but are not limited to, human papillomavirus, influenza, hepatitis A, B, C or D virus, adenovirus, poxvirus, herpes simplex virus, human cytomegalovirus, severe acute respiratory syndrome virus, Ebola virus, measles virus, herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), flavivirus, echovirus, rhinovirus, coxsackievirus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, infectious virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0130] Pathogenic bacteria causing infections treatable by the methods of the present disclosure include, but are not limited to, Chlamydia, Rickettsia bacteria, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Conococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, and Lyme Disease bacteria.

[0131] Pathogenic fungi causing infections treatable by the methods of the present disclosure include, but are not limited to, Candida (such as albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (such as fumigatus and niger), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0132] Pathogenic parasites causing infections treatable by the methods of the present disclosure include, but are not limited to, Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis.

[0133] When more than one pharmaceutical agent is administered to a patient, they may be administered simultaneously, separately, sequentially, or in combination (eg, in the case of more than two agents).

[0134] Methods for safely and effectively administering most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in standard texts. For example, the administration of many chemotherapeutic agents is described in the "Physicians' Desk Reference" (PDR, e.g., 1996 edition, Medical Economics Company, Montvale, NJ), the disclosure of which is incorporated herein by reference as if set forth in its entirety.

[0135] II. Immune checkpoint therapy The compounds of the present disclosure may be used in combination with one or more immune checkpoint inhibitors to treat diseases, such as cancer or infectious diseases. Exemplary immune checkpoint inhibitors include inhibitors against immune checkpoint molecules, such as CBL-B, CD20, CD28, CD40, CD70, CD122, CD96, CD73, CD47, CDK2, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, HPK1, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, TLR (TLR7 / 8), TIGIT, CD112R, VISTA, PD-1, PD-L1, and PD-L2. In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40, GITR, and CD137. In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, TIGIT, and VISTA. In some embodiments, the compounds provided herein may be used in combination with one or more agents selected from KIR inhibitors, TIGIT inhibitors, LAIR1 inhibitors, CD160 inhibitors, 2B4 inhibitors, and TGFR beta inhibitors.

[0136] In some embodiments, the compounds provided herein may be used in combination with one or more agonists of immune checkpoint molecules, such as OX40, CD27, GITR, and CD137 (also known as 4-1BB).

[0137] In some embodiments, the inhibitor of an immune checkpoint molecule is an anti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0138] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1 or PD-L1, such as an anti-PD-1 or anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-1 or anti-PD-L1 antibody is selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, cemiplimab, atezolizumab, avelumab, tislelizumab, spartalizumab (PDR001), cetrelimab (JNJ-63723283), toripalimab (JS001), camrelizumab (SHR-1210), sintilimab (IBI308), AB122 (GLS-010), AMP-224, AMP-514 / MEDI- 0680, BMS936559, JTX-4014, BGB-108, SHR-1210, MEDI4736, FAZ053, BCD-100, KN035, CS1001, BAT1306, LZM009, AK105, HLX10, SHR-1316, CBT-502(TQB2450), A167(KL-A167), STI-A101(ZKAB001), CK-301, BGB-A333, MSB-2311, HLX20, TSR-042, or LY3300054.In some embodiments, the PD-1 or PD-L1 inhibitor is a compound described in U.S. Pat. Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, or 10,308,644; U.S. Publication Nos. 2017 / 0145025, 2017 / 0174671, and 2017 / 0174679. , No. 2017 / 0320875, No. 2017 / 0342060, No. 2017 / 0362253, No. 2018 / 0016260, No. 2018 / 0057486, No. 2018 / 0177784, 2018 / 0177870, 2018 / 0179179, 2018 / 0179201, 2018 / 0179202, 2018 No. 2019 / 0273519, No. 2019 / 0040082, No. 2019 / 0062345, No. 2019 / 0071439, No. 2019 / 0127467, No. 2019 / 0144439, No. 2019 / 0202824, No. 2019 / 0225601, No. 2019 / 0300524, or No. 2019 / 0345170, or PCT Publication No. and / or WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, or WO2011161699, each of which is incorporated by reference in its entirety. In some embodiments, the inhibitor of PD-L1 is INCB086550.

[0139] In some embodiments, the PD-L1 inhibitor is selected from the compounds of Table A, or a pharma- ceutically acceptable salt thereof. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

[0140] In some embodiments, the antibody is an anti-PD-1 antibody, e.g., an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, cetrelimab, toripalimab, sintilimab, AB122, AMP-224, JTX-4014, BGB-108, BCD-100, BAT1306, LZM009, AK105, HLX10, or TSR-042. In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, cetrelimab, toripalimab, or sintilimab. In some embodiments, the anti-PD-1 antibody is pembrolizumab. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is cemiplimab. In some embodiments, the anti-PD-1 antibody is spartalizumab. In some embodiments, the anti-PD-1 antibody is camrelizumab. In some embodiments, the anti-PD-1 antibody is cetrelimab. In some embodiments, the anti-PD-1 antibody is toripalimab. In some embodiments, the anti-PD-1 antibody is sintilimab. In some embodiments, the anti-PD-1 antibody is AB122. In some embodiments, the anti-PD-1 antibody is AMP-224. In some embodiments, the anti-PD-1 antibody is JTX-4014. In some embodiments, the anti-PD-1 antibody is BGB-108. In some embodiments, the anti-PD-1 antibody is BCD-100. In some embodiments, the anti-PD-1 antibody is BAT1306. In some embodiments, the anti-PD-1 antibody is LZM009. In some embodiments, the anti-PD-1 antibody is AK105. In some embodiments, the anti-PD-1 antibody is HLX10. In some embodiments, the anti-PD-1 antibody is TSR-042. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD-1 monoclonal antibody is MGA012 (INCMGA0012, retifanlimab). In some embodiments, the anti-PD1 antibody is SHR-1210.Other anti-cancer agent(s) include antibody therapeutics such as 4-1BB (e.g., urelumab, utomirumab). In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, e.g., an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is atezolizumab, avelumab, durvalumab, tislelizumab, BMS-935559, MEDI4736, atezolizumab (also known as MPDL3280A; RG7446), avelumab (MSB0010718C), FAZ053, KN035, CS1001, SHR-1316, CBT-502, A167, STI-A101, CK-301, BGB-A333, MSB-2311, HLX20, or LY3300054. In some embodiments, the anti-PD-L1 antibody is atezolizumab, avelumab, durvalumab, or tislelizumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is durvalumab. In some embodiments, the anti-PD-L1 antibody is tislelizumab. In some embodiments, the anti-PD-L1 antibody is BMS-935559. In some embodiments, the anti-PD-L1 antibody is MEDI4736. In some embodiments, the anti-PD-L1 antibody is FAZ053. In some embodiments, the anti-PD-L1 antibody is KN035. In some embodiments, the anti-PD-L1 antibody is CS1001. In some embodiments, the anti-PD-L1 antibody is SHR-1316. In some embodiments, the anti-PD-L1 antibody is CBT-502. In some embodiments, the anti-PD-L1 antibody is A167. In some embodiments, the anti-PD-L1 antibody is STI-A101. In some embodiments, the anti-PD-L1 antibody is CK-301. In some embodiments, the anti-PD-L1 antibody is BGB-A333. In some embodiments, the anti-PD-L1 antibody is MSB-2311. In some embodiments, the anti-PD-L1 antibody is HLX20. In some embodiments, the anti-PD-L1 antibody is LY3300054.

[0141] In some embodiments, the inhibitor of an immune checkpoint molecule is a small molecule that binds to PD-L1, or a pharma- ceutically acceptable salt thereof. In some embodiments, the inhibitor of an immune checkpoint molecule is a small molecule that binds to and internalizes PD-L1, or a pharma- ceutically acceptable salt thereof. In some embodiments, the inhibitor of an immune checkpoint molecule is a compound selected from among those described in US2018 / 0179201, US2018 / 0179197, US2018 / 0179179, US2018 / 0179202, US2018 / 0177784, US2018 / 0177870, US Ser. No. 16 / 369,654 (filed Mar. 29, 2019), and US Ser. No. 62 / 688,164, each of which is incorporated herein by reference in its entirety, or a pharma- ceutically acceptable salt thereof.

[0142] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of KIR, TIGIT, LAIR1, CD160, 2B4, and TGFRbeta.

[0143] In some embodiments, the inhibitor is MCLA-145.

[0144] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab, AGEN1884, or CP-675,206.

[0145] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of LAG3, such as an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016, LAG525, INCAGN2385, or eftiragimodo alpha (IMP321).

[0146] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD73. In some embodiments, the inhibitor of CD73 is oleclumab.

[0147] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIGIT. In some embodiments, the inhibitor of TIGIT is OMP-31M32.

[0148] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of VISTA. In some embodiments, the inhibitor of VISTA is JNJ-61610588 or CA-170.

[0149] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of B7-H3. In some embodiments, the inhibitor of B7-H3 is enoblituzumab, MGD009, or 8H9.

[0150] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of KIR. In some embodiments, the inhibitor of KIR is lirilumab or IPH4102.

[0151] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of A2aR. In some embodiments, the inhibitor of A2aR is CPI-444.

[0152] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TGF-beta. In some embodiments, the inhibitor of TGF-beta is travedersen, galcertinib, or M7824.

[0153] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PI3K-gamma. In some embodiments, the inhibitor of PI3K-gamma is IPI-549.

[0154] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD47. In some embodiments, the inhibitor of CD47 is Hu5F9-G4 or TTI-621.

[0155] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD73. In some embodiments, the inhibitor of CD73 is MEDI9447.

[0156] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD70. In some embodiments, the inhibitor of CD70 is cusatuzumab or BMS-936561.

[0157] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of TIM3, such as an anti-TIM3 antibody. In some embodiments, the anti-TIM3 antibody is INCAGN2390, MBG453, or TSR-022.

[0158] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD20, such as an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is obinutuzumab or rituximab.

[0159] In some embodiments, the agonist of an immune checkpoint molecule is an agonist of OX40, CD27, CD28, GITR, ICOS, CD40, TLR7 / 8, and CD137 (also known as 4-1BB).

[0160] In some embodiments, the agonist of CD137 is urelumab. In some embodiments, the agonist of CD137 is utomirumab.

[0161] In some embodiments, the agonist of the immune checkpoint molecule is an inhibitor of GITR. In some embodiments, the agonist of GITR is TRX518, MK-4166, INCAGN1876, MK-1248, AMG228, BMS-986156, GWN323, MEDI1873, or MEDI6469. In some embodiments, the agonist of the immune checkpoint molecule is an agonist of OX40, such as an OX40 agonist antibody or an OX40L fusion protein. In some embodiments, the anti-OX40 antibody is INCAGN01949, MEDI0562 (tavolimab), MOXR-0916, PF-04518600, GSK3174998, BMS-986178, or 9B12. In some embodiments, the OX40L fusion protein is MEDI6383.

[0162] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of CD40. In some embodiments, the agonist of CD40 is CP-870893, ADC-1013, CDX-1140, SEA-CD40, RO7009789, JNJ-64457107, APX-005M, or Chi Lob7 / 4.

[0163] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of ICOS. In some embodiments, the agonist of ICOS is GSK-3359609, JTX-2011, or MEDI-570.

[0164] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of CD28. In some embodiments, the agonist of CD28 is ceralizumab.

[0165] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of CD27. In some embodiments, the agonist of CD27 is varlilumab.

[0166] In some embodiments, the agonist of the immune checkpoint molecule is an agonist of TLR7 / 8. In some embodiments, the agonist of TLR7 / 8 is MEDI9197.

[0167] The compounds of the present disclosure may be used in combination with bispecific antibodies. In some embodiments, one of the domains of the bispecific antibody targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3, CD137, ICOS, CD3, or TGFβ receptor. In some embodiments, the bispecific antibody binds to PD-1 and PD-L1. In some embodiments, the bispecific antibody that binds to PD-1 and PD-L1 is MCLA-136. In some embodiments, the bispecific antibody binds to PD-L1 and CTLA-4. In some embodiments, the bispecific antibody that binds to PD-L1 and CTLA-4 is AK104.

[0168] In some embodiments, the compounds of the present disclosure may be used in combination with one or more metabolic enzyme inhibitors.In some embodiments, the metabolic enzyme inhibitor is an inhibitor of IDO1, TDO, or arginase.Examples of IDO1 inhibitors include epacadostat, NLG919, BMS-986205, PF-06840003, IOM2983, RG-70099, and LY338196.Inhibitors of arginase inhibitors include INCB1158.

[0169] As provided throughout, the additional compounds, inhibitors, agents, etc. may be combined with the compounds of the present invention in a single or continuous dosage form, or they may be administered simultaneously or sequentially as separate dosage forms.

[0170] Formulation, Dosage Forms, and Administration When used as a pharmaceutical, the compounds of the present disclosure can be administered in the form of a pharmaceutical composition. Thus, the present disclosure provides a composition comprising a compound of any of formula I, II or formulas described herein, any of the compounds cited in any of the claims and described herein, or a pharma- ceutically acceptable salt thereof, or any of the embodiments thereof, and at least one pharma- ceutically acceptable carrier or excipient. These compositions can be prepared by methods well known in the pharmaceutical art, and can be administered by various routes, depending on whether local or systemic treatment is indicated and depending on the area to be treated. Administration can be topical (including transdermal, epidermal, intraocular, and mucous membranes, including intranasal, intravaginal, and rectal delivery), pulmonary (e.g., by inhalation or filling of powder or aerosol, including by nebulizer, intratracheal or intranasal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, or injection or infusion, or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose or, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like, may be necessary or desirable.

[0171] The present invention also includes pharmaceutical compositions that contain the disclosed compound or its pharma- ceutically acceptable salt as an active ingredient in combination with one or more pharma- ceutically acceptable carriers or excipients. In some embodiments, the compositions are suitable for topical administration. In making the compositions of the present invention, the active ingredient is usually mixed with an excipient, diluted by an excipient, or enclosed in such a carrier, for example, in the form of a capsule, sachet, paper, or other container. When the excipient functions as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the composition can be in the form of a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium), for example, ointment containing up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injection liquid, and sterile packaged powder.

[0172] When preparing formulations, active compound can be milled to provide suitable particle size before being combined with other components.If active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh.If active compound is substantially water-soluble, its particle size can be adjusted by milling to, for example, about 40 mesh to provide substantially uniform distribution in formulation.

[0173] The compounds of the present invention can be milled using known milling procedures, such as wet milling, to obtain a particle size suitable for tableting and other formulation types. Micronized (nanoparticulate) preparations of the compounds of the present invention can be prepared by processes known in the art (see, for example, WO2002 / 000196).

[0174] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose.The formulation can further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl benzoate and propyl hydroxybenzoate; sweeteners; and flavoring agents.The composition of the present invention can be formulated to provide quick, sustained, or delayed release of active ingredient after administration to a patient by using procedures known in the art.

[0175] In some embodiments, the pharmaceutical composition comprises silicified microcrystalline cellulose (SMCC) and at least one compound described herein or a pharma- ceutically acceptable salt thereof. In some embodiments, the silicified microcrystalline cellulose is composed of about 98% by weight microcrystalline cellulose and about 2% by weight silicon dioxide.

[0176] In some embodiments, the composition is a sustained release composition comprising at least one compound described herein or a pharma- ceutically acceptable salt thereof and at least one pharma- ceutically acceptable carrier or excipient. In some embodiments, the composition comprises at least one compound described herein or a pharma- ceutically acceptable salt thereof and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropyl methylcellulose, and polyethylene oxide. In some embodiments, the composition comprises at least one compound described herein or a pharma- ceutically acceptable salt thereof and microcrystalline cellulose, lactose monohydrate, and hydroxypropyl methylcellulose. In some embodiments, the composition comprises at least one compound described herein or a pharma- ceutically acceptable salt thereof and microcrystalline cellulose, lactose monohydrate, and polyethylene oxide. In some embodiments, the composition further comprises magnesium stearate or silicon dioxide. In some embodiments, the microcrystalline cellulose is Avicel PH102™. In some embodiments, the lactose monohydrate is Fast-flo 316™. In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208 K4M (e.g., Methocel K4 M Premier™) and / or hydroxypropyl methylcellulose 2208 K100LV (e.g., Methocel K00LV™). In some embodiments, the polyethylene oxide is polyethylene oxide WSR 1105 (e.g., Polyox WSR 1105™).

[0177] In some embodiments, the compositions are manufactured using a wet granulation process. In some embodiments, the compositions are manufactured using a dry granulation process.

[0178] The compositions may be formulated in unit dosage form, each dosage containing from about 5 to about 1,000 mg (1 g), more usually from about 100 mg to about 500 mg, of the active ingredient. In some embodiments, each dosage contains about 10 mg of active ingredient. In some embodiments, each dosage contains about 50 mg of active ingredient. In some embodiments, each dosage contains about 25 mg of active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0179] The components used to formulate the pharmaceutical composition are of high purity and substantially free of potentially harmful contaminants (e.g., at least national food grade, generally at least analytical grade, and more usually at least pharmaceutical grade). Particularly for human consumption, the composition is preferably manufactured or formulated under the standards of the Good Manufacturing Practices for Drugs and Quasi-drugs defined in the applicable regulations of the United States Food and Drug Administration. For example, suitable formulations can be sterile and / or substantially isotonic, and / or can be in full compliance with all provisions of the Good Manufacturing Practices for Drugs and Quasi-drugs of the United States Food and Drug Administration.

[0180] The active compounds can be effective over a wide dosage range and are generally administered in a therapeutically effective amount. However, it will be understood that the amount of compound actually administered will usually be determined by the physician according to the relevant circumstances, including the condition being treated, the selected route of administration, the actual compound being administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0181] The therapeutic dosage of the compounds of the invention may vary according to, for example, the particular application for which the treatment is being given, the manner in which the compound is administered, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of the compound of the invention in a pharmaceutical composition can vary depending on a number of factors, including the dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w / v of the compound for parenteral administration. Some typical dosage ranges are about 1 μg / kg to about 1 g / kg of body weight per day. In some embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg of body weight per day. The dosage may depend on such variables as the type and extent of progression of the disease or disorder, the overall health of the particular patient, the relative bioefficacy of the selected compound, the formulation of excipients, and its route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0182] To prepare solid compositions such as tablets, the primary active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention. When these preformulation compositions are referred to as homogenous, the active ingredient is usually dispersed evenly throughout the composition so that the composition can be readily divided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation is then divided into unit dosage forms of the type described above, containing, for example, about 0.1 to about 1000 mg of the active ingredient of the present invention.

[0183] The tablet or pill of the present invention can be coated or otherwise compounded to provide a dosage form that provides the advantage of long-term action.For example, the tablet or pill can comprise an inner dose and an outer dose component, the latter being in the form of an envelope on the former.The two components can be separated by an enteric layer that resists disintegration in the stomach and helps the inner component pass intact into the duodenum or delay release.Various materials can be used for such enteric layer or coating, including a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0184] Liquid forms into which the compounds and compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0185] Compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered orally or nasally from a device that delivers the formulation in an appropriate manner.

[0186] Topical formulations may contain one or more conventional carriers. In some embodiments, ointments may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petrolatum, and the like. Cream carrier compositions may be based on water in combination with glycerol and one or more other components, for example, glycerol monostearate, PEG-glycerol monostearate, and cetylstearyl alcohol. Gels may be formulated using isopropyl alcohol and water, preferably in combination with other components, such as, for example, glycerol, hydroxyethylcellulose, and the like. In some embodiments, topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5 wt % of the compound of the present invention. Topical formulations may be suitably packaged in, for example, 100 g tubes, optionally associated with instructions for the treatment of a selected indication, for example, psoriasis or other skin conditions.

[0187] The amount of compound or composition administered to a patient will vary depending on what is being administered, the purpose of the administration, such as prophylaxis or treatment, the condition of the patient, the mode of administration, etc. In therapeutic applications, the compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. The effective dose will depend on the condition being treated and the judgment of the attending physician depending on such factors as the severity of the disease, the age, weight, and general condition of the patient, etc.

[0188] The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques or can be sterile filtered. Aqueous solutions can be packaged for use as is or lyophilized, the lyophilized preparations being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations will typically be 3-11, more preferably 5-9, most preferably 7-8. It will be understood that the use of some of the aforementioned excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

[0189] The therapeutic dosage of the compounds of the invention may vary depending, for example, on the particular application for which the treatment is being given, the manner in which the compound is administered, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of the compound of the invention in a pharmaceutical composition can vary depending on a number of factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w / v of the compound for parenteral administration. Some typical dosage ranges are about 1 μg / kg to about 1 g / kg of body weight per day. In some embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg of body weight per day. The dosage may depend on such variables as the type and extent of progression of the disease or disorder, the overall health of the particular patient, the relative bioefficacy of the selected compound, the formulation of excipients, and its route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0190] Labeled Compounds and Assay Methods Another aspect of the present invention relates to the labeled compounds (radiolabeled, fluorescently labeled, etc.) of the present disclosure that may be useful in both in vitro and in vivo assays, as well as imaging techniques, to locate and quantify KRAS protein in tissue samples, including humans, and to identify KRAS ligands, by the inhibitory binding of the labeled compounds.Substitution of one or more atoms of the compounds of the present disclosure may also be useful in producing different ADME (adsorption, distribution, metabolism, and excretion).Therefore, the present invention includes KRAS binding assays that contain such labeled or substituted compounds.

[0191] The present disclosure further includes isotopically labeled compounds of the present disclosure. An "isotopically labeled" or "radiolabeled" compound is a compound of the present disclosure in which one or more atoms have been replaced or substituted with an atom having an atomic mass or mass number different from that normally found in nature (i.e., naturally occurring). Suitable radionuclides that can be incorporated into the compounds of the present disclosure include: 2 H (also written as D for deuterium), 3 H (also written as T for tritium), 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 18 F, 35 S, 36 Cl, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I, and 131 For example, one or more hydrogen atoms in the compounds of the present disclosure can be replaced with a deuterium atom (e.g., C of Formula I, II, or any formula provided herein). 1~6One or more hydrogen atoms of an alkyl group can be optionally replaced with a deuterium atom, such as -CD3 substituted for -CH3.) In some embodiments, an alkyl group in Formula I, II, or any formula provided herein can be perdeuterated.

[0192] One or more constituent atoms of the compounds presented herein can be replaced or substituted with an isotope of the atom at natural or non-natural abundance. In some embodiments, the compound contains at least one deuterium atom. In some embodiments, the compound contains two or more deuterium atoms. In some embodiments, the compound contains 1-2, 1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all hydrogen atoms in the compound can be replaced or substituted with deuterium atoms.

[0193] Synthetic methods for incorporating isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, NY, Appleton-Century-Crofts, 1971; The Renaissance of H / D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in a variety of studies, such as NMR spectroscopy, metabolic experiments, and / or assays.

[0194] Substitution with heavier isotopes, such as deuterium, may provide certain therapeutic advantages due to higher metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some circumstances (see, for example, A. Kerekes et.al. J. Med. Chem. 2011, 54, 201-210; R. Xu et.al. J. Label Compd. Radiopharm. 2015, 58, 308-312). In particular, substitution at one or more metabolic sites may provide one or more therapeutic advantages.

[0195] The radionuclide that is incorporated into the present radiolabeled compounds will depend on the particular application of that radiolabeled compound. For example, for in vitro adenosine receptor labeling and competition assays: 3 H, 14 C. 82 Br, 125 I, 131 I or 35 Compounds incorporating S may be useful. For radioimaging applications, 11 C. 18 F, 125 I, 123 I, 124 I, 131 I, 75 Br, 76 Br, or 77 Br may be useful.

[0196] It is understood that a "radiolabel" or "labeled compound" is a compound that incorporates at least one radionuclide. In some embodiments, the radionuclide is 3 H, 14 C. 125 I, 35 S, and 82 Br.

[0197] The present disclosure may further include synthetic methods for incorporating radioisotopes into the compounds of the present disclosure. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and one of ordinary skill in the art will readily recognize methods applicable to the compounds of the present disclosure.

[0198] The labeled compounds of the present invention can be used in screening assays to identify and / or evaluate compounds. For example, a newly synthesized or identified compound that is labeled (i.e., a test compound) can be evaluated for its ability to bind to KRAS protein by monitoring its concentration change upon contact with KRAS via tracking the label. For example, a (labeled) test compound can be evaluated for its ability to reduce the binding of another compound known to bind to KRAS protein (i.e., a standard compound). Thus, the ability of a test compound to compete with a standard compound for binding to KRAS protein is directly correlated with its binding affinity. Conversely, in some other screening assays, the standard compound is labeled and the test compound is not labeled. Thus, the concentration of the labeled standard compound is monitored to evaluate the competition between the standard compound and the test compound, and thus the relative binding affinity of the test compound is confirmed.

[0199] kit The present disclosure also includes pharmaceutical kits useful for treating or preventing diseases or disorders associated with the activity of KRAS, such as, for example, cancer or infectious diseases, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, II, or any of its embodiments. Such kits can further include one or more of a variety of conventional pharmaceutical kit components, such as, for example, a container with one or more pharma- ceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Instructions, either as inserts or labels, indicating the amounts of components to be administered, guidelines for administration, and / or guidelines for mixing the components, can also be included in the kit.

[0200] The present invention will be described in more detail by specific examples.The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way.Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to essentially produce the same results.The compounds of the examples have been found to inhibit the activity of KRAS according to at least one assay described herein. EXAMPLES

[0201] The experimental procedures for the compounds of the present invention are provided below. Preparative LC-MS purification of some of the compounds prepared was carried out on Waters mass directed fractionation systems. The basic instrument setup, protocols, and control software for the operation of these systems are described in detail in the literature. See, for example, "Two-Pump At Column Dilution Configuration for Preparative LC-MS", K. Blom, J. Combi. Chem., 4, 295 (2002), "Optimizing Preparative LC-MS Configurations and Methods for Parallel Synthesis Purification", K. Blom, R. Sparks, J. Doughty, G. Everlof, T. Haque, A. Combs, J. Combi. Chem., 5, 670 (2003), and "Preparative LC-MS Purification: Improved Compound Specific Method Optimization", K. Blom, B. Glass, R. Sparks, A. Combs, J. Combi. Chem., 6, 874-883 (2004). Separated compounds were typically subjected to analytical liquid chromatography mass spectrometry (LCMS) for purity checks.

[0202] The separated compounds were typically subjected to analytical liquid chromatography mass spectrometry (LCMS) for purity check under the following conditions: Instrument: Agilent 1100 series, LC / MSD, Column: Waters Sunfire™ C 18 5 μm particle size, 2.1 × 5.0 mm, buffer: mobile phase A: 0.025% TFA in water and mobile phase B: acetonitrile, gradient 2% to 80% of B in 3 min at a flow rate of 2.0 mL / min.

[0203] Some of the prepared compounds were also separated on a preparative scale by reversed-phase high performance liquid chromatography (RP-HPLC) or flash chromatography (silica gel) with MS detection as indicated in the examples. Typical preparative reversed-phase high performance liquid chromatography (RP-HPLC) column conditions are as follows: pH=2 purification: Waters Sunfire(TM) C 18 The elution was performed with a 5 μm particle size, 19×100 mm column, mobile phase A: 0.1% TFA (trifluoroacetic acid) in water and mobile phase B: acetonitrile, the flow rate was 30 mL / min, and the separation gradient was optimized for each compound using the Compound Specific Method Optimization protocol described in the literature [see “Preparative LCMS Purification: Improved Compound Specific Method Optimization”, K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem., 6, 874-883 (2004)]. The flow rate typically used with the 30×100 mm column was 60 mL / min.

[0204] pH=10 Purification: Waters XBridge C 18The elution was performed with a 5 μm particle size, 19×100 mm column, mobile phase A: 0.15% NH4OH in water and mobile phase B: acetonitrile, the flow rate was 30 mL / min, and the separation gradient was optimized for each compound using the Compound Specific Method Optimization protocol described in the literature [see "Preparative LCMS Purification: Improved Compound Specific Method Optimization", K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem., 6, 874-883 (2004)]. The flow rate typically used with the 30×100 mm column was 60 mL / min.

[0205] The following abbreviations may be used herein:AcOH (acetic acid);AcO (acetic anhydride);aq. (aqueous);atm. (atmosphere(s));Boc (t-butoxycarbonyl);BOP ((benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate);br (broad);Cbz (carboxybenzyl);calc. (calculated);d (doublet);dd (doublet of doublet);DBU (1,8-diazabichloro[5.4.0]undec-7-ene);DCM (dichloromethane);DIAD (N,N'-diisopropyl azidodicarboxylate);DIEA (N,N -Diisopropylethylamine;DIPEA(N,N-Diisopropylethylamine);DIBAL(Diisobutylaluminum hydride);DMF(N,N-Dimethylformamide);Et(Ethyl);EtOAc(Ethyl acetate);FCC(Flash column chromatography);g(Gram(s));h(Hour(s));HATU(N,N,N',N'-Tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate);HCl(Hydrogen acid);HPLC(High performance liquid chromatography);Hz(Hertz) ;J (coupling constant);LCMS (liquid chromatography-mass spectrometry);LDA (lithium diisopropylamide);m (multiplet);M (molar);mCPBA (3-chloroperoxybenzoic acid);MS (mass spectrometry);Me (methyl);MeCN (acetonitrile);MeOH (methanol);mg (milligram(s));min (minute(s));mL (milliliter(s));mmol (millimoles(s));N (normal);NCS (N-chlorosuccinimide);NEt3 (triethylamine);nM (nanomole);NMP (N-methyl pyrrolidinone); NMR (nuclear magnetic resonance spectroscopy); OTf (trifluoromethanesulfonic acid); Ph (phenyl); pM (picomole); PPT (precipitate); RP-HPLC (reverse-phase high performance liquid chromatography); rt (room temperature), s (singlet); t (triplet or tertiary); TBS (tert-butyldimethylsilyl); tert (tertiary); tt (triplet of triplets); TFA (trifluoroacetic acid); THF (tetrahydrofuran); μg (microgram(s)); μL (microliter(s)); μM (micromole); wt% (weight percent).Brine is a saturated aqueous solution of sodium chloride. in vacuo is under vacuum.

[0206] Example 1a and Example 1b. 1-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-8-chloro-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile [ka] Step 1. Ethyl 2-amino-4-bromo-3-fluorobenzoate [ka] To a solution of 2-amino-4-bromo-3-fluorobenzoic acid (22.7 g, 92 mmol) in ethanol (184 ml) was slowly added sulfuric acid (9.82 ml, 184 mmol). The resulting mixture was heated to reflux for 2 days. After cooling to room temperature, the reaction mixture was diluted with water and the pH was adjusted to 7 with 6 M NaOH (22 mL). The organic solvents were removed in vacuo. The resulting mixture was diluted with ethyl acetate and water. The organic layer was washed with 0.5 N NaOH solution, brine, dried over Na2SO4, filtered and concentrated under vacuum to give the desired product (23.2 g, 96%). C9H 10 BrFNO2(M+H) + LCMS calculated m / z=262.0, 264.0; found 262.0, 264.0.

[0207] Step 2. Ethyl 2-amino-3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate [ka] A mixture of ethyl 2-amino-4-bromo-3-fluorobenzoate (21.8 g, 83 mmol), bis(pinacolato)diboron (25.3 g, 100 mmol), dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (6.79 g, 8.32 mmol), acetic acid, anhydrous potassium salt (17.96 g, 183 mmol), and dioxane (416 ml) was stirred for 5 h under nitrogen at 100° C. The crude was filtered through Celite and washed with ethyl acetate. The filtrate was concentrated. The residue was purified by flash chromatography to give the desired product (24 g, 93%). C 15 H 22 BFNO4(M+H) + LCMS calculated m / z=310.2; found 310.1.

[0208] Step 3. 8-Cyanoisoquinoline 2-oxide [ka] To a solution of isoquinoline-8-carbonitrile (3.70 g, 24.00 mmol) in CHCl (240 ml) was added m-CPBA (7.10 g, 28.8 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 2 h. The reaction mixture was diluted with saturated NaHCO3 solution. The aqueous layer was extracted with DCM (×3). The combined organic layers were dried over NaSO4, filtered and concentrated. The crude was purified by flash chromatography (eluting with a gradient of 0-100% ethyl acetate in hexanes) to give the desired product (3.2 g, 78%). 10 H7N2O(M+H) + LC-MS calculated for: m / z=171.1; found 171.1.

[0209] Step 4. 1-Chloroisoquinoline-8-carbonitrile [ka] To a solution of 8-cyanoisoquinoline 2-oxide (5.30 g, 31.1 mmol), 2,6-lutidine (7.26 ml, 62.3 mmol) in CHCl (62.3 ml) was added POCl (5.81 ml, 62.3 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched by adding saturated NaHCO (80 mL). The organic layer was dried over MgSO and concentrated to give the crude product. The crude product was triturated with ethyl acetate in hexane to give the desired product as a white solid (4.0 g, 68%). C 10 H6ClN2(M+H) + LC-MS calculated for: m / z=189.0; found 189.0.

[0210] Step 5. Ethyl 2-amino-4-(8-cyanoisoquinolin-1-yl)-3-fluorobenzoate [ka] A mixture of 1-chloroisoquinoline-8-carbonitrile (6.60 g, 35.0 mmol), ethyl 2-amino-3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (11.36 g, 36.7 mmol), SPhos Pd G4 (1.390 g, 1.750 mmol), and tripotassium phosphate hydrate (17.73 g, 77 mmol) in 1,4-dioxane (120 mL) and water (24 mL) was stirred at 80° C. for 2 h. The solution was diluted with ethyl acetate and water. The organic layer was concentrated. The crude was used in the next step without purification. C 19 H 15 FN3O2(M+H) + LC-MS calculated for: m / z=336.1; found 336.1.

[0211] Step 6. Ethyl 2-amino-5-chloro-4-(8-cyanoisoquinolin-1-yl)-3-fluorobenzoate [ka] To a solution of ethyl 2-amino-4-(8-cyanoisoquinolin-1-yl)-3-fluorobenzoate (11.7 g, 34.9 mmol) in DMF (116 ml) was added NCS (5.12 g, 38.4 mmol) at room temperature. The mixture was heated at 80° C. for 15 h. The reaction mixture was cooled to room temperature and diluted with water. The precipitate was collected by filtration and washed with water and ethyl acetate / hexane (1:2). The filtrate was extracted with ethyl acetate. The organic layer was concentrated. The solid was collected by filtration and washed with ethyl acetate / hexane (1:2) to give the desired product (10.2 g, 79%). C 19 H 14 ClFN3O2(M+H) + LC-MS calculated for: m / z=370.1; found 370.1.

[0212] Step 7. 5-Chloro-4-(8-cyanoisoquinolin-1-yl)-2-(3-ethoxy-3-oxopropanamido)-3-fluorobenzoate ethyl [ka] To a solution of ethyl 2-amino-5-chloro-4-(8-cyanoisoquinolin-1-yl)-3-fluorobenzoate (10.3 g, 27.9 mmol) and TEA (5.05 ml, 36.2 mmol) in DCM (280 mL) was added ethyl 3-chloro-3-oxopropanoate (3.92 ml, 30.6 mmol) dropwise at 0° C. The resulting mixture was stirred at 0° C. and monitored by LC-MS. Another equivalent of ethyl 3-chloro-3-oxopropanoate (3.92 ml, 30.6 mmol) was added dropwise and stirred for 1 h. The reaction was diluted with water and DCM. The organic layer was separated, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography to give the desired product (9.5 g, 70%). C 24 H 20 ClFN3O5(M+H) + LC-MS calculated for: m / z=484.1; found 484.1.

[0213] Step 8. Ethyl 6-chloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-2,4-dihydroxyquinoline-3-carboxylate [ka] A solution of 21% sodium ethoxide in EtOH (19.91 ml, 53.3 mmol) was added dropwise to a solution of ethyl 5-chloro-4-(8-cyanoisoquinolin-1-yl)-2-(3-ethoxy-3-oxopropanamido)-3-fluorobenzoate (8.6 g, 17.77 mmol) in EtOH (80 ml). The resulting mixture was stirred at room temperature for 2 hours. The pH was adjusted to 3 by adding 1N HCl to the reaction flask. The solvent was removed under vacuum. The resulting precipitate was collected and washed with ethyl acetate to give the desired product as a white solid (7.4 g, 95%). C 22 H 14 ClFN3O4(M+H) + LC-MS calculated for: m / z=438.1; found 438.1.

[0214] Step 9. Ethyl 6-chloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-2,4-dihydroxyquinoline-3-carboxylate [ka] To a reaction flask, ethyl 6-chloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-2,4-dihydroxyquinoline-3-carboxylate (7.4 g, 16.90 mmol) and POCl3 (31.5 ml, 338 mmol) were added, and the resulting mixture was stirred at 110° C. for 2 h. POCl3 was removed by azeotroping with toluene (3 times), and the residue was diluted with DCM and saturated NaHCO3 solution. The organic layer was separated, dried over Na2SO4, filtered, and concentrated. The crude was triturated with ethyl acetate / hexane (1:1) to give the desired product as a white solid (7.24 g, 90%). C 22 H 12 Cl3FN3O2(M+H) +LC-MS calculated for: m / z=474.0, 476.0; found 474.0, 476.0.

[0215] Step 10. Ethyl 4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoroquinoline-3-carboxylate [ka] To a solution of ethyl 2,4,6-trichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoroquinoline-3-carboxylate (7.24 g, 15.25 mmol) in DMF (100 ml) was added tert-butyl (2S,4S)-4-amino-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate (6.56 g, 18.30 mmol) and DIEA (5.3 ml, 30.5 mmol). The resulting mixture was stirred at 65° C. overnight. The reaction mixture was diluted with ethyl acetate and water. The organic layer was washed with water and brine, dried over Na2SO4, filtered and concentrated. The crude was purified on a silica gel column (eluted with a gradient of 0-30% ethyl acetate in hexanes) to give the desired product as a pale yellow foam (11.5 g, 95%). C 40 H 49 Cl2FN5O5Si(M+H) + LC-MS calculated for: m / z=796.3, 798.3; found 796.3, 798.3.

[0216] Step 11. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-(hydroxymethyl)quinolin-4-yl)amino)piperidine-1-carboxylate [ka] To a solution of ethyl 4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoroquinoline-3-carboxylate (2.45 g, 3.07 mmol) in toluene (30.7 ml) at -78°C was added 1.0 M DIBAL-H in DCM (9.84 ml, 9.84 mmol). The resulting mixture was warmed to -20°C over 2 h and quenched with methanol (1.3 mL). Rochelle salt solution (prepared from 14.7 g (6 wt) of Rochelle salt and 50 mL of water) was added to the solution below 10°C. The biphasic mixture was stirred vigorously at 15-25°C for 1 h or more and separated to obtain the organic layer. The organic layer was dried over Na2SO4, filtered, concentrated and used as is. 38 H 47 Cl2FN5O4Si(M+H) + LC-MS calculated for: m / z=754.3, 756.3; found 754.3, 756.3.

[0217] Step 12. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-formylquinolin-4-yl)amino)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-(hydroxymethyl)quinolin-4-yl)amino)piperidine-1-carboxylate (2.32 g, 3.07 mmol) in DCM (23 ml) and acetonitrile (7.7 ml) was added acetic acid (0.53 ml, 9.22 mmol) and IBX (2.58 g, 9.22 mmol). The resulting mixture was stirred at 38° C. for 22 h, and the reaction mixture was filtered and washed with DCM. The filtrate was concentrated and purified on a silica gel column (eluted with a gradient of 0-20% ethyl acetate in hexs) to give the desired product as two peaks.

[0218] Diastereomer 1 (1.05 g, 45%). Peak 1. C 38 H 45 Cl2FN5O4Si(M+H) + LC-MS calculated for: m / z=752.3, 754.3; found 752.3, 754.3.

[0219] Diastereomer 2 (1.05 g, 45%). Peak 2. C 38 H 45 Cl2FN5O4Si(M+H) + LC-MS calculated for: m / z=752.3, 754.3; found 752.3, 754.3.

[0220] Step 13. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-((E)-(hydroxyimino)methyl)quinolin-4-yl)amino)piperidine-1-carboxylate [ka] To a mixture of tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-formylquinolin-4-yl)amino)piperidine-1-carboxylate (0.85 g, 1.13 mmol) (peak 1 from the last step), DCM (11 ml) and EtOH (11 ml) was added hydroxylamine hydrochloride (0.259 g, 3.73 mmol) and pyridine (0.30 ml, 3.73 mmol). The mixture was stirred at 40° C. for 16 h. The solvent was evaporated in vacuum. The residue contained DCM and water. The aqueous layer was extracted with DCM. The combined organic layers were washed with CuSO4 solution, brine, dried over MgSO4, filtered and concentrated in vacuum. The residue was purified by column chromatography on silica gel to give the desired product (0.5 g, 57%). 38 H 46 Cl2FN6O4Si(M+H) + LC-MS calculated for: m / z=767.3, 769.3; found 767.3, 769.3.

[0221] Diastereomer 2 was prepared in a similar manner to diastereomer 1 using peak 2 from the last step. 38 H 46 Cl2FN6O4Si(M+H) + LC-MS calculated for: m / z=767.3, 769.3; found 767.3, 769.3.

[0222] Step 14. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(4,8-dichloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate [ka] To a solution of (tert-butyl(2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-((E)-(hydroxyimino)methyl)quinolin-4-yl)amino)piperidine-1-carboxylate (486 mg, 0.633 mmol) (diastereomer 1 from the last step) in CHCl (5 mL) was added 2-aminopyridine (113 mg, 1.203 mmol) and Ms-Cl (84 μl, 1.076 mmol) at 0° C. The resulting mixture was stirred at 0° C. for 2 h and then allowed to warm to room temperature overnight. The reaction mixture was diluted with water. The organic layer was washed with brine, dried over MgSO, filtered, concentrated, and used as is. 38 H 44 Cl2FN6O3Si(M+H) + LC-MS calculated for: m / z=749.3, 751.3; found 749.3, 751.3.

[0223] Diastereomer 2 was prepared in a similar manner to diastereomer 1 using peak 2 from the last step. 38 H 44 Cl2FN6O3Si(M+H) + LC-MS calculated for: m / z=749.3, 751.3; found 749.3, 751.3.

[0224] Step 15 tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate [ka] To a mixture of tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(4,8-dichloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate (475 mg, 0.634 mmol) (diastereomer 1 from the last step) in MeOH (6.3 ml) / 1,4-dioxane (6.3 ml) was added sodium thiomethoxide (133 mg, 1.901 mmol) and stirred at 90° C. for 18 h. The mixture was diluted with saturated NH4Cl and extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered, concentrated and used as is. C 39 H 47 ClFN6O3SSi(M+H) + LC-MS calculated for: m / z=761.3, found 761.3.

[0225] Diastereomer 2 was prepared in a similar manner to diastereomer 1 using peak 2 from the last step. 39 H 47 ClFN6O3SSi(M+H) + LC-MS calculated for: m / z=761.3, found 761.3.

[0226] Step 16. tert-Butyl (2S,4S)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate (482 mg, 0.633 mmol) (diastereomer 1 from the last step) in THF (6.33 ml) was added 1.0 M TBAF in THF (633 μl, 0.633 mmol). The resulting mixture was stirred at 60° C. for 1 h. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate. The organic layer was separated, washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography to give the desired product (0.39 g, 95%). C 33 H 33 ClFN6O3S(M+H) + LC-MS calculated for: m / z = 647.2; found 647.2.

[0227] Diastereomer 2 was prepared in a similar manner to diastereomer 1 using peak 2 from the last step. 33 H 33 ClFN6O3S(M+H) + LC-MS calculated for: m / z = 647.2; found 647.2.

[0228] Step 17. tert-Butyl (2S,4S)-4-(8-chloro-7-(8-chloronaphthalen-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate (392 mg, 0.606 mmol) in DCM (6.0 ml) was added Dess-Martin periodinane (283 mg, 0.666 mmol). The resulting mixture was stirred for 1 h and saturated NaHCO3 was added to the reaction flask and stirred for 10 min. The organic layer was separated, dried over Na2SO4, filtered and concentrated. The crude was dissolved in THF (20 mL) and ammonium hydroxide (1.37 ml, 9.81 mmol) was added to the reaction flask followed by iodine (157 mg, 0.618 mmol). The resulting mixture was stirred at room temperature for 2 hours, and the reaction solution was diluted with ethyl acetate and saturated NaSO solution. The organic layer was separated, washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography to give the desired product (0.32 g, 82%). 33 H 30 ClFN7O2S(M+H) + LC-MS calculated for: m / z=641.2; found 641.2.

[0229] Diastereomer 2 was prepared in a similar manner to diastereomer 1 using peak 2 from the last step. 33 H 30 ClFN7O2S(M+H) + LC-MS calculated for: m / z=641.2; found 641.2.

[0230] Step 18. 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile [ka] m-CPBA (43.9 mg, 0.254 mmol) was added to a solution of tert-butyl (2S,4S)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (142 mg, 0.221 mmol) in CHCl (2.211 ml) at 0° C., and the reaction was then stirred at this temperature for 20 minutes. The reaction was quenched by adding saturated NaSO, diluted with ethyl acetate, washed with saturated NaHCO solution, brine, filtered, concentrated, and the crude was used directly in the next step.

[0231] LiHMDS (318 μl, 0.318 mmol) was added to a solution of (S)-1-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (41.0 mg, 0.318 mmol) in THF (1 mL). The resulting mixture was stirred at room temperature for 30 min. The first solution was added to a solution of tert-butyl (2S,4S)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylsulfinyl)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (95 mg, 0.144 mmol) in THF (2.0 ml), and the reaction was then stirred at 60° C. for 2 h. The reaction mixture was diluted with ethyl acetate and water. The organic layer was separated, dried over Na2SO4, filtered, and concentrated. The residue was treated with 1:1 DCM / TFA (2 mL) for 1 h. The solvent was evaporated in vacuo. The residue was purified by preparative LCMS (XBridge C18 column, eluting with a gradient of acetonitrile / water containing 0.1% TFA at a flow rate of 60 mL / min) to give the desired product as two peaks (60 mg, 58%). 34 H 33 ClFNO(M+H) + LC-MS calculated for: m / z=623.2; found 623.2.

[0232] Diastereomer 2 was prepared in a similar manner to diastereomer 1 using peak 2 from the last step. 34 H 33 ClFNO(M+H) + LC-MS calculated for: m / z=623.2; found 623.2.

[0233] Step 19. 1-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-8-chloro-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile To a solution of but-2-ynic acid (0.711 mg, 8.46 μmol) and 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile bis(2,2,2-trifluoroacetate) (6.0 mg, 7.05 μmol) in DMF (1.0 ml), HATU (3.4 mg, 8.81 μmol) and DIEA (4.9 μl, 0.028 mmol) were added. The resulting mixture was stirred at room temperature for 2 hours. The reaction was diluted with methanol and 1N HCl (0.1 mL) and then purified using preparative LCMS (XBridge C18 column, eluting with a gradient of acetonitrile / water containing 0.1% TFA at a flow rate of 60 mL / min) to give the desired diastereomer 1.

[0234] Diastereomer 2 was synthesized in a similar manner using 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile bis(2,2,2-trifluoroacetate) (peak 2 from the last step).

[0235] Example 1a. Diastereomer 1. Peak 1.C 38 H 35 ClFN8O2(M+H) + LCMS calculated for m / z=689.3; found 689.3.

[0236] Example 1b. Diastereomer 2. Peak 2.C 38 H 35 ClFN8O2(M+H) + LCMS calculated for m / z=689.3; found 689.3.

[0237] Example 2a and Example 2b. 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-((E)-4-methoxybut-2-enoyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile [ka] This compound was prepared according to the procedure described in Example 1a and Example 1b, step 19, substituting (E)-4-methoxybut-2-enoic acid for but-2-ynoic acid. 39 H 39 ClFN8O3(M+H) + LCMS calculated for: m / z=721.3; Found: 721.3.

[0238] Example 3a and Example 3b. 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile [ka] This compound was prepared according to the procedure described in Example 1a and Example 1b, step 19, substituting 2-fluoroacrylic acid for but-2-ynoic acid. 37 H 34 ClF2N8O2(M+H) + LCMS calculated for: m / z=695.2; Found: 695.2.

[0239] Example 4a and Example 4b. 2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile [ka] Step 1: Methyl 2-amino-4-bromo-5-chloro-3-fluorobenzoate [ka] Sulfuric acid (7.76 ml, 146 mmol) was slowly added to a solution of 2-amino-4-bromo-5-chloro-3-fluorobenzoic acid (19.5 g, 72.8 mmol) in MeOH (146 ml) at room temperature. The resulting mixture was heated to 80° C. overnight. The mixture was then cooled to room temperature and slowly poured into saturated NaHCO3. The mixture was stirred at room temperature for 30 minutes and then extracted with EtOAc. The organic layer was dried over MgSO4, filtered, concentrated and used in the next step without further purification. C8H7BrClFNO2 (M+H) + LC-MS calculated for: m / z = 281.9, 283.9; found 281.9, 283.9.

[0240] Step 2: Ethyl 7-bromo-6-chloro-8-fluoro-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate [ka] Ethyl 3-chloro-3-oxopropanoate (9.60 ml, 75.0 mmol) was added dropwise to a solution of methyl 2-amino-4-bromo-5-chloro-3-fluorobenzoate (19.25 g, 68.1 mmol) and TEA (14.25 ml, 102 mmol) in DCM (150 mL) at room temperature. After stirring for 1 h, additional ethyl 3-chloro-3-oxopropanoate (1.745 ml, 13.63 mmol) was added. After stirring for an additional hour, the reaction was quenched with water and then extracted with ethyl acetate. The organic layer was dried, filtered, and concentrated. The concentrated residue was redissolved in EtOH (150 ml) and sodium ethoxide in ethanol (53.4 ml, 143 mmol) was added and stirred at room temperature for 1 h. The reaction mixture was poured into water (1 L), acidified to pH 3, and the resulting precipitate was collected by filtration to give the desired product (18.39 g, 74.0%). 12 H9BrClFNO4(M+H) + LC-MS calculated for: m / z=363.9, 365.9; found 363.9, 365.9.

[0241] Step 3: Ethyl 7-bromo-2,4,6-trichloro-8-fluoroquinoline-3-carboxylate [ka] Ethyl 7-bromo-6-chloro-8-fluoro-2,4-dihydroxyquinoline-3-carboxylate (2.0 g, 5.49 mmol) was dissolved in POCl3 (10.2 ml, 110 mmol) and DIEA (1.92 ml, 10.97 mmol) was added. The resulting mixture was stirred at 100° C. for 2 h. After cooling to room temperature, the reaction was quenched by slowly pouring into rapidly stirred ice water (approximately 250 mL), stirred for 30 min, and then the solid was collected by filtration to give the desired product as a brown solid (1.66 g, 75%). C 12 H7BrCl3FNO2(M+H) + LC-MS calculated for: m / z=399.9, 401.9, 403.9; found 399.9, 401.9, 403.9.

[0242] Step 4. tert-Butyl (R)-6-cyano-5-hydroxy-3-oxohexanoate [ka] A solution of 2.0M LDA (100ml, 200mmol) in anhydrous THF (223ml) was cooled to -78°C for 1 hour, then tert-butyl acetate (26.9ml, 200mmol) was added dropwise with stirring over 20 minutes. After an additional 40 minutes at -78°C, a solution of ethyl (R)-4-cyano-3-hydroxybutanoate (10.5g, 66.8mmol) was added dropwise. The mixture was stirred at -40°C for 4 hours, then an appropriate amount of HCl (2M) was added to the mixture while maintaining the pH at about 6. The temperature of the mixture was maintained at -10°C during this quench. Upon completion, the temperature of the mixture was cooled to 0°C. The mixture was extracted with ethyl acetate (3x100mL). The combined organic layers were washed with NaHCO3 (100 mL) and brine (100 mL), dried over anhydrous Na2SO4 and evaporated to give the material as a yellow oil (15.0 g, 99%).

[0243] Step 5. tert-Butyl (2S,4R)-2-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxypiperidine-1-carboxylate [ka] A solution of tert-butyl (R)-6-cyano-5-hydroxy-3-oxohexanoate (15.0 g, 66.0 mmol) in acetic acid (110 ml) was treated with platinum(IV) oxide hydrate (0.868 g, 3.30 mmol). The Parr bottle was evacuated and back-filled with H2 three times and stirred at 22 °C under H2 atmosphere (45 psi, refilled four times) for 3 h. The mixture was filtered through Celite and the filter cake was washed with EtOH. The filtrate was concentrated to give the product with a cis:trans diastereomeric ratio of approximately 9:1. The residue was dissolved in methanol (100 mL) and Boc anhydride (15.3 ml, 66.0 mmol), sodium carbonate (13.99 g, 132 mmol) were added. The reaction mixture was stirred at room temperature overnight. The mixture was filtered and concentrated. The residue was purified on a silica gel column to give the desired product (11.7 g, 56%). 16 H 29 NNaO5(M+Na) + LCMS calculated value for (product + Na + ): m / z = 338.2; Measured value: 338.2.

[0244] Step 6. tert-Butyl (2S,4S)-4-azido-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4R)-2-(2-(tert-butoxy)-2-oxoethyl)-4-hydroxypiperidine-1-carboxylate (2.10 g, 6.66 mmol) in DCM (33 ml) at 0° C., Ms-Cl (0.67 mL, 8.66 mmol) was added and after stirring for 1 h, the reaction was diluted with water, the organic layer was separated, dried over Na2SO4, filtered and concentrated. The resulting residue was dissolved in DMF, sodium azide (1.3 g, 20 mmol) was added and the reaction mixture was heated at 70° C. for 5 h. After cooling to room temperature, the reaction was diluted with EtOAc and water. The organic layer was separated, dried over Na2SO4, filtered and concentrated. The residue was purified on a silica gel column to give the desired product (1.90 g, 84%). (Product-Boc)C 11 H 21 N4O2(M+H) + LCMS calculated for: m / z=241.2; Found: 241.2.

[0245] Step 7. tert-Butyl (2S,4S)-4-azido-2-(2-hydroxyethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-azido-2-(2-(tert-butoxy)-2-oxoethyl)piperidine-1-carboxylate (21.4 g, 62.9 mmol) in DCM (400 ml) at -78 °C was added 1.0 M DIBAL-H in DCM (113 ml, 113 mmol). The resulting mixture was stirred at -78 °C for 2 h. The reaction was quenched with methanol (38.1 ml, 943 mmol) at -78 °C. Aqueous Rochelle salt solution (prepared from 126 g (6 wt) of Rochelle salt and 300 mL of water) was added to the solution below 10 °C. The biphasic mixture was vigorously stirred at 15-25 °C for more than 1 h and separated to obtain the organic layer. The biphasic mixture was separated. The organic layer was washed with aqueous NaCl (×2) at 15-25° C., the organic layer was dried over Na2SO4, filtered, concentrated and used as is. The residue was dissolved in methanol (300 mL) and sodium borohydride (1.43 g, 37.7 mmol) was added at 0° C. The reaction mixture was stirred at 0° C. for 1 h. The reaction was quenched with water and the methanol was evaporated under reduced pressure. The reaction mixture was extracted with ethyl acetate (×2) and the organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The crude was purified by flash chromatography (eluted with a gradient of 0-50% ethyl acetate in hexanes) to give the desired product as a colorless oil (14.8 g, 87%). (Product-Boc)C7H 15 LCMS calculated for NO(M+H)+: m / z=171.1; Found: 171.1.

[0246] Step 8. tert-Butyl (2S,4S)-4-azido-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-azido-2-(2-hydroxyethyl)piperidine-1-carboxylate (4.0 g, 14.80 mmol) in DMF (74.0 ml) was added imidazole (1.51 g, 22.2 mmol) and TBS-Cl (2.90 g, 19.24 mmol). The resulting mixture was stirred at 60° C. for 1 h 15 min. The reaction mixture was diluted with EtOAc and water. The organic layer was washed with water (2×), brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (0-20% ethyl acetate in hexanes) to give the desired product as a colorless oil (5.30 g, 93%). (Product-Boc)C 13 H 29 LCMS calculated for N4OSi(M+H)+: m / z=285.2, found: 285.2.

[0247] Step 9. tert-Butyl (2S,4S)-4-amino-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-azido-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-piperidine-1-carboxylate (5.30 g, 13.78 mmol) in methanol (70 ml) was added 10% palladium on carbon (1.47 g, 1.38 mmol). The reaction mixture was evacuated under vacuum, recharged with H2 and stirred at room temperature for 2 h. The reaction mixture was filtered through a pad of Celite and washed with methanol. The filtrate was concentrated to give the desired product (4.5 g, 91%). (Product-Boc)C 13 H 31 LCMS calculated for N2OSi(M+H)+: m / z=259.2, found: 259.2.

[0248] Step 10. Ethyl 7-bromo-4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2,6-dichloro-8-fluoroquinoline-3-carboxylate [ka] To a solution of ethyl 7-bromo-2,4,6-trichloro-8-fluoroquinoline-3-carboxylate (8.7 g, 21.7 mmol) in DMF (80 ml) was added tert-butyl (2S,4S)-4-amino-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate (9.33 g, 26.0 mmol) and DIEA (7.6 ml, 43.3 mmol). The resulting mixture was stirred at 65° C. for 5 h. After cooling to room temperature, ethyl acetate and water were added. The organic layer was washed with water (twice) and brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (eluted with 0% to 25% ethyl acetate in hexanes) to give the desired product as a foam (14.6 g, 93%). C 30 H 44 BrCl2FN3O5Si(M+H) + LC-MS calculated for: m / z=722.2, 724.2; found 722.2, 724.2.

[0249] Step 11. tert-Butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-(hydroxymethyl)quinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] To a solution of ethyl 7-bromo-4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2,6-dichloro-8-fluoroquinoline-3-carboxylate (14.6 g, 20.18 mmol) in toluene (200 ml) at 78° C. was added 1.0 M DIBAL-H in -DCM (60.5 ml, 60.5 mmol). The resulting mixture was stirred at -78° C. for 40 min, warmed to 0° C. for 1 h 20 min, and quenched with methanol (6.8 ml, 167 mmol). Aqueous Rochelle salt solution (prepared from 88 g (6 wt) Rochelle salt and 200 mL water) was added to the solution below 10° C. The biphasic mixture was stirred vigorously at 15-25 °C for at least 1 h and separated to give the organic layer. The biphasic mixture was separated. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was used as is. 28 H 42 BrCl2FN3O4Si(M+H) + LC-MS calculated for: m / z = 680.1, 682.1; found 680.1, 682.1.

[0250] Step 12. tert-Butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-formylquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-(hydroxymethyl)quinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate (13.0 g, 19.07 mmol) in DCM (150 ml) and acetonitrile (50 ml) was added IBX (16.02 g, 57.2 mmol) and acetic acid (3.28 ml, 57.2 mmol). The resulting reaction mixture was stirred at 35° C. for 16 h. The reaction mixture was filtered and the filtrate was concentrated. The resulting residue was triturated with EtOAc and the resulting precipitate was collected by filtration and dried under vacuum to give the desired product as a pale yellow solid (9.4 g, 73% for two steps). C 28 H 40 BrCl2FN3O4Si(M+H) + LC-MS calculated for: m / z=678.1, 680.1; found 678.1, 680.1.

[0251] Step 13. tert-Butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-((E)-(hydroxyimino)methyl)quinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] To a mixture of tert-butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-formylquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate (7.67 g, 11.29 mmol), DCM (56 ml) and EtOH (56 ml) was added hydroxylamine hydrochloride (2.35 g, 33.9 mmol) and pyridine (2.8 ml, 34.4 mmol). The reaction mixture was stirred at 40° C. for 16 h. Another portion of pyridine (2.8 ml, 34.4 mmol) and hydroxylamine hydrochloride (2.35 g, 33.9 mmol) was added and stirred for 4 h. The solvent was evaporated in vacuum. The residue contained DCM and water. The aqueous layer was extracted with DCM. The combined organic layers were washed with aqueous CuSO4, brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel to give the desired product (4.5 g, 57%). 28 H 41 BrCl2FN4O4Si(M+H) + LC-MS calculated for: m / z=693.1, 695.1; found 693.1, 695.1.

[0252] Step 14. tert-Butyl (2S,4S)-4-(7-bromo-4,8-dichloro-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] To a solution of (tert-butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-((E)-(hydroxyimino)methyl)quinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate (4.53 g, 6.52 mmol) in CHCl (75 mL) was added 2-aminopyridine (0.798 g, 8.48 mmol) and Ms-Cl (0.610 ml, 7.83 mmol) at 0° C. The resulting mixture was stirred at 0° C. for 2 h. The reaction mixture was allowed to warm to room temperature overnight. The reaction was diluted with water. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (eluted with a gradient of 0-40% ethyl acetate in hexanes) to give the desired product (1.80 g, 41%). C 28 H 39 BrCl2FN4O3Si(M+H) + LC-MS calculated for: m / z=675.1, 677.1; found 675.1, 677.1.

[0253] Step 15. tert-Butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] Sodium thiomethoxide (0.56 g, 8.00 mmol) was added to a mixture of tert-butyl (2S,4S)-4-(7-bromo-4,8-dichloro-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)-oxy)ethyl)piperidine-1-carboxylate (1.80 g, 2.67 mmol) in MeOH (26 ml) / DCM (26 ml) and then stirred at room temperature for 1 h. The mixture was diluted with saturated NH4Cl and extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography on silica gel to give the desired product (1.75 g, 95%). C 29 H 42 BrClFN4O3SSi(M+H) + LC-MS calculated for: m / z = 687.2, 689.2; found 687.2, 689.2.

[0254] Step 16. tert-Butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate (1.96 g, 2.84 mmol) in THF (28 ml) was added 1.0 M TBAF in THF (4.27 ml, 4.27 mmol). The resulting mixture was stirred at 60° C. for 1 h. After cooling to room temperature, the reaction mixture was diluted with water and ethyl acetate. The organic layer was separated, washed with brine, dried over Na2SO4, filtered and concentrated. The crude was used as is. C 23 H 28 BrClFN4O3S(M+H) +LC-MS calculated for: m / z=573.1, 575.1; found 573.1, 575.1.

[0255] Step 17. tert-Butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate (0.50 g, 0.871 mmol) in DCM (8 ml) was added Dess-Martin periodinane (0.406 g, 0.958 mmol). The resulting mixture was stirred for 1 h and saturated NaHCO3 was added to the reaction flask and stirred for 10 min. The organic layer was separated, dried over Na2SO4, filtered and concentrated. The crude was dissolved in THF (10 mL) and ammonium hydroxide (1.96 ml, 14.11 mmol) was added to the reaction flask followed by iodine (0.243 g, 0.958 mmol). The resulting mixture was stirred at room temperature for 3 hours, and the reaction solution was diluted with ethyl acetate and saturated NaSO solution. The organic layer was separated, washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography to give the desired product (0.40 g, 80%). 23 H 25 BrClFN5O2S(M+H) + LC-MS calculated for: m / z=568.1, 570.1; found 568.1, 570.1.

[0256] Step 18. tert-Butyl (2S,4S)-4-(8-chloro-7-(6-chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate [ka] A vial charged with tert-butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (401 mg, 0.705 mmol), 6-chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (319 mg, 0.846 mmol), tetrakis(triphenylphosphine)palladium(0) (122 mg, 0.106 mmol), sodium carbonate (299 mg, 2.82 mmol), and 5:1 dioxane / water (6 ml) was heated at 105° C. overnight. The mixture was diluted with brine and EtOAc, and the organic layer was separated, dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography to give the desired product (0.39 g, 75%). 36 H 39 Cl2FN7O3S(M+H) + LC-MS calculated for: m / z=738.2; found 738.2.

[0257] Step 19. tert-Butyl (2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-(8-chloro-7-(6-chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (0.73 g, 0.988 mmol) in DCM (10 ml) at 0° C. was added m-CPBA (0.196 g, 1.136 mmol). The reaction mixture was stirred at this temperature for 20 min. The reaction was quenched by adding saturated Na2S2O3, diluted with ethyl acetate, washed with saturated NaHCO3, brine, filtered, dried and concentrated. The crude was dissolved in acetonitrile (8 ml) and triethylamine (0.607 ml, 4.36 mmol), N-ethyl-N-methylazetidin-3-amine dihydrochloride (0.306 g, 1.634 mmol) was added to the reaction vial, and the resulting mixture was stirred at 80° C. for 2 h. The crude was concentrated, and the residue was purified by silica gel column (eluted with a gradient of 0-20% DCM in MeOH) to give the desired product (0.65 g, 76%). 42 H 52 ClFN9O3(M+H) + LC-MS calculated for: m / z=784.4; found 784.4.

[0258] Step 20. 2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidin-2-yl)acetonitrile [ka] To a solution of tert-butyl (2S,4S)-4-(8-chloro-7-(6-chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)-4-(3-(dimethylamino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (0.65 g, 0.771 mmol) in DCM (5 ml) was added TFA (4.8 ml, 61.7 mmol). After stirring for 0.5 h, the solvent was removed in vacuo and the residue was purified by preparative LCMS (XBridge C18 column, eluting with a gradient of acetonitrile / water containing 0.1% TFA at a flow rate of 60 mL / min) to give the desired product as two peaks (0.40 g, 85%).

[0259] Diastereomer 1. Peak 1.C 32 H 36 ClFN9(M+H) + LC-MS calculated for: m / z=600.3; found 600.3.

[0260] Diastereomer 2. Peak 2.C 32 H 36 ClFN9(M+H) + LC-MS calculated for: m / z=600.3; found 600.3.

[0261] Step 21. 2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile To a solution of 2-fluoroacrylic acid (1.1 mg, 0.012 mmol) and 2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidin-2-yl)acetonitrile bis(2,2,2-trifluoroacetate) (8.5 mg, 10.3 μmol) (peak 2 from the last step) in DMF (1.0 ml) was added HATU (5.1 mg, 0.013 mmol) and DIEA (9.0 μl, 0.051 mmol). The resulting mixture was stirred at room temperature for 2 h. The reaction was diluted with methanol and 1N HCl (0.1 mL) and purified using preparative LCMS (XBridge C18 column, eluting with a gradient of acetonitrile / water containing 0.1% TFA at a flow rate of 60 mL / min) to give the desired diastereomer 1.

[0262] Diastereomer 2 was synthesized in a similar manner using 2-((2S,4S)-4-(8-chloro-7-(6-chloro-5-methyl-1H-indazol-4-yl)-4-(3-(dimethylamino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidin-2-yl)acetonitrile bis(2,2,2-trifluoroacetic acid) (peak 1 from the last step).

[0263] Example 4a. Diastereomer 1. Peak 1.C 35 H 37 ClF2NO (M+H) + LCMS calculated m / z = 672.3; found 672.3. 1H NMR (500 MHz, DMSO) δ 8.34 (m, 2H), 7.48 (s, 1H), 7.38 (s, 1H), 5.70 (m, 1H), 5.35 (m, 2H), 4.98-4.27 (m, 9H), 3.54 (m, 2H), 3.27 (m, 2H), 2.86 (m, 2H), 2.49 (s, 3H), 2.37 - 2.27 (m, 4H), 2.09 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H).

[0264] Example 4b. Diastereomer 2. Peak 2.C 35 H 37 ClF2NO(M+H) + LCMS calculated for m / z=672.3; found 672.3.

[0265] Example 5a and Example 5b. 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile [ka] Step 1. Ethyl 2-amino-4-(8-cyanonaphthalen-1-yl)-3-fluorobenzoate [ka] The title compound was synthesized according to the procedures described in Step 5 of Example 1a and Example 1b, utilizing 8-bromo-1-naphthonitrile instead of 1-chloroisoquinoline-8-carbonitrile. 20 H 16 FN2O2(M+H) + LCMS calculated for m / z=335.1; found 335.1.

[0266] Step 2. Ethyl 2-amino-5-chloro-4-(8-cyanonaphthalen-1-yl)-3-fluorobenzoate [ka] The title compound was synthesized according to the procedures described in Step 6 of Example 1a and Example 1b, using ethyl 2-amino-4-(8-cyanoisoquinolin-1-yl)-3-fluorobenzoate instead of ethyl 2-amino-4-(8-cyanonaphthalen-1-yl)-3-fluorobenzoate. 20 H 15 ClFN2O2(M+H) + LCMS calculated for m / z=369.1; found 369.1.

[0267] Step 3. Ethyl 5-chloro-4-(8-cyanonaphthalen-1-yl)-2-(3-ethoxy-3-oxopropanamido)-3-fluorobenzoate [ka] This compound was prepared according to the procedure described in Step 7 of Example 1a and Example 1b, substituting ethyl 2-amino-5-chloro-4-(8-cyanoisoquinolin-1-yl)-3-fluorobenzoate for ethyl 2-amino-5-chloro-4-(8-cyanonaphthalen-1-yl)-3-fluorobenzoate. 25 H 21 ClFN2O5(M+H) + LC-MS calculated for: m / z=483.1; found 483.1.

[0268] Step 4. Ethyl 6-chloro-7-(8-cyanonaphthalen-1-yl)-8-fluoro-2,4-dihydroxyquinoline-3-carboxylate [ka] This compound was prepared according to the procedure described in step 8 of Example 1a and Example 1b, substituting ethyl 5-chloro-4-(8-cyanoisoquinolin-1-yl)-2-(3-ethoxy-3-oxopropanamido)-3-fluorobenzoate for ethyl 5-chloro-4-(8-cyanonaphthalen-1-yl)-2-(3-ethoxy-3-oxopropanamido)-3-fluorobenzoate. 23 H 15 ClFN2O4(M+H) + LC-MS calculated for: m / z=437.1; found 437.1.

[0269] Step 5. Ethyl 2,4,6-trichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoroquinoline-3-carboxylate [ka] This compound was prepared according to the procedure described in step 9 of Example 1a and Example 1b, substituting ethyl 6-chloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-2,4-dihydroxyquinoline-3-carboxylate for ethyl 6-chloro-7-(8-cyanonaphthalen-1-yl)-8-fluoro-2,4-dihydroxyquinoline-3-carboxylate. 23 H 13 Cl3FN2O2(M+H) + LC-MS calculated for: m / z=473.0, 475.0; found 473.1, 475.1.

[0270] Step 6. Ethyl 4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2,6-dichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoroquinoline-3-carboxylate [ka] This compound was prepared according to the procedure described in step 10 of Example 1a and Example 1b, substituting ethyl 2,4,6-trichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoroquinoline-3-carboxylate for ethyl 2,4,6-trichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoroquinoline-3-carboxylate. 41 H 50 Cl2FN4O5Si(M+H) + LC-MS calculated for: m / z=795.3, 797.3; found 795.5, 797.5.

[0271] Step 7. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoro-3-(hydroxymethyl)quinolin-4-yl)amino)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 11 of Example 1a and Example 1b, substituting ethyl 4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoroquinoline-3-carboxylate for ethyl 4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2,6-dichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoroquinoline-3-carboxylate. 39 H 48 Cl2FN4O4Si(M+H) + LC-MS calculated for: m / z=753.3, 755.3; found 753.4, 755.5.

[0272] Step 8. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoro-3-formylquinolin-4-yl)amino)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 12 of Example 1a and Example 1b, substituting tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-(hydroxymethyl)quinolin-4-yl)amino)piperidine-1-carboxylate for tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoro-3-(hydroxymethyl)quinolin-4-yl)amino)piperidine-1-carboxylate. 39 H6Cl2FN4O4Si(M+H) + LC-MS calculated for: m / z=751.3, 753.3; found 751.4, 753.4.

[0273] Step 9. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoro-3-((E)-(hydroxyimino)methyl)quinolin-4-yl)amino)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 13 of Example 1a and Example 1b, substituting tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-formylquinolin-4-yl)amino)piperidine-1-carboxylate for tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoro-3-formylquinolin-4-yl)amino)piperidine-1-carboxylate. 39 H 47 Cl2FN5O4Si(M+H) + LC-MS calculated for: m / z=766.3, 768.3; found 766.4, 768.4.

[0274] Step 10. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(4,8-dichloro-7-(8-cyanonaphthalen-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in Example 1a and Step 14 of Example 1b, substituting tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanoisoquinolin-1-yl)-8-fluoro-3-((E)-(hydroxyimino)methyl)quinolin-4-yl)amino)piperidine-1-carboxylate for tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-((2,6-dichloro-7-(8-cyanonaphthalen-1-yl)-8-fluoro-3-((E)-(hydroxyimino)methyl)quinolin-4-yl)amino)piperidine-1-carboxylate. 39 H 45Cl2FN5O3Si(M+H) + LC-MS calculated for: m / z=748.3, 750.3; found 748.4, 750.4.

[0275] Step 11. tert-Butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(8-chloro-7-(8-cyanonaphthalen-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 15 of Example 1a and Example 1b, substituting tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(4,8-dichloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate for tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(4,8-dichloro-7-(8-cyanonaphthalen-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate. 40 H 48 ClFN5O3SSi(M+H) + LC-MS calculated for: m / z=760.3; found 760.3.

[0276] Step 12. tert-Butyl (2S,4S)-4-(8-chloro-7-(8-cyanonaphthalen-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 16 of Example 1a and Example 1b, substituting tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate for tert-butyl (2S,4S)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(8-chloro-7-(8-cyanonaphthalen-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate. 34 H 34 ClFN5O3S(M+H) + LC-MS calculated for: m / z = 646.2; found 646.2.

[0277] Step 13. tert-Butyl (2S,4S)-4-(8-chloro-7-(8-cyanonaphthalen-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 17 of Example 1a and Example 1b, substituting tert-butyl (2S,4S)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate for tert-butyl (2S,4S)-4-(8-chloro-7-(8-cyanonaphthalen-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate. 34 H 31 ClFN6O2S(M+H) +LC-MS calculated for: m / z = 641.2; found 641.2.

[0278] Step 14. 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile [ka] This compound was prepared according to the procedure described in step 18 of Example 1a and Example 1b, substituting tert-butyl (2S,4S)-4-(8-chloro-7-(8-cyanoisoquinolin-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate for tert-butyl (2S,4S)-4-(8-chloro-7-(8-cyanonaphthalen-1-yl)-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate. 35 H 34 ClFNO(M+H) + LC-MS calculated for: m / z=622.2; found 622.2.

[0279] Step 15. 8-(1-((2S,4S)-1-acetyl-2-(cyanomethyl)piperidin-4-yl)-8-chloro-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile To a solution of 2-fluoroacrylic acid (0.9 mg, 9.88 μmol) and 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile bis(2,2,2-trifluoroacetate) (7.0 mg, 8.23 ​​μmol) in DMF (1.0 ml), HATU (3.9 mg, 10.3 μmol) and DIEA (1.4 μl, 8.23 ​​μmol) were added. The resulting mixture was stirred at room temperature for 2 hours. The reaction was diluted with methanol and 1N HCl (0.1 mL) and then purified using preparative LCMS (XBridge C18 column, eluting with a gradient of acetonitrile / water containing 0.1% TFA at a flow rate of 60 mL / min) to give the desired diastereomer 1.

[0280] Diastereomer 2 was synthesized in a similar manner using 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile bis(2,2,2-trifluoroacetate) (peak 2 from the last step).

[0281] Example 5a. Diastereomer 1. Peak 1.C 37 H 36 ClFN7O2(M+H) + LCMS calculated for m / z=664.3; found 664.3.

[0282] Example 5b. Diastereomer 2. Peak 2.C 37 H 36 ClFN7O2(M+H) + LCMS calculated m / z = 664.3; found 664.3. 1H NMR (600 MHz, DMSO) δ 9.86 (m, 1H), 8.52 (m, 3H), 8.34 (m, 1H), 8.17 (m, 1H), 7.88 (m, 1H), 7.78 (m, 1H), 7.73 (m, 1H), 5.83 (m, 1H), 5.50 (m, 1H), 5.39 (m, 1H), 5.27 (s, 1H), 3.84 (m, 1H), 3.56 (m, 1H), 3.30 (m, 2H), 3.18 (m, 1H), 3.09 (m, 3H), 2.30 (m, 1H), 2.11 (m, 1H), 1.92 (m, 2H), 1.53 (d, J = 6.0 Hz, 3H).

[0283] Example 6a and Example 6b. 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-((E)-4-methoxybut-2-enoyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile [ka] This compound was prepared according to the procedure described in step 15 of Example 5a and Example 5b, substituting (E)-4-methoxybut-2-enoic acid for 2-fluoroacrylic acid. 40 H 40 ClFN7O3(M+H) + LCMS calculated for: m / z=720.3; Found: 720.3.

[0284] Example 7 8-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile [ka] Step 1: Ethyl 7-bromo-2,4-dichloro-8-fluoro-6-iodoquinoline-3-carboxylate [ka] The title compound was synthesized according to the procedures described in steps 1-3 of Example 4a and Example 4b, except that 2-amino-4-bromo-3-fluoro-5-iodobenzoic acid was used in place of 2-amino-4-bromo-5-chloro-3-fluorobenzoic acid in step 1. 12 H7BrCl2FINO2(M+H) + LCMS calculated m / z=491.80, 493.80; found 491.80, 493.80.

[0285] Step 2. Ethyl 7-bromo-4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2-chloro-8-fluoro-6-iodoquinoline-3-carboxylate [ka] This compound was prepared according to the procedures described in step 10 of Example 4a and Example 4b, substituting ethyl 7-bromo-2,4,6-trichloro-8-fluoroquinoline-3-carboxylate with ethyl 7-bromo-2,4-dichloro-8-fluoro-6-iodoquinoline-3-carboxylate. 30 H 44 BrClFIN3O5Si(M+H) + LC-MS calculated for: m / z=814.1, 816.1; found 814.1, 816.2.

[0286] Step 3. tert-Butyl (2S,4S)-4-((7-bromo-2-chloro-8-fluoro-3-(hydroxymethyl)-6-iodoquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in Example 4a and Step 11 of Example 4b, substituting ethyl 7-bromo-4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2,6-dichloro-8-fluoroquinoline-3-carboxylate with ethyl 7-bromo-4-(((2S,4S)-1-(tert-butoxycarbonyl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)amino)-2-chloro-8-fluoro-6-iodoquinoline-3-carboxylate. 28 H 42 BrClFIN3O4Si(M+H) + LC-MS calculated for: m / z=772.1, 774.1; found 772.1, 774.1.

[0287] Step 4. tert-Butyl (2S,4S)-4-((7-bromo-2-chloro-8-fluoro-3-formyl-6-iodoquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 12 of Example 4a and Example 4b, substituting tert-butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-(hydroxymethyl)quinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate for tert-butyl (2S,4S)-4-((7-bromo-2-chloro-8-fluoro-3-(hydroxymethyl)-6-iodoquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate. 28 H 40 BrClFIN3O4Si(M+H) +LC-MS calculated for: m / z=770.1, 772.1; found 770.1, 772.1.

[0288] Step 5. tert-Butyl (2S,4S)-4-((7-bromo-2-chloro-8-fluoro-3-((E)-(hydroxyimino)methyl)-6-iodoquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 13 of Example 4a and Example 4b, substituting tert-butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-formylquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate for tert-butyl (2S,4S)-4-((7-bromo-2-chloro-8-fluoro-3-formyl-6-iodoquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate. 28 H 41 BrClFIN4O4Si(M+H) + LC-MS calculated for: m / z=785.1, 787.1; found 785.2, 787.2.

[0289] Step 6. tert-Butyl (2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in Example 4a and Step 14 of Example 4b, substituting tert-butyl (2S,4S)-4-((7-bromo-2,6-dichloro-8-fluoro-3-((E)-(hydroxyimino)methyl)quinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate for tert-butyl (2S,4S)-4-((7-bromo-2-chloro-8-fluoro-3-((E)-(hydroxyimino)methyl)-6-iodoquinolin-4-yl)amino)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate. 28 H 39 BrClFIN4O3Si(M+H) + LC-MS calculated for: m / z=767.1, 769.1; found 767.1, 769.1.

[0290] Step 7. tert-Butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 15 of Example 4a and Example 4b, substituting tert-butyl (2S,4S)-4-(7-bromo-4,8-dichloro-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate for tert-butyl (2S,4S)-4-(7-bromo-4-chloro-6-fluoro-8-iodo-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate. 29 H 42 BrFIN4O3SSi(M+H) +LC-MS calculated for: m / z=779.1, 781.1; found 779.1, 781.1.

[0291] Step 8. tert-Butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 16 of Example 4a and Example 4b, substituting tert-butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate for tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidine-1-carboxylate. 23 H 28 BrFIN4O3S(M+H) + LC-MS calculated for: m / z=665.0, 667.0; found 665.1, 667.1.

[0292] Step 9. tert-Butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate [ka] This compound was prepared according to the procedure described in step 17 of Example 4a and Example 4b, substituting tert-butyl (2S,4S)-4-(7-bromo-8-chloro-6-fluoro-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate for tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(2-hydroxyethyl)piperidine-1-carboxylate. 23 H 25 BrFIN5O2S(M+H) + LC-MS calculated for: m / z = 660.0, 662.0; found 660.0, 662.0.

[0293] Step 10. tert-Butyl (2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate [ka] To a solution of tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-iodo-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (2.75 g, 4.16 mmol) in 1,4-dioxane (36 ml), water (6.0 ml), methylboronic acid (1.496 g, 24.99 mmol), K2CO3 (1.151 g, 8.33 mmol) and Pd(PPh3)2CI2 (0.292 g, 0.416 mmol) were added at room temperature. The reaction mixture was stirred at 90° C. for 10 hours under N2 atmosphere. After the reaction was completed, the reaction mixture was quenched with water and extracted with EtOAc. The organic phase was dried over anhydrous Na2SO4, concentrated, and then purified by column chromatography on silica gel (eluent: hexane:ethyl acetate=5:1) to give the compound (1.9 g, 83%) as a white solid. 24 H 28BrFN5O2S(M+H) + LC-MS calculated for: m / z=548.1, 550.1; found 548.2, 550.2.

[0294] Step 11. tert-Butyl (2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate [ka] m-CPBA (57.9 mg, 0.335 mmol) was added to a solution of tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylthio)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (160 mg, 0.29 mmol) in CHCl (2.9 ml) at 0° C., and the reaction was then stirred at this temperature for 20 minutes. The reaction was quenched by adding saturated NaSO, diluted with ethyl acetate, washed with saturated NaHCO, brine, filtered, dried and concentrated. LiHMDS 1.0M in THF (753 μl, 0.753 mmol) was added to a solution of (S)-1-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (97 mg, 0.753 mmol) in THF (1 mL). The resulting mixture was stirred at room temperature for 30 min. A solution of tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-(methylsulfinyl)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (170 mg, 0.301 mmol) in THF (2.0 mL) was added to the reaction vial and the reaction was then stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and water. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified on a silica gel column (eluted with a gradient of 0-20% methanol in DCM) to give the desired product as a yellow foam (150 mg, 79%). 30 H39 BrFN6O3(M+H) + LC-MS calculated for: m / z=629.2, 631.2; found 629.3, 631.3.

[0295] Step 12. tert-Butyl (2S,4S)-2-(cyanomethyl)-4-(7-(8-cyanonaphthalen-1-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-1-yl)piperidine-1-carboxylate [ka] A mixture of tert-butyl (2S,4S)-4-(7-bromo-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-1-yl)-2-(cyanomethyl)piperidine-1-carboxylate (150 mg, 0.238 mmol), 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthonitrile (86 mg, 0.31 mmol), SPhos Pd G4 (19 mg, 0.024 mmol) and tripotassium phosphate hydrate (121 mg, 0.524 mmol) in 1,4-dioxane (2.0 mL) / water (0.400 mL) was stirred at 80 °C for 2 h under N2 atmosphere. The solution was diluted with ethyl acetate and water, the organic layer was concentrated and the residue was purified by preparative LCMS (XBridge C18 column, eluting with a gradient of acetonitrile / water containing 0.1% TFA at a flow rate of 60 mL / min) to give the desired product as two peaks (105 mg, 63%).

[0296] Diastereomer 1. Peak 1.C 41 H 45 FN7O3(M+H) + LC-MS calculated for: m / z=702.3; found 702.3.

[0297] Diastereomer 2. Peak 2.C 41 H 45FN7O3(M+H) + LC-MS calculated for: m / z=702.3; found 702.3.

[0298] Step 13. 8-(1-((2S,4S)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile [ka] The two diastereomers from the last step were treated with 1:1 DCM / TFA (2 mL) for 40 min, the volatiles were removed in vacuo and the residue was used directly in the next step.

[0299] Diastereomer 1. Peak 1.C 36 H 37 FN7O(M+H) + LC-MS calculated for: m / z=602.3; found 602.3.

[0300] Diastereomer 2. Peak 2.C 36 H 37 FN7O(M+H) + LC-MS calculated for: m / z=602.3; found 602.3.

[0301] Step 14. 8-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile To a solution of but-2-yneoic acid (0.730 mg, 8.68 μmol) and 8-(1-((2S,4S)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-8-methyl-4-(((S)-1-methylpyrrolidin-2-yl)methoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile bis(2,2,2-trifluoroacetate) (6.0 mg, 7.36 μmol) (diastereomer 2 peak 2 from the last step) in DMF (1.0 ml) was added HATU (3.5 mg, 9.19 μmol) and DIEA (6.4 μl, 0.037 mmol). The resulting mixture was stirred at room temperature for 1 h. The reaction was diluted with methanol and 1N HCl (0.1 mL) and purified using preparative LCMS (XBridge C18 column, eluting with a gradient of acetonitrile / water containing 0.1% TFA at a flow rate of 60 mL / min) to give the desired product (2.5 mg, 52%). 40 H 39 FN7O2(M+H) + LCMS calculated for m / z=668.3; found 668.3.

[0302] Example A. GDP-GTP Exchange Assay Inhibitor potency of exemplified compounds was determined by a fluorescence-based guanine nucleotide exchange assay that measures the exchange of GppNHp (a non-hydrolyzable GTP analog) with Bodipy-GDP (fluorescently labeled GDP) to generate an active state of KRAS in the presence of SOS1 (a guanine nucleotide exchange factor). Inhibitors were serially diluted in DMSO and 0.1 μL volumes were transferred to wells of a black low-volume 384-well plate. A 5 μL / well volume of Bodipy-loaded KRAS G12C diluted to 5 nM in assay buffer (25 mM Hepes pH 7.5, 50 mM NaCl, 10 mM MgCl2, and 0.01% Brij-35) was added to the plate and pre-incubated with inhibitor for 2 hours at ambient temperature. Appropriate controls (enzyme without inhibitor or with G12C inhibitor (AMG-510)) were included on the plate. Exchange was initiated by adding a 5 μL / well volume containing 1 mM GppNHp and 300 nM SOS1 in assay buffer. The 10 μL / well reaction concentrations of Bodipy-loaded KRAS G12C, GppNHp, and SOS1 were 2.5 nM, 500 uM, and 150 nM, respectively. The reaction plate was incubated at ambient temperature for 2 hours, the time estimated for complete GDP-GTP exchange in the absence of inhibitors. For KRAS G12D and G12V mutants, a similar guanine nucleotide exchange assay was used with 2.5 nM as the final concentration of Bodipy-loaded KRAS protein and 4 and 3 hours of incubation after addition of the GppNHp-SOS1 mixture to G12D and G12V, respectively. A cyclic peptide described to selectively bind to the G12D mutant (Sakamoto et al., BBRC, 484.3 (2017), 605-611) or an internal compound with confirmed binding was used as a positive control in the assay plate. Fluorescence intensity was measured on a PheraStar plate reader instrument (BMG Labtech) using excitation at 485 nm and emission at 520 nm.

[0303] Data were analyzed using either GraphPad prism or Genedata Screener SmartFit. IC was estimated by fitting data to a four-parameter logistic equation to generate a sigmoidal dose-response curve with variable Hill coefficient. 50 derived the value.

[0304] KRAS_G12C exchange assay IC 50 Data, KRAS_G12C pERK assay IC 50 Data, KRAS_G12C WB pERK assay IC 50 The data are provided in Table 1 below. The symbol "†" indicates IC 50 ≤ 100 nM, and "††" indicates IC 50 >100 nM but ≤1 μM, and "†††" indicates IC 50 indicates IC > 1 μM but ≦ 5 μM, "††††" indicates IC 50 >5 μM but ≦10 μM. "NA" indicates data not available. [Table 4]

[0305] Example B: Luminescent viability assay MIA PaCa-2 (KRAS G12C; ATCC® CRL-1420), NCI-H358 (KRAS G12C; ATCC® CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC® CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells are cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco / Life Technologies). 800 cells per well are seeded into white clear bottom 384-well Costar tissue culture plates containing 50 nL dots of test compound (final concentration 1:500 dilution, final concentration in 0.2% DMSO) in RPMI1640 medium supplemented with 2% FBS. Plates are incubated at 370° C., 5% CO2 for 3 days. At the end of the assay, 25 μl / well CellTiter-Glo reagent (Promega) is added. Luminescence is read on a PHERAstar (BMG) after 15 min. Data are expressed as IC 50 Values ​​are analyzed with Genedata Screener using SmartFit.

[0306] Example C: Cellular pERK HTRF Assay MIA PaCa-2 (KRAS G12C; ATCC® CRL-1420), NCI-H358 (KRAS G12C; ATCC® CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC® CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells were purchased from ATCC and maintained in RPMI 1640 medium supplemented with 10% FBS (Gibco / Life Technologies). Cells are seeded at 5000 cells (8 uL) per well in Greiner 384-well low volume, flat bottom, tissue culture treated white plates and incubated overnight at 370° C., 5% CO2. The next morning, test compound stock solutions are diluted to 3× final concentration in medium and 4 μL is added to cells, resulting in a final concentration of 0.1% DMSO. Cells are incubated with test compounds for 4 hours (G12C and G12V) or 2 hours (G12D) at 37° C., 5% CO2. 4 uL of 4× lysis buffer containing blocking reagent (Cisbio) is added to each well and plates are gently rotated (300 rpm) for 30 minutes at room temperature. 4 uL / well of Cisbio anti-phospho-ERK1 / 2d2 is mixed with anti-phospho-ERK1 / 2 cryptate (1:1) and added to each well and incubated overnight at room temperature in the dark. Plates are read on a Pherastar plate reader at wavelengths of 665 nm and 620 nm. Data are expressed as IC 50 Values ​​are analyzed with Genedata Screener using SmartFit.

[0307] Example D: Whole Blood pERK1 / 2 HTRF Assay MIA PaCa-2 cells (KRAS G12C; ATCC® CRL-1420), HPAF-II (KRAS G12D; ATCC® CRL-1997) and YAPC (KRAS G12V; DSMZ ACC382) are maintained in RPMI 1640 with 10% FBS (Gibco / Life Technologies). For MIA PaCa-2 assays, cells are seeded in 96-well tissue culture plates (Corning #3596) at 25,000 cells per well in 100 μL of medium and cultured at 37° C., 5% CO2 for 2 days prior to assay. For HPAF-II and YAPC assays, cells are seeded in 96-well tissue culture plates at 50,000 cells per well in 100 μL of medium and cultured for 1 day prior to assay. Whole blood is added to a 1 uL dot of compound (prepared in DMSO) in a 96-well plate and mixed gently by pipetting up and down so that the concentration of compound in the blood is 1x the desired concentration in 0.5% DMSO. Media is aspirated from the cells and 50 uL / well whole blood containing test compound is added and incubated at 37°C, 5% CO2 for 4 hours for MIA PaCa and YAPC assays; or 2 hours for HPAF-II assays, respectively. After discarding the blood, the plate is gently washed twice by adding PBS to the side of the well, discarding the PBS from the plate onto a paper towel, and tapping the plate to drain thoroughly. Then, 50 ul / well of 1x lysis buffer #1 (Cisbio) containing blocking reagent (Cisbio) and benzonase nuclease (Sigma catalog number E1014-5KU, final concentration of 1:10000) is added and incubated at room temperature for 30 minutes with shaking (250 rpm). After lysis, 16 uL of lysate is transferred to a 384-well Greiner small volume white plate using Assist Plus (Integra Biosciences, NH). 4 uL of a 1:1 mixture of anti-phospho-ERK1 / 2 d2 and anti-phospho-ERK1 / 2 cryptate (Cisbio) is added to the wells using Assist Plus and incubated overnight at room temperature in the dark. Plates are read on a Pherastar plate reader at wavelengths of 665 nm and 620 nm.Data is IC. 50 Values ​​are analyzed with Genedata Screener using SmartFit.

[0308] Example E: Ras Activation ELISA The 96-well Ras Activation ELISA Kit (Cell Biolabs Inc; #STA441) uses Raf1 RBD (Rho-binding domain) bound to a 96-well plate to selectively pull down active Ras from cell lysates. The captured GTP-Ras is then detected by a pan-Ras antibody and an HRP-conjugated secondary antibody.

[0309] MIA PaCa-2 (KRAS G12C; ATCC® CRL-1420), NCI-H358 (KRAS G12C; ATCC® CRL-5807), A427 (KRAS G12D; ATCC® HTB53), HPAFII (KRAS G12D; ATCC® CRL-1997), YAPC (KRAS G12V; DSMZ ACC382), SW480 (KRAS G12V; ATCC® CRL-228) and NCI-H838 (KRAS WT; ATCC® CRL-5844) cells were maintained in RPMI 1640 medium with 10% FBS (Gibco / Life Technologies). Cells are seeded in 96-well tissue culture plates (Corning #3596) at 25,000 cells per well in 100 μL medium and cultured at 37° C., 5% CO2 for 2 days to be approximately 80% confluent at the start of the assay. Cells are treated with compounds for 4 hours or overnight at 37° C., 5% CO2. At the time of harvest, cells are washed with PBS, drained thoroughly, and then lysed with 50 uL of 1× lysis buffer (provided by the kit) plus Halt protease and phosphatase inhibitors (1:100) for 1 hour on ice.

[0310] Dilute Raf-1 RBD 1:500 in assay diluent (provided in the kit) and add 100 μL of diluted Raf-1 RBD to each well of the RAf-1RBD capture plate. Cover the plate with plate sealing film and incubate on an orbital shaker at room temperature for 1 h. Wash the plate three times with 250 μL of 1x wash buffer per well, performing a complete aspiration between each wash. Add 50 μL of Ras lysate sample (10-100 μg) per well in duplicate. Add "no cell lysate" controls to some wells for background measurements. For all wells, immediately add 50 μL of assay diluent to each well and incubate the plate at room temperature for 1 h on an orbital shaker. Wash the plate five times with 250 μL of 1x wash buffer per well, performing a complete aspiration between each wash. Add 100 μL of diluted anti-pan-Ras antibody to each well and incubate the plate at room temperature for 1 h on an orbital shaker. The plate is washed five times as before. 100 μL of diluted secondary antibody, HRP conjugate, is added to each well and the plate is incubated on an orbital shaker at room temperature for 1 hour. The plate is washed five times as before and thoroughly drained. 100 μL of chemiluminescence reagent (provided in the kit) is added to each well, including the blank wells. After incubating the plate on an orbital shaker at room temperature for 5 minutes, the luminescence of each microwell is read on a plate luminometer. % inhibition is calculated relative to DMSO control wells after subtracting the background level of the "no lysate control" from all values. IC 50 is determined by curve fitting of percent inhibition of inhibitor versus the logarithm of inhibitor concentration using GraphPad Prism7 software.

[0311] Example F: Inhibition of the RAS-RAF and PI3K-AKT pathways The cellular potency of the compounds is determined by measuring the phosphorylation of the KRAS downstream effectors extracellular signal-regulated kinase (ERK), ribosomal S6 kinase (RSK), AKT (also known as protein kinase B, PKB), and the downstream substrate S6 ribosomal protein.

[0312] To measure phosphorylated extracellular signal-regulated kinase (ERK), ribosomal S6 kinase (RSK), AKT and S6 ribosomal protein, cells (details regarding the cell lines and the type of data generated are described in more detail in Table 2) are seeded in Corning 96-well tissue culture treated plates at 4×104 cells / well in RPMI medium with 10% FBS overnight. The next day, cells are incubated for 4 hours at 37° C., 5% CO2 in the presence or absence of a range of test compounds at concentrations. Cells are washed with PBS and lysed in 1× lysis buffer (Cisbio) containing protease and phosphatase inhibitors (Thermo Fisher, 78446). Ten or 20 μg of total protein lysates are subjected to SDS-PAGE and immunoblot analysis using the following antibodies from Cell Signaling Technologies (Danvers, Mass.): phospho-ERK1 / 2-Thr202 / Tyr204 (#9101L), total-ERK1 / 2 (#9102L), phosphor-AKT-Ser473 (#4060L), phospho-p90RSK-Ser380 (#11989S), and phospho-S6 ribosomal protein-Ser235 / Ser236 (#2211S). [Table 5]

[0313] Example G: In vivo efficacy testing Mia-Paca-2 (KRAS G12C), H358 (KRAS G12C), HPAF-II (KRAS G12D), AGS (KRAS G12D), SW480 (KRAS G12V) or YAPC (KRAS G12V) human cancer cells are obtained from the American Type Culture Collection and maintained in RPMI medium supplemented with 10% FBS. For efficacy testing experiments, 5 × 106 cells are inoculated subcutaneously into the right hind flank of 6- to 8-week-old BALB / c nude mice (Charles River Laboratories, Wilmington, MA, USA). Tumor volumes of approximately 150–250 mm 3 Mice are randomized by tumor volume and orally administered compounds when tumor volume reaches 100 mg / kg / day. Tumor volume is calculated using the formula (L×W2) / 2, where L and W refer to the length and width dimensions, respectively. Tumor growth inhibition is calculated using the formula (1-(VT / VC))×100, where VT is the tumor volume of the treatment group on the last day of treatment, and VC is the tumor volume of the control group on the last day of treatment. Two-way ANOVA with Dunnett's multiple comparison test is used to determine statistical differences between treatment groups (GraphPad Prism). Mice are housed 10-12 per cage, enriched, and exposed to a 12-hour light / dark cycle. Mice with tumor volumes exceeding the limit (10% of body weight) are humanely euthanized by inhalation of CO2. Animals are maintained in a barrier facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. All procedures are performed in accordance with the US Public Service Policy on Human Care and Use of Laboratory Animals and Incyte Animal Care and Use Committee guidelines.

[0314] Example H: Caco2 Assay Caco-2 cells were grown at 37 °C in a 5% CO2 atmosphere in DMEM growth medium supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) non-essential amino acids, penicillin (100 U / mL), and streptomycin (100 μg / mL). Confluent cell monolayers were subcultured for Caco-2 every 7 days or every 4 days by treating with 0.05% trypsin containing 1 μM EDTA. Caco-2 cells were seeded in 96-well Transwell plates. The seeding density of Caco-2 cells was 14,000 cells / well. DMEM growth medium was changed every other day after seeding. Cell monolayers were used for transport assays for 22–25 days for Caco-2 cells.

[0315] The cell culture medium was removed and replaced with HBSS. To measure TEER, HBSS was added to the donor compartment (apical side) and receiver compartment (basolateral side). TEER was measured using a REMS Autosampler to ensure the integrity of the cell monolayer. TEER values ​​were 300 Ω cm. 2 The above Caco-2 cell monolayers were used for transport experiments. app To determine the permeability, a solution of test compound (50 μM) in HBSS was added to the donor compartment (apical side) and a HBSS solution containing 4% BSA was added to the receiver compartment (basolateral side). The apical volume was 0.075 mL and the basal volume was 0.25 mL. The incubation period was 120 min at 37° C. in an atmosphere of 5% CO2. At the end of the incubation period, samples were removed from the donor and receiver sides and an equal volume of acetonitrile was added for protein precipitation. The supernatant was collected after centrifugation (Allegra X-14R Centrifuge, 3000 rpm, Beckman Coulter, Indianapolis, IN) for LCMS analysis. The permeability value was determined according to the following formula: P app (cm / s)=(F*VD) / (SA*MD), where the flux rate (F, mass / time) is calculated from the slope of the cumulative amount of the compound of interest on the receiver side, SA is the surface area of ​​the cell membrane, VD is the donor volume, and MD is the initial amount of solution in the donor chamber.

[0316] Caco-2 permeability assay data is provided below in Table 3. The symbol "†" indicates Caco2 < 0.5, "††" indicates Caco-2 > 0.5 but < 1, and "†††" indicates Caco-2 > 1. "NA" indicates Caco-2 data is not available. [Table 6]

[0317] Example I: Human Whole Blood Stability Whole blood stability of exemplary compounds was determined by LC-MS / MS. A 96-well Flexi-Tier™ block (Analytical Sales & Services, Inc, Flanders, NJ) was used for incubation plates containing 1.0 mL glass vials with 0.5 mL of blood per vial (pooled gender, human whole blood sourced from BIOIVT, Hicksville, NY, etc.). Blood was pre-warmed to 37° C. in a water bath for 30 minutes. A 96-deep well analytical plate was prepared by adding 100 μL of ultrapure water / well. 50 μL of chilled ultrapure water / well was added to a 96-deep well sample collection plate and covered with a sealing mat. 1 μL of 0.5 mM compound working solution (DMSO:water) was added to the blood in the incubation plate to reach a final concentration of 1 μM, mixed thoroughly by pipetting, and 50 μL was transferred to the T=0 well of the sample collection plate. The blood was left in the water for 2 minutes, then 400 μL of stop solution / well was added (acetonitrile containing internal standard). The incubation plate was placed in an Incu-Shaker CO2 Mini incubator (Benchmark Scientific, Sayreville, NJ) at 37°C with shaking at 150 rpm. At 1, 2, and 4 hours, the blood samples were mixed thoroughly by pipetting and 50 μL was transferred to the corresponding wells of the sample collection plate. The blood was left in the water for 2 minutes, then 400 μL of stop solution / well was added. The collection plate was sealed and vortexed at 1700 rpm for 3 minutes (VX-2500 Multi-Tube Vortexer, VWR International, Radnor, PA), then the samples were centrifuged in the collection plate at 3500 rpm for 10 minutes (Allegra X-14R Centrifuge Beckman Coulter, Indianapolis, IN). 100 μL of supernatant / well was transferred from the sampling plate to the corresponding wells of the analysis plate. The final plate was vortexed at 1700 rpm for 1 min and samples were analyzed by LC-MS / MS. Peak area ratios of 1, 2, and 4 hour samples relative to T=0 were used to determine percent remaining.The natural logarithm of percent remaining versus time was used to determine the slope for calculating the half-life of the compound in blood (t 1 / 2 = 0.693 / slope).

[0318] Human whole blood stability data is provided below in Table 4. The symbol "†" indicates that the WBS is ≦70%, "††" indicates that the WBS is >70% but ≦90%, and "†††" indicates that the WBS is >90%. "NA" indicates that the WBS data is not available. [Table 7]

[0319] Example J: In Vitro Intrinsic Clearance Protocol Test compounds are incubated with human liver microsomes at 37° C. for in vitro metabolic stability experiments. The incubation mixture contains test compound (1 μM), NADPH (2 mM), and human liver microsomes (0.5 mg protein / mL) in 100 mM phosphate buffer (pH 7.4). The mixture is preincubated at 37° C. for 2 min before adding NADPH. The reaction is initiated with the addition of NADPH and quenched with ice-cold methanol at 0, 10, 20, and 30 min. The completed incubation mixture is analyzed using an LC-MS / MS system. The analysis system consisted of a Sciex Triple Quad6500+ mass spectrometer from Applied Biosystems (Foster City, CA) coupled with a Shimadzu LC-30AD binary pump system and a SIL-30AC autosampler (Shimadzu Scientific Instruments, Columbia, MD). Chromatographic separation of test compounds and internal standards is achieved using a Hypersil Gold C18 column (50 x 2.1 mm, 5 μΜ, 175 Å) from ThermoFisher Scientific (Waltham, MA). Mobile phase A consists of 0.1% formic acid in water and mobile phase B consists of 0.1% formic acid in acetonitrile. Total LC-MS / MS run time can be 2.75 min at a flow rate of 0.75 mL / min. Peak area integration and peak area ratio calculations are performed using Analyst software (version 1.6.3) from Applied Biosystems.

[0320] In vitro intrinsic clearance, CL int,in vitro CL int,in vitro =(0.693 / t 1 / 2 )×(1 / C protein t of disappearance of the test compound as 1 / 2 is calculated from 、 In the formula, C protein is the protein concentration during incubation, and t 1 / 2is determined by the slope (k) of the log-linear regression analysis of the concentration versus time profile; therefore, t 1 / 2 = ln2 / k. CL int,in vitro Values ​​are scaled to human in vivo values ​​by using physiologically based scaling factors, liver microsomal protein concentration (45 mg protein / g liver), and liver weight (21 g / kg body weight). The formula CL int =CL int,in vitro × (mg protein / g liver weight) × (g liver weight / kg body weight). Next, in vivo hepatic clearance (CL H ) in a well-stirred liver model, int and hepatic blood flow, Q (20 mL min -1 ·kg -1 ) and CL H =(Q × CL int ) / (Q+CL int ) ignoring all binding. The liver extraction ratio is calculated from CL H was calculated as divided by Q.

[0321] Example K: In vivo Pharmacokinetic Protocol For in vivo pharmacokinetic studies, test compounds are administered intravenously or via oral gavage to male Sprague Dawley rats or male and female cynomolgus monkeys. For intravenous (IV) administration, test compounds are administered at 0.5-1 mg / kg using a formulation of 10% dimethylacetamide (DMAC) in acidified saline via an IV bolus for rats and a 5- or 10-minute IV infusion for monkeys. For oral (PO) administration, test compounds are administered at 1.0-3.0 mg / kg using a formulation of 5% DMAC in 0.5% methylcellulose in citrate buffer (pH 2.5). Blood samples are collected pre-dose and at various time points up to 24 hours post-dose. All blood samples are collected using EDTA as an anticoagulant and centrifuged to obtain plasma samples. Plasma concentrations of the test compounds are determined by LC-MS methods. Measured plasma concentrations are used to calculate PK parameters by standard non-compartmental methods using the Phoenix® WinNonlin software program (version 8.0, Pharsight Corporation).

[0322] In rats and monkeys, cassette dosing of test compounds is performed to obtain preliminary PK parameters.

[0323] In vivo pharmacokinetic studies using male beagle dogs may be carried out under the conditions described above.

[0324] Example L: Time-Dependent Inhibition (TDI) of CYP Protocol This assay is designed to characterize the increase in CYP inhibition as the test compound is metabolized over time. Possible mechanisms for this include the formation of tightly bound quasi-irreversible inhibitory metabolite complexes or inactivation of P450 enzymes by covalent adduct formation of metabolites. Although this experiment employs 10-fold dilutions to reduce metabolite concentrations and thus the effect of reversible inhibition, it is possible (but not common) that metabolites that are very potent CYP inhibitors may produce positive results.

[0325] Results are from a cocktail of CYP-specific probe substrates at 4x their Km concentrations for CYP2C9, 2C19, 2D6 and 3A4 (midazolam) using human liver microsomes (HLM). HLM can be preincubated for 30 min with test compounds at a concentration of 10 μM in the presence (+N) or absence (-N) of an NADPH regenerating system, diluted 10-fold, and incubated for 8 min in the presence of the substrate cocktail with the addition of a new aliquot of NADPH regenerating system. Using a calibration curve of metabolite standards, enzyme activity can be quantitatively measured using LC-MS / MS. In addition, incubations with known time-dependent inhibitors, tienilic acid (CYP2C9), ticlopidine (CYP2C19), paroxetine (CYP2D6), and troleandomycin (CYP3A4), used as positive controls, are preincubated for 30 min with or without an NADPH regenerating system.

[0326] The analytical system consists of a Sciex Triple Quad 6500+ mass spectrometer from Applied Biosystems (Foster City, CA) coupled with a Shimadzu LC-30AD binary pump system and a SIL-30AC autosampler (Shimadzu Scientific Instruments, Columbia, MD). Chromatographic separation of test compounds and internal standards can be achieved using an ACQUITY UPLC BEH 130A, 2.1×50 mm, 1.7 μm HPLC column (Waters Corp, Milford, MA). Mobile phase A consists of 0.1% formic acid in water and mobile phase B consists of 0.1% formic acid in acetonitrile. The total LC-MS / MS run time is 2.50 min at a flow rate of 0.9 mL / min. Peak area integration and peak area ratio calculations are performed using Applied Biosystems Analyst software (version 1.6.3).

[0327] The percentage of control CYP2C9, CYP2C19, CYP2D6, and CYP3A4 remaining activity following preincubation of compound with NADPH is corrected for the corresponding control vehicle activity and then calculated with 0 min set to 100%. A linear regression plot of the natural log of the % remaining activity for each isoenzyme versus time is used to calculate the slope. - The slope represents the rate of enzyme loss, or K obs is equal to.

[0328] In addition to those described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Each reference cited in this application, including, but not limited to, all patents, patent applications, and publications, is hereby incorporated by reference in its entirety.

Claims

1. Formula (I) 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein R １ But Cl, CH ３ , C.H. ２ F, CHF ２ , and CF ３ is selected from Cy １ is selected from the following: 【Chemistry 2】 R ２ is selected from F and Cl; R ３ is selected from the following: 【Chemistry 3】 and, Cy ２ is selected from the following: 【Chemistry 4】 The compound of formula I, or a pharmaceutically acceptable salt thereof, with the proviso that said compound is other than: 【Table 1】

2. R １ But Cl, CH ２ F, CHF ２ , and CF ３ is selected from Cy １ is selected from the following: 【Chemistry 5】 R ２ is selected from F and Cl; R ３ is selected from the following: 【Chemistry 6】 and, Cy ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: 【Chemistry 7】

3. R １ But CH ３ , C.H. ２ F, CHF ２ , and CF ３ 2. The compound of claim 1, selected from:

4. R １ But Cl, CH ３ , and CF ３ 2. The compound of claim 1, selected from:

5. R １ But CH ２ F, CHF ２ , and CF ３ 2. The compound of claim 1, selected from:

6. R １ is Cl and CH ３ 2. The compound of claim 1, selected from:

7. R １ but Cl and CF ３ 2. The compound of claim 1, selected from:

8. R １ But CH ３ and CF ３ 2. The compound of claim 1, selected from:

9. Cy １ But Cy １ -a and Cy １ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

10. Cy １ But Cy １ -a and Cy １ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

11. Cy １ But Cy １ -b and Cy １ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

12. R ２ 2. The compound of claim 1, wherein is F, or a pharmaceutically acceptable salt thereof.

13. R ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

14. R ３ But, R ３ -a, or a pharmaceutically acceptable salt thereof.

15. R ３ But, R ３ -b, or a pharmaceutically acceptable salt thereof.

16. Cy ２ But Cy ２ -a and Cy ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

17. Cy ２ But Cy ２ -a and Cy ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

18. Cy ２ But Cy ２ -b and Cy ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

19. Cy １ But Cy １ -a, Cy ２ But Cy ２ -a, or a pharmaceutically acceptable salt thereof.

20. The compound is 1-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-8-chloro-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-((E)-4-methoxybut-2-enoyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 1-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)isoquinoline-8-carbonitrile, 2-((2S,4S)-4-(8-chloro-7-(5,6-dimethyl-1H-indazol-4-yl)-4-(3-(ethyl(methyl)amino)azetidin-1-yl)-6-fluoro-1H-pyrazolo[4,3-c]quinolin-1-yl)-1-(2-fluoroacryloyl)piperidin-2-yl)acetonitrile, 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-(2-fluoroacryloyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, 8-(8-chloro-1-((2S,4S)-2-(cyanomethyl)-1-((E)-4-methoxybut-2-enoyl)piperidin-4-yl)-6-fluoro-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, and 8-(1-((2S,4S)-1-(but-2-ynoyl)-2-(cyanomethyl)piperidin-4-yl)-6-fluoro-8-methyl-4-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)-1H-pyrazolo[4,3-c]quinolin-7-yl)-1-naphthonitrile, 2. The compound of claim 1 selected from: or a pharmaceutically acceptable salt thereof.

21. A pharmaceutical composition comprising a compound according to any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

22. A pharmaceutical for inhibiting KRAS activity, comprising the compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof.

23. A medicament for treating a disease or disorder associated with the activity of KRAS, comprising a compound according to any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof.

24. A pharmaceutical for treating a disease or disorder associated with the activity of a KRAS protein having a G12C mutation, the pharmaceutical comprising a compound according to any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof.

25. A medicament for treating cancer in a patient, said medicament comprising a compound according to any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof.

26. The method of claim 25, wherein the cancer is selected from carcinoma, blood cancer, sarcoma, and glioblastoma.

27. 27. The pharmaceutical composition of claim 26, wherein the cancer is a blood cancer selected from myeloproliferative neoplasms, myelodysplastic syndromes, chronic and juvenile myelomonocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, and multiple myeloma.

28. 27. The pharmaceutical composition of claim 26, wherein the cancer is a carcinoma selected from pancreatic carcinoma, colorectal carcinoma, lung carcinoma, bladder carcinoma, gastric carcinoma, esophageal carcinoma, breast carcinoma, head and neck carcinoma, cervical carcinoma, skin carcinoma, and thyroid carcinoma.

29. 25. The method of claim 24, wherein the disease or disorder is an immunological or inflammatory disorder.

30. The pharmaceutical composition of claim 29, wherein the immunological or inflammatory disorder is a Ras-associated lymphoproliferative disorder or juvenile myelomonocytic leukemia caused by a somatic mutation of KRAS.

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