RAS inhibitors for cancer treatment

Abstract

The present invention relates to macrocyclic compounds capable of inhibiting Ras proteins, as well as pharmaceutical compositions and protein complexes thereof, and their use in the treatment of cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 184,618, filed May 5, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The vast majority of small molecule drugs act by binding to functionally important pockets on target proteins, thereby regulating their activity. For example, cholesterol-lowering drugs known as statins bind to the enzyme active site of HMG-CoA reductase, thereby preventing the enzyme from engaging its substrate. The fact that many such drug / target interaction pairs are known may mislead some into believing that, given a reasonable amount of time, effort, and resources, small molecule modulators can be discovered for most, if not all, proteins. This is far from the case. Current estimates suggest that only about 10% of all human proteins are targetable by small molecules. (Non-Patent Document 1) The other 90% are currently considered refractory or intractable to small molecule drug discovery as described above. Such targets are commonly referred to as "undruggable." These undruggable targets represent a vast and abundant, untapped reservoir of clinically important human proteins. Therefore, there is much interest in discovering novel molecular modalities that can control the function of such undruggable targets.

[0003] It has been well established in the literature that Ras proteins (K-Ras, H-Ras, and N-Ras) play essential roles in various human cancers and are therefore suitable targets for anticancer therapeutics. Indeed, mutations in Ras proteins account for approximately 30% of all human cancers in the United States, many of which are fatal. Dysregulation of Ras proteins through activating mutations, overexpression, or upstream expression is common in human tumors, and activating mutations in Ras are frequently found in human cancers. For example, activating mutations at codon 12 in Ras proteins function by inhibiting both the GTPase-activating protein (GAP)-dependent and intrinsic hydrolysis rates of GTP, significantly skewing the population of Ras mutant proteins toward the "on" (GTP-bound) state (Ras(ON)), resulting in oncogenic MAPK signaling. Notably, Ras exhibits picomolar affinity for GTP, allowing it to be activated even in the presence of low concentrations of this nucleotide. Mutations at codon 13 (eg, G13D) and codon 61 (eg, Q61K) of Ras are also responsible for oncogenic activity in some cancers.

[0004] Although extensive drug discovery efforts have been made against Ras in recent decades, no drugs that directly target Ras remain approved, and further efforts are needed to identify additional drugs for cancers driven by various Ras mutations. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Bojadzic and Buchwald,Curr Top Med Chem 18:674-699(2019) Summary of the Invention

[0006] Ras inhibitors are provided herein. The approach described herein requires the formation of a high-affinity ternary complex or conjugate between a synthetic ligand and two intracellular proteins that do not interact under normal physiological conditions: the protein of interest (e.g., Ras) and a cytosolic chaperone (presenter protein) (e.g., cyclophilin A) that is ubiquitously expressed in the cell. More specifically, in some embodiments, the Ras inhibitors described herein contain a novel cytosolic binding pocket in Ras by driving the formation of a high-affinity tri-complex or conjugate between the Ras protein and the ubiquitously expressed cytosolic chaperone cyclophilin A (CYPA). Without being bound by theory, the inventors believe that one way in which the compounds and complexes or conjugates of the present invention affect their inhibitory effects at Ras is by forming steric blockage of the interaction site between Ras and downstream effector molecules, such as RAF and PI3K, which is necessary for oncogenic signaling to propagate.

[0007] Thus, in some embodiments, the present invention relates to compounds of structural formula I, or a pharmaceutically acceptable salt thereof:

[0008] [ka]

[0009] wherein A is an optionally substituted 3- to 6-membered heterocycloalkylene, an optionally substituted 3- to 6-membered cycloalkylene, an optionally substituted 6-membered arylene, or an optionally substituted 5- to 10-membered heteroarylene; W is a bridging group comprising an aziridine, epoxide, carbodiimide, oxazoline, thiazoline, chloroethylurea, chloroethylthiourea, chloroethylcarbamate, chloroethylthiocarbamate, trifluoromethylketone, boronic acid, boronic ester, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isoEEDQ or other EEDQ derivatives, oxazolium, or glycal; X 1 is CH2 or O; m is 1 or 2, n is 0 or 1, R 1 is hydrogen or an optionally substituted 3- to 10-membered heterocycloalkyl; R 2 is an optionally substituted C1-C6 alkyl, and R 3 is an optionally substituted C1-C6 alkyl or an optionally substituted 3- to 6-membered cycloalkyl.

[0010] In some embodiments, the present invention also relates to compounds of structural formula III, or a pharmaceutically acceptable salt thereof:

[0011] [ka]

[0012] wherein P is —(CO)R 9 , -(PO)(OH)2, or -Si(R 10 )3, W is a bridging group comprising an aziridine, epoxide, carbodiimide, oxazoline, thiazoline, chloroethylurea, chloroethylthiourea, chloroethylcarbamate, chloroethylthiocarbamate, trifluoromethylketone, boronic acid, boronic ester, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isoEEDQ or other EEDQ derivatives, oxazolium, or glycal; L 1is an optionally substituted 3- to 9-membered heterocycloalkylene or an optionally substituted C2-C4 heteroalkyl; R 4 is an optionally substituted C1-C6 alkyl; R 5 is an optionally substituted C1-C6 alkyl or an optionally substituted 3- to 6-membered cycloalkyl.

[0013] R 9 is an optionally substituted C1-C3 alkyl or an optionally substituted C1-C3 heteroalkyl, and Each R 10 are independently optionally substituted C1-C3 alkyl.

[0014] In some embodiments, the present invention also relates to a compound selected from Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6, or a pharmaceutically acceptable salt thereof. Also provided is a pharmaceutical composition comprising a compound of Formula I or Formula III, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Also provided is a pharmaceutical composition comprising a compound of Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0015] Also provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, there is provided a method for treating a Ras protein-associated disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof.

[0017] Further provided is a method for inhibiting a Ras protein in a cell, said method comprising contacting said cell with an effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof.

[0018] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any compound or composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or utilize any compound or composition of the invention.

[0019] Definitions and Chemical Terms In this application, unless otherwise clear from the context, (i) the term "a" means "one or more," (ii) is used to mean "and / or" unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive, but the present invention supports definitions that refer to alternatives only and to "and / or," (iii) the terms "comprising" and "including" are understood to encompass the itemized components or steps, whether presented by themselves or with one or more additional components or steps, and (iv) when ranges are presented, the endpoints are included.

[0020] As used herein, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being used to determine the value. In certain embodiments, the term "about" refers to a range of values ​​that is included by 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (above or below) the stated value, unless otherwise stated or otherwise apparent from the context (e.g., where such number may exceed 100% of the possible values).

[0021] As used herein, the term "adjacent" in the context of describing adjacent atoms means divalent atoms that are directly joined by a covalent bond. As used herein, "compounds of the invention" and similar terms refer to the Ras inhibitors described herein, including compounds of Formula I, Formula III, or subformulas thereof, and compounds of Table 1, Table 2, Table 3, Table 4, Table 5, or Table 6, as well as salts (e.g., pharmaceutically acceptable salts), solvates, hydrates, stereoisomers (including atropisomers), and tautomers thereof, whether or not explicitly stated.

[0022] The term "wild-type" refers to an entity having a structure or activity that is found in a "normal" (as opposed to a mutant, diseased, altered, etc.) state or context in nature. Those skilled in the art will understand that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0023] Those of skill in the art will understand that certain compounds described herein can exist in one or more different isomeric (e.g., stereoisomers, geometric isomers, atropisomers, tautomers) or isotopic (e.g., one or more atoms replaced with a different isotope of that atom, such as hydrogen replaced with deuterium) forms. Unless otherwise specified, or apparent from context, the depicted structures can be understood to represent any such isomeric or isotopic forms, individually or in combination.

[0024] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise specified. 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 active starting materials, such as by resolution of racemic mixtures or stereoselective synthesis, are known in the art. 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.

[0025] In some embodiments, one or more compounds described herein can exist in different tautomeric forms. Unless explicitly excluded, as is clear from the context, reference to such a compound encompasses all such tautomeric forms. In some embodiments, a tautomeric form results from the exchange of a single bond with an adjacent double bond and the accompanying migration of a proton. In certain embodiments, a tautomeric form can be a prototropic tautomer, which is an isomeric protonation state having the same empirical formula and total charge as the referenced form. Examples of moieties having prototropic tautomeric forms are ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position in a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. In some embodiments, the tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. In certain embodiments, the tautomeric forms arise from acetal interconversion.

[0026] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. Illustrative isotopes that can be incorporated into the compounds of the invention include: 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 32 P, 33 P, 35 S, 18 F, 36 Cl, 123 I, and 125 Isotopically labeled compounds (e.g., 3 H and 14 C) can be useful in compound or substrate tissue distribution assays. 3 H), and carbon-14 (i.e., 14 C) isotopes can be useful for their ease of preparation and detectability. Additionally, heavier isotopes, such as deuterium (i.e., 2 Substitution with, for example, H, may result in greater metabolic stability and may confer certain therapeutic advantages (e.g., longer in vivo half-life or reduced dosage requirements). In some embodiments, one or more hydrogen atoms may be 2 H or 3 H or one or more carbon atoms are replaced by 13 C or 14 It is replaced by C-enriched carbon. 15 O. 13 N, 11 C and 18Positron-emitting isotopes, such as F, are useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. The preparation of isotopically labeled compounds is known to those skilled in the art. For example, isotopically labeled compounds can generally be prepared by following procedures similar to those disclosed for the compounds of the invention described herein, substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0027] Non-limiting examples of moieties that may contain one or more deuterium substitutions in the compounds of the invention (any position "R" may be deuterium (D)) include:

[0028] [ka]

[0029] Additional examples include:

[0030] [ka]

[0031] and similar R 1 Deuteration of the R 1 The definitions of are found herein (e.g., in Formulas I, Ia, and V). Deuteration of moieties within substituent W in the compounds of the invention is also contemplated, where W is defined herein (e.g., in general Formulas I-III, and subformulas thereof, as well as specific examples of W described herein, such as

[0032] [ka]

[0033] (See also

[0034] [ka]

[0035] Also contemplated is deuteration at any available position in any A moiety of compounds of the formulae described herein, such as:

[0036] [ka]

[0037] can occur in the compounds of the present invention at linker positions such as: In further embodiments, silylated substitutions are also contemplated, such as in linkers such as:

[0038] [ka]

[0039] As is known in the art, many chemical entities can be adopted in a variety of different solid forms, such as amorphous or crystalline forms (e.g., polymorphs, hydrates, solvates), etc. In some embodiments, the compounds of the present invention can be utilized in any such form, including any solid form. In some embodiments, the compounds described or illustrated herein can be provided or utilized in hydrate or solvate form.

[0040] At various places herein, substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual subcombination of the members of such groups and ranges. For example, the term "C1-C6 alkyl" is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl. Furthermore, when a compound contains multiple positions where a substituent is disclosed in a group or range, unless otherwise specified, the invention is intended to cover individual compounds containing each and every individual subcombination of the elements at each position, as well as groups (e.g., genera and subgenera) of compounds.

[0041] The term "optionally substituted X" (e.g., optionally substituted alkyl) is intended to be equivalent to "X, where X is optionally substituted" (e.g., "alkyl, where the alkyl is optionally substituted"). The feature "X" (e.g., alkyl) itself is not intended to imply optionality. As described herein, certain compounds of interest can contain one or more "optionally substituted" moieties. Generally, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified moiety are replaced with a suitable substituent, e.g., any of the substituents or groups described herein. Unless otherwise specified, an "optionally substituted" group can have a suitable substituent at each suitable position of the group. Also, when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituents can be either the same or different at each position. For example, in the term "optionally substituted C-C alkyl-C-C heteroaryl," the alkyl portion, the heteroaryl portion, or both can be optionally substituted. Combinations of substituents contemplated by the present invention are preferably those that result in the formation of stable or chemically suitable compounds. The term "stable," as used herein, refers to compounds that remain substantially unchanged when subjected to conditions that foresee their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0042] Suitable monovalent substituents at a substitutable carbon atom of an "optionally substituted" group are independently deuterium, halogen, -(CH) 0-4 R°, -(CH2) 0-4 OR°, -O(CH2) 0-4 R°, -O-(CH2) 0-4 C(O)OR°, -(CH2) 0-4 CH(OR°)2, -(CH2) 0-4 SR°, -(CH2) 0-4Ph [may be substituted with R°], -(CH2) 0-4 O(CH2) 0-1 Ph [may be substituted with R°], -CH=CHPh [may be substituted with R°], -(CH2) 0-4 O(CH2) 0-1 -pyridyl [optionally substituted with R°], 4-8 membered saturated or unsaturated heterocycloalkyl (e.g., pyridyl), 3-8 membered saturated or unsaturated cycloalkyl (e.g., cyclopropyl, cyclobutyl, or cyclopentyl), -NO2, -CN, -N3, -(CH2) 0-4 N(R°)2, -(CH2) 0-4 N(R°)C(O)R°, -N(R°)C(S)R°, -(CH2) 0-4 N(R°)C(O)NR°2, -N(R°)C(S)NR°2, -(CH2) 0-4 N(R°)C(O)OR°, -N(R°)N(R°)C(O)R°, -N(R°)N(R°)C(O)NR°2, -N(R°)N(R°)C(O)OR°, -(CH2) 0-4 C(O)R°, -C(S)R°, -(CH2) 0-4 C(O)OR°, -(CH2) 0-4 -C(O)-N(R°)2, -(CH2) 0-4 -C(O)-N(R°)-S(O)2-R°, -C(NCN)NR°2, -(CH2) 0-4 C(O)SR°, -(CH2) 0-4 C(O)OSiR°3, -(CH2) 0-4 OC(O)R°, -OC(O)(CH2) 0-4 SR°, -SC(S)SR°, -(CH2) 0-4 SC(O)R°, -(CH2) 0-4 C(O)NR°2, -C(S)NR°2, -C(S)SR°, -(CH2) 0-4 OC(O)NR°2, -C(O)N(OR°)R°, -C(O)C(O)R°, -C(O)CH2C(O)R°, -C(NOR°)R°, -(CH2) 0-4 SSR°, -(CH2) 0-4 S(O)2R°, -(CH2) 0-4 S(O)2OR°, -(CH2) 0-4OS(O)2R°, -S(O)2NR°2, -(CH2) 0-4 S(O)R°, —N(R°)S(O)NR°2, —N(R°)S(O)R°, —N(OR°)R°, —C(NOR°)NR°2, —C(NH)NR°2, —P(O)R°, —P(O)R°2, —P(O)(OR°)2, —OP(O)R°2, —OP(O)(OR°)2, —OP(O)(OR°)R°, —SiR°3, —(C1-C4 straight or branched chain alkylene)ON(R°)2, or —(C1-C4 straight or branched chain alkylene)C(O)ON(R°)2, wherein each R° is optionally substituted as defined below and independently represents hydrogen, —C1-C6 aliphatic, —CH2Ph, —O(CH2) 0-1 Ph, -CH2- (a 5-6 membered heteroaryl ring), or a 3-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definitions, two independent R° together with the atom(s) between them form a 3-12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0043] Suitable monovalent substituents on R° (or the ring formed by taking two independently occurring R° together with the atoms between them) are independently halogen, —(CH) 0-2 R ● ,-(Halo R ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● )2, -O(HaloR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2SR ● , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● , -(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● , -(C 1-4 Straight or branched chain alkylene)C(O)OR ● , or -SSR ● wherein each R ● is unsubstituted or, where preceded by "halo", is substituted only with one or more halogens, independently selected from C1-C4 aliphatic, -CH2Ph, -O(CH2) 0-1 or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0044] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O-, or -S(C(R * 2)) 2-3 S-, wherein each R * is selected from hydrogen, a C1-C6 aliphatic, which may be substituted as defined below, or an unsubstituted 5-6 membered, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Preferred divalent substituents attached to a vicinal substitutable carbon of an "optionally substituted" group include -O(CR * 2)2-3 O-, wherein each R * is selected from hydrogen, a C1-C6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0045] R * Suitable substituents on the aliphatic group include -R ● , (Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, wherein each R ● is unsubstituted or, where preceded by "halo", is substituted only with one or more halogens, independently C1-C4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0046] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † wherein each R †are independently hydrogen, C1-C6 aliphatic, which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 3-6 membered, saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, regardless of the above definition, two independently occurring R † together with the intervening atom(s), form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0047] R † Suitable substituents on the aliphatic group are independently halogen, —R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, where preceded by "halo", is substituted only with one or more halogens, independently selected from C1-C4 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. † Suitable divalent substituents on a saturated carbon atom of include ═O and ═S.

[0048] As used herein, the term "acetyl" refers to the group -C(O)CH3. As used herein, the term "alkoxy" refers to an -O-C 20 It means that the alkyl and alkoxy groups are attached to the remainder of the compound through an oxygen atom.

[0049] As used herein, the term "alkyl" refers to a saturated, straight- or branched-chain monovalent hydrocarbon group containing 1 to 20 (e.g., 1 to 10, or 1 to 6) carbons. In some embodiments, alkyl groups are unbranched (i.e., straight-chain), and in some embodiments, alkyl groups are branched. Alkyl groups are exemplified by, but not limited to, methyl, ethyl, n- and isopropyl, n-, sec-, iso- and tert-butyl, and neopentyl.

[0050] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by removing two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene, and the like. x -C y "Alkylene" refers to an alkylene group having x to y carbons. Exemplary values ​​of x are 1, 2, 3, 4, 5, and 6, and exemplary values ​​of y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., C1-C6, C1-C6). 10 , C2-C 20 , C2-C6, C2-C 10 , or C2-C 20 In some embodiments, the alkylene can be further substituted with 1, 2, 3, or 4 substituents, as defined herein.

[0051] As used herein, the term "alkenyl," unless otherwise specified, represents a monovalent straight- or branched-chain group of 2 to 20 carbons (e.g., 2 to 6, or 2 to 10 carbons) containing one or more carbon-carbon double bonds, and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl. Alkenyl includes both cis and trans isomers. As used herein, the term "alkenylene," unless otherwise specified, represents a divalent straight- or branched-chain group of 2 to 20 carbons (e.g., 2 to 6, or 2 to 10 carbons) containing one or more carbon-carbon double bonds.

[0052] As used herein, the term "alkynyl" refers to a monovalent straight or branched chain group of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons) containing a carbon-carbon triple bond and is exemplified by ethynyl and 1-propynyl.

[0053] As used herein, the term "alkynyl sulfone" refers to a group having the structure

[0054] [ka]

[0055] wherein R is any suitable chemical substituent as described herein. As used herein, the term "amino" refers to -N(R † )2, for example, -NH2 and -N(CH3)2.

[0056] As used herein, the term "aminoalkyl" refers to an alkyl moiety substituted on one or more carbon atoms with one or more amino moieties. As used herein, the term "amino acid" refers to a molecule having a side chain, an amino group, and an acidic group (e.g., -COH or -SOH), where the amino acid is attached to the parent molecular group by the side chain, the amino group, or the acidic group (e.g., the side chain). As used herein, the term "amino acid" in its broadest sense refers to any compound or substance that can be incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, optionally substituted hydroxylnorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine.

[0057] As used herein, the term "aryl" refers to a monovalent monocyclic, bicyclic, or polycyclic ring system formed by carbon atoms, wherein the ring attached to the pendant group is aromatic. Examples of aryl groups are phenyl, naphthyl, phenanthrenyl, and anthracenyl. An aryl ring can be attached to its pendant group at any heteroatom or carbon ring atom that results in a stable structure, and unless otherwise specified, any of the ring atoms can be optionally substituted.

[0058] As used herein, the term "C" represents a bond. For example, part of the term -N(C(O)-(C-C alkylene-H)- includes -N(C(O)-(C alkylene-H)-, which is also represented by -N(C(O)-H)-.

[0059] As used herein, the terms "carbocyclic" and "carbocyclyl" refer to a monovalent, optionally substituted, 3- to 12-membered, monocyclic, bicyclic, or tricyclic ring system, which may be optionally bridged, fused, or spirocyclic, in which all rings are formed by carbon atoms and at least one ring is non-aromatic. Carbocyclic systems include cycloalkyl, cycloalkenyl, and cycloalkynyl groups. Examples of carbocyclyl groups are cyclohexyl, cyclohexenyl, cyclooctynyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indenyl, indanyl, decalinyl, and the like. A carbocyclic ring can be attached to its pendant group at any ring atom that results in a stable structure, and unless otherwise specified, any of the ring atoms can be optionally substituted.

[0060] As used herein, the term "carbonyl" refers to a C(O) group, which can also be represented as C=O. As used herein, the term "carboxyl" refers to -CO2H, (C=O)(OH), COOH, or C(O)OH, or the unprotonated corresponding groups.

[0061] As used herein, the term "cyano" refers to a -CN group. As used herein, the term "cycloalkyl" refers to a monovalent saturated cyclic hydrocarbon group, which, unless otherwise specified, may be bridged, fused, or spirocyclic having 3 to 8 carbons, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cycloheptyl.

[0062] As used herein, the term "cycloalkenyl" refers to a monovalent non-aromatic saturated cyclic hydrocarbon group, which, unless otherwise specified, may be bridged, fused, or spirocyclic having 3 to 8 carbons and containing one or more carbon-carbon double bonds.

[0063] As used herein, the term "diastereomers" means stereoisomers that are not mirror images of each other and are not superimposable with respect to one another. As used herein, "enantiomer" means each individual optically active form of a compound of the invention having an optical purity or enantiomeric excess (as measured by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and up to 10% of the other enantiomer), preferably at least 90%, and more preferably at least 98%.

[0064] The term "guanidyl" means a compound having the structure

[0065] [ka]

[0066] wherein each R is independently any chemically suitable substituent described herein. As used herein, the term "guanidinoalkylalkyl" refers to an alkyl moiety substituted on one or more carbon atoms with one or more guanidyl moieties.

[0067] As used herein, the term "haloacetyl" means an acetyl group in which at least one hydrogen has been replaced by a halogen. As used herein, the term "haloalkyl" refers to an alkyl moiety substituted on one or more carbon atoms with one or more of the same or different halogen moieties.

[0068] As used herein, the term "halogen" refers to a halogen selected from bromine, chlorine, iodine, or fluorine. As used herein, the term "heteroalkyl" refers to an "alkyl" group (as defined herein) in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom). The heteroatom can occur at the center or end of the radical.

[0069] As used herein, the term "heteroaryl" refers to a monovalent monocyclic or polycyclic ring system containing at least one fully aromatic ring, i.e., they contain 4n+2 pi electrons in the monocyclic or polycyclic ring system and at least one ring heteroatom selected from N, O, or S in the aromatic ring. Exemplary unsubstituted heteroaryl groups are those of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term "heteroaryl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heteroaromatic rings are fused to one or more aryl or carbocyclic rings, such as phenyl or cyclohexane rings. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrazolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, imidazolyl, thiazolyl, quinolinyl, tetrahydroquinolinyl, and 4-azaindolyl. A heteroaryl ring can be attached to its pendant group at any ring atom that results in a stable structure, and unless otherwise specified, any ring atom can be optionally substituted. In some embodiments, a heteroaryl is substituted with 1, 2, 3, or 4 substituents.

[0070] As used herein, the term "heterocycloalkyl" refers to a monovalent monocyclic, bicyclic, or polycyclic ring system, which may be bridged, fused, or spirocyclic, in which at least one ring is non-aromatic and the non-aromatic ring contains 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Five-membered rings have 0 to 2 double bonds, and six- and seven-membered rings have 0 to 3 double bonds. Exemplary unsubstituted heterocycloalkyl groups are those of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term "heterocycloalkyl" also refers to heterocyclic compounds having bridged polycyclic structures in which one or more carbons or heteroatoms bridge two non-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group. The term "heterocycloalkyl" includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one or more aromatic, carbocyclic, heteroaromatic, or heterocyclic rings, such as an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, a pyridine ring, or a pyrrolidine ring. Examples of heterocycloalkyl groups are pyrrolidinyl, piperidinyl, 1,2,3,4-tetrahydroquinolinyl, decahydroquinolinyl, dihydropyrrolopyridine, and decahydronaphthyridinyl. A heterocycloalkyl ring can be attached to its pendant group at any ring atom that results in a stable structure, and unless otherwise specified, any of the ring atoms can be optionally substituted.

[0071] As used herein, the term "hydroxy" refers to an --OH group. As used herein, the term "hydroxyalkyl" refers to a group having one or more hydroxy groups on one or more carbon atoms. represents an alkyl moiety substituted with one or more —OH moieties.

[0072] As used herein, "isomer" refers to any tautomer, stereoisomer, atropisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention can have one or more chiral centers or double bonds and therefore exist as stereoisomers, e.g., double bond isomers (i.e., geometric E / Z isomers), or diastereomers (e.g., enantiomers (i.e., (+) or (-), or cis / trans isomers)). In accordance with the present invention, the chemical structures depicted herein, and therefore the compounds of the invention, may be present in all their corresponding stereoisomers, i.e., in stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomeric pure), as well as in their corresponding stereoisomers. Both enantiomeric and stereoisomeric mixtures (e.g., racemates) are encompassed. Enantiomeric and stereoisomeric mixtures of the compounds of the invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0073] As used herein, the term "linker" refers to a divalent organic moiety that connects a first moiety (e.g., a macrocyclic moiety) to a second moiety (e.g., a bridging group). In some embodiments, the linker results in compounds that are capable of achieving an IC50 of 2 uM or less in the Examples below and in the Ras-RAF disruption assay protocol provided herein.

[0074] The purpose of this biochemical assay is to measure the ability of a test compound to facilitate ternary complex formation between a nucleotide-loaded Ras isoform and cyclophilin A, and the resulting ternary complex is a BRAF RBD disrupting binding to the construct and inhibiting Ras signaling through RAF effectors.

[0075] In an assay buffer containing 25 mM HEPES (pH 7.3), 0.002% Tween 20, 0.1% BSA, 100 mM NaCl, and 5 mM MgCl, untagged cyclophilin A, His6-K-Ras-GMPPNP (or other Ras variants), and GST-BRAF RBD are combined in a 384-well assay plate at final concentrations of 25 μM, 12.5 nM, and 50 nM, respectively. Compounds are present in the plate wells as a 10-point, 3-fold dilution series starting at a final concentration of 30 μM. After 3 hours of incubation at 25°C, a mixture of anti-His Eu-W1024 and anti-GST allophycocyanin was added to the assay sample wells at final concentrations of 10 nM and 50 nM, respectively, and the reactions were incubated for an additional 1.5 hours. TR-FRET signals were read in a microplate reader (excitation 320 nm, emission 665 / 615 nm). Compounds that promote disruption of the Ras:RAF complex are identified as those that induce a decrease in the TR-FRET ratio relative to DMSO control wells.

[0076] In some embodiments, the linker comprises 20 or fewer linear chain atoms. In some embodiments, the linker comprises 15 or fewer linear chain atoms. In some embodiments, the linker comprises 10 or fewer linear chain atoms. In some embodiments, the linker has a molecular weight of less than 500 g / mol. In some embodiments, the linker has a molecular weight of less than 400 g / mol. In some embodiments, the linker has a molecular weight of less than 300 g / mol. In some embodiments, the linker has a molecular weight of less than 200 g / mol. In some embodiments, the linker has a molecular weight of less than 100 g / mol. In some embodiments, the linker has a molecular weight of less than 50 g / mol.

[0077] As used herein, a "monovalent organic moiety" is less than 500 kDa. In some embodiments, a "monovalent organic moiety" is less than 400 kDa. In some embodiments, a "monovalent organic moiety" is less than 300 kDa. In some embodiments, a "monovalent organic moiety" is less than 200 kDa. In some embodiments, a "monovalent organic moiety" is less than 100 kDa. In some embodiments, a "monovalent organic moiety" is less than 50 kDa. In some embodiments, a "monovalent organic moiety" is less than 25 kDa. In some embodiments, a "monovalent organic moiety" is less than 20 kDa. In some embodiments, a "monovalent organic moiety" is less than 15 kDa. In some embodiments, a "monovalent organic moiety" is less than 10 kDa. In some embodiments, a "monovalent organic moiety" is less than 1 kDa. In some embodiments, a "monovalent organic moiety" is less than 500 g / mol. In some embodiments, the "monovalent organic moiety" is in the range of 500 g / mol to 500 kDa.

[0078] As used herein, the term "stereoisomer" refers to all possible different isomeric forms and structural forms that a compound (e.g., a compound of any formula described herein) may possess, in particular all possible stereochemical and structural isomeric forms of the basic molecular structure, including atropisomers, all diastereomers, enantiomers, or conformational isomers. Some compounds of the present invention can exist in different tautomeric forms, all of the latter being within the scope of the present invention.

[0079] As used herein, the term "sulfonyl" refers to the group -S(O)2-. As used herein, the term "thiocarbonyl" refers to a -C(S)- group. As used herein, the term "vinyl ketone" refers to a group containing a carbonyl group attached directly to a carbon-carbon double bond.

[0080] As used herein, the term "vinyl sulfone" refers to a group containing a sulfonyl group attached directly to a carbon-carbon double bond. As used herein, the term "ynone" refers to a compound having the structure

[0081] [ka]

[0082] wherein R is any suitable optional substituent as described herein. Those skilled in the art reading this disclosure will understand that certain compounds described herein can be provided or utilized in any of a variety of forms, such as, for example, salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical or structural isomers), isotopic forms, etc. In some embodiments, reference to a particular compound may refer to a particular form of that compound. In some embodiments, reference to a particular compound may refer to that compound in any form. In some embodiments, for example, a preparation of a single stereoisomer of a compound may be considered a form of that compound that is different from a racemic mixture of that compound, a particular salt of a compound may be considered a form that is different from another salt form of that compound, a preparation containing a structural isomer of a double bond ((Z) or (E)) may be considered a form that is different from one containing the other structural isomer of that double bond ((E) or (Z)), and a preparation in which one or more atoms are isotopically different from those present in a reference preparation may be considered a different form. DETAILED DESCRIPTION OF THE INVENTION

[0083] compound Ras inhibitors are provided herein. The approach described herein requires the formation of a high-affinity ternary complex or conjugate between a synthetic ligand and two intracellular proteins that do not interact under normal physiological conditions: the protein of interest (e.g., Ras) and a cytosolic chaperone (presenter protein) (e.g., cyclophilin A) that is ubiquitously expressed in the cell. More specifically, in some embodiments, the Ras inhibitors described herein contain a novel cytosolic binding pocket in Ras by driving the formation of a high-affinity tri-complex or conjugate between the Ras protein and the ubiquitously expressed cytosolic chaperone, cyclophilin A (CYPA). Without being bound by theory, the inventors believe that one way in which the compounds and complexes or conjugates of the present invention affect their inhibitory effects at Ras is by forming steric blockage of the interaction site between Ras and downstream effector molecules, such as RAF, which is necessary for the propagation of oncogenic signals.

[0084] Without being bound by theory, the inventors hypothesize that both covalent and noncovalent interactions of the compounds of the present invention with Ras and chaperone proteins (e.g., cyclophilin A) may contribute to the inhibition of Ras activity. In some embodiments, the compounds of the present invention form covalent adducts with side chains of Ras proteins (e.g., the -CH2-COOH or -CH2-COO- side chain of aspartic acid at positions 12 or 13 of mutant Ras proteins). Covalent adducts may also be formed with other side chains of Ras. Additionally or alternatively, noncovalent interactions may be involved: for example, van der Waals interactions, hydrophobic interactions, hydrophilic interactions, and hydrogen-bonding interactions, as well as combinations thereof, may contribute to the ability of the compounds of the present invention to form complexes and act as RAS inhibitors. Thus, various Ras proteins can be inhibited by the compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and their mutants at positions 12, 13, and 61, e.g., G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, as well as others described herein).

[0085] Methods for measuring covalent adduct formation are known in the art. Another method for measuring covalent adduct formation is to perform a "crosslinking" assay, such as that described in the Examples, as follows: Note: The following protocol describes the procedure for monitoring crosslinking of K-Ras G12C(GMP-PNP) to compounds of the invention. This protocol can also be performed by substituting other Ras proteins or nucleotides, such as K-Ras G12D.

[0086] The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. A stock solution of 5 μM GMP-PNP-loaded K-Ras(1-169)G12C was diluted 10-fold in assay buffer containing 12.5 mM HEPES (pH 7.4), 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 μM cyclophilin A, and 2 μM test compound, to obtain a final concentration of 0.5 μM. The final sample volume was 100 μL.

[0087] Samples are incubated at 25°C for up to 24 hours and then quenched by the addition of 10 μL of 5% formic acid. After centrifugation of the quenched samples at 15,000 rpm for 15 minutes in a benchtop centrifuge, a 10 μL aliquot is injected onto a reversed-phase C4 column and eluted into a mass spectrometer using an increasing acetonitrile gradient in the mobile phase. Raw data analysis can be performed using Waters MassLynx MS software, and percent binding is calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.

[0088] In some embodiments, the compounds of the present invention are useful in treating other K-Ras mutants or K-Ras WT In some embodiments, the compounds of the present invention inhibit K-Ras G12D more potently than compounds known in the art. In some embodiments, the compounds of the present invention crosslink K-Ras G12D to a greater extent than other K-Ras mutants. In some embodiments, the compounds of the present invention crosslink K-Ras G12D to a greater extent than compounds known in the art.

[0089] Thus, provided herein are compounds having the structure of Formula I, or a pharmaceutically acceptable salt thereof:

[0090] [ka]

[0091] wherein A is an optionally substituted 3- to 6-membered heterocycloalkylene, an optionally substituted 3- to 6-membered cycloalkylene, an optionally substituted 6-membered arylene, or an optionally substituted 5- to 10-membered heteroarylene; W is a bridging group comprising an aziridine, epoxide, carbodiimide, oxazoline, thiazoline, chloroethylurea, chloroethylthiourea, chloroethylcarbamate, chloroethylthiocarbamate, trifluoromethylketone, boronic acid, boronic ester, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isoEEDQ or other EEDQ derivatives, oxazolium, or glycal; X 1 is CH2 or O; m is 1 or 2, n is 0 or 1, R 1 is hydrogen or an optionally substituted 3- to 10-membered heterocycloalkyl; R 2 is an optionally substituted C1-C6 alkyl, and R 3 is an optionally substituted C1-C6 alkyl or an optionally substituted 3- to 6-membered cycloalkyl.

[0092] In some embodiments, W is an aziridine or epoxide-containing bridging group. In some embodiments, A is optionally substituted thiazole, optionally substituted oxazole, optionally substituted morpholino, optionally substituted pyrrolidinyl, optionally substituted piperidinyl, or optionally substituted phenyl.

[0093] In some embodiments, the compound of the invention has the structure of Formula Ia, or a pharmaceutically acceptable salt thereof:

[0094] [ka]

[0095] In some embodiments, the compounds of the invention have the structure of Formula II-1, or a pharmaceutically acceptable salt thereof:

[0096] [ka]

[0097] In some embodiments, the compounds of the invention have the structure of Formula II-2, or a pharmaceutically acceptable salt thereof:

[0098] [ka]

[0099] [In the formula, R 6 , R 7 , and R 8 are each independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted 3- to 6-membered cycloalkyl, and optionally substituted 3- to 6-membered heterocycloalkyl; R 6 and R 7 combine with the atom to which they are attached to form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 8-membered heterocycloalkyl; or R 6 and R 8 combine with the atom to which they are attached to form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 8-membered heterocycloalkyl.

[0100] In some embodiments, the compound of the invention has the structure of Formula II-3, or a pharmaceutically acceptable salt thereof:

[0101] [ka]

[0102] In some embodiments, the compound of the invention has the structure of Formula II-4, or a pharmaceutically acceptable salt thereof:

[0103] [ka]

[0104] [where, X 2 is CH2 or O, and o is 1 or 2]. In some embodiments of the compounds of the present invention, X 2 is CH. In some embodiments, o is 1. In some embodiments, o is 2.

[0105] In some embodiments of the compounds of the present invention, X 2 is O. In some embodiments, o is 1. In some embodiments, o is 2. In some embodiments of the compounds of the present invention, R 2 teeth,

[0106] [ka]

[0107] is. In some embodiments of the compounds of the present invention, R 3 is an optionally substituted C1-C6 alkyl. In some embodiments, R 3 teeth,

[0108] [ka]

[0109] is. In some embodiments of the compounds of the present invention, R 3 is an optionally substituted 3- to 6-membered cycloalkyl. In some embodiments, R3 teeth,

[0110] [ka]

[0111] is. In some embodiments of the compounds of the present invention, A is an optionally substituted 5-10 membered heteroarylene.

[0112] [ka]

[0113] is. In some embodiments of the compounds of the present invention, A is optionally substituted phenyl. In some embodiments, A is

[0114] [ka]

[0115] is. In some embodiments of the compounds of the present invention, A is an optionally substituted 3-6 membered heterocycloalkylene. In some embodiments, A is selected from the following, or a stereoisomer thereof:

[0116] [ka]

[0117] In some embodiments of the compounds of the present invention, m is 1. In some embodiments, n is 1. In some embodiments, X 1 is CH. In some embodiments, X 1 is O. In some embodiments, m is 1, n is 1, and X 1 In some embodiments, m is 1, n is 1, and X1 is O.

[0118] In some embodiments of the compounds of the present invention, m is 2. In some embodiments, X 1 In some embodiments, n is 1. In some embodiments, n is 0. In some embodiments, m is 2 and X 1 is CH2 and n is 1. In some embodiments, m is 2 and X 1 is O. In some embodiments, m is 2 and X 1 is O and n is 1. In some embodiments, m is 2 and X 1 is O and n is 0.

[0119] In some embodiments of the compounds of the present invention, W comprises an aziridine. In some embodiments, W comprises an optionally substituted cyclopropyl-aziridinyl moiety. In some embodiments, W is selected from the following, or a stereoisomer thereof:

[0120] [ka]

[0121] In some embodiments of the compounds of the present invention, W comprises an epoxide.

[0122] [ka]

[0123] Or, it is selected from these stereoisomers. In some embodiments, the compound of the present invention is selected from Table 1, or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the compound of the present invention is selected from Table 1, or a pharmaceutically acceptable salt or atropisomer thereof.

[0124]

Table 1-1

[0125]

Table 1-2

[0126]

Table 1-3

[0127]

Table 1-4

[0128]

Table 1-5

[0129]

Table 1-6

[0130]

Table 1-7

[0131]

Table 1-8

[0132]

Table 1-9

[0133]

Table 1-10

[0134]

Table 1-11

[0135] [Table 1-12]

[0136] [Table 1-13]

[0137] [Table 1-14]

[0138] Note that some compounds depict bonds as flat or wedged. In some cases, the relative stereochemistry of stereoisomers has been determined. In some cases, the absolute stereochemistry has been determined. In some cases, a single example number corresponds to a mixture of stereoisomers. All stereoisomers of the compounds in the foregoing tables are contemplated by the present invention. In certain embodiments, atropisomers of the compounds in the foregoing tables are contemplated. In some embodiments, a compound of Table 2, or a pharmaceutically acceptable salt thereof, is provided. In some embodiments, a compound of the present invention is selected from Table 2, or a pharmaceutically acceptable salt or atropisomer thereof.

[0139] [Table 2-1]

[0140] [Table 2-2]

[0141] [Table 2-3]

[0142] [Table 2-4]

[0143]

Table 2-5

[0144]

Table 2-6

[0145]

Table 2-7

[0146]

Table 2-8

[0147]

Table 2-9

[0148]

Table 2-10

[0149]

Table 2-11

[0150]

Table 2-12

[0151]

Table 2-13

[0152]

Table 2-14

[0153]

Table 2-15

[0154]

Table 2-16

[0155]

Table 2-17

[0156]

Table 2-18

[0157]

Table 2-19

[0158]

Table 2-20

[0159]

Table 2-21

[0160]

Table 2-22

[0161]

Table 2-23

[0162]

Table 2-24

[0163]

Table 2-25

[0164]

Table 2-26

[0165]

Table 2-27

[0166]

Table 2-28

[0167]

Table 2-29

[0168]

Table 2-30

[0169]

Table 2-31

[0170]

Table 2-32

[0171]

Table 2-33

[0172]

Table 2-34

[0173] [Table 2-35]

[0174] [Table 2-36]

[0175] [Table 2-37]

[0176] [Table 2-38]

[0177] [Table 2-39]

[0178] Also provided herein are compounds having the structure of Formula III, or a pharmaceutically acceptable salt thereof:

[0179] [ka]

[0180] wherein P is —(CO)R 9 , -(PO)(OH)2, or -Si(R 10 )3, W is a bridging group comprising an aziridine, epoxide, carbodiimide, oxazoline, thiazoline, chloroethylurea, chloroethylthiourea, chloroethylcarbamate, chloroethylthiocarbamate, trifluoromethylketone, boronic acid, boronic ester, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isoEEDQ or other EEDQ derivatives, oxazolium, or glycal; L 1is an optionally substituted 3- to 9-membered heterocycloalkylene or an optionally substituted C2-C4 heteroalkyl; R 4 is an optionally substituted C1-C6 alkyl; R 5 is optionally substituted C1-C6 alkyl or optionally substituted 3- to 6-membered cycloalkyl.

[0181] R 9 is an optionally substituted C1-C3 alkyl or an optionally substituted C1-C3 heteroalkyl, and Each R 10 are independently optionally substituted C1-C3 alkyl.

[0182] In some embodiments, W is an aziridine-containing bridging group. In some embodiments of the compounds of the present invention, R 4 teeth

[0183] [ka]

[0184] is. In some embodiments of the compounds of the present invention, R 5 is an optionally substituted C1-C6 alkyl. In some embodiments, R 5 teeth,

[0185] [ka]

[0186] is. In some embodiments of the compounds of the present invention, R 5 is an optionally substituted 3- to 6-membered cycloalkyl. In some embodiments, R 5 teeth,

[0187] [ka]

[0188] is. In some embodiments of the compounds of the present invention, P is —(CO)R 9 In some embodiments, P is

[0189] [ka]

[0190] is selected from. In some embodiments of the compounds of the present invention, P is —(PO)(OH) 2 . In some embodiments of the compounds of the present invention, P is —Si(R 10 )3. In some embodiments, P is

[0191] [ka]

[0192] is selected from. In some embodiments of the compounds of the present invention, L 1 is a 3- to 9-membered heterocycloalkylene. In some embodiments, L 1 or its stereoisomers:

[0193] [ka]

[0194] is. In some embodiments of the compounds of the present invention, L 1 is an optionally substituted C2-C4 heteroalkyl. In some embodiments, L 1 teeth,

[0195] [ka]

[0196] is. In some embodiments of the compounds of the present invention, W is an optionally substituted cyclopropyl-aziridinyl moiety. In some embodiments, W is: or a stereoisomer thereof:

[0197] [ka]

[0198] In some embodiments, W is or a stereoisomer thereof:

[0199] [ka]

[0200] In some embodiments, W is

[0201] [ka]

[0202] In some embodiments, W is

[0203] [ka]

[0204] In some embodiments, W is or a stereoisomer thereof:

[0205] [ka]

[0206] In some embodiments, W is: or a stereoisomer thereof:

[0207] [ka]

[0208] In some embodiments, W is: or a stereoisomer thereof:

[0209] [ka]

[0210] In some embodiments, W is

[0211] [ka]

[0212] In some embodiments, W is

[0213] [ka]

[0214] In some embodiments, W is or a stereoisomer thereof:

[0215] [ka]

[0216] In some embodiments, W is: or a stereoisomer thereof:

[0217] [ka]

[0218] In some embodiments, W is

[0219] [ka]

[0220] In some embodiments, W is

[0221] [ka]

[0222] In some embodiments, W is or a stereoisomer thereof:

[0223] [ka]

[0224] In some embodiments, W is: or a stereoisomer thereof:

[0225] [ka]

[0226] In some embodiments, W is: or a stereoisomer thereof:

[0227] [ka]

[0228] In some embodiments, W is

[0229] [ka]

[0230] In some embodiments, W is

[0231] [ka]

[0232] is. In some embodiments, the compound of the present invention is selected from Table 3, or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the compound of the present invention is selected from Table 3, or a pharmaceutically acceptable salt or atropisomer thereof.

[0233] [Table 3-1]

[0234] [Table 3-2]

[0235] [Table 3-3]

[0236] Also provided herein is a compound selected from Table 4, or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the compound of the invention is selected from Table 4, or a pharmaceutically acceptable salt or atropisomer thereof.

[0237] [Table 4-1]

[0238] [Table 4-2]

[0239] In some embodiments, provided is a compound of Table 5, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of the invention is selected from Table 5, or a pharmaceutically acceptable salt or atropisomer thereof.

[0240] [Table 5-1]

[0241] [Table 5-2]

[0242] In some embodiments, the compound of the present invention is selected from Table 6, or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the compound of the present invention is selected from Table 6, or a pharmaceutically acceptable salt or atropisomer thereof.

[0243] [Table 6-1]

[0244] [Table 6-2]

[0245] [Table 6-3]

[0246] [Table 6-4]

[0247] [Table 6-5]

[0248] [Table 6-6]

[0249] In some embodiments, W is a crosslinking group attached to the organic moiety, i.e., RBM-W, that is a Ras-binding moiety. When the RBM-W compound contacts a Ras protein, the RBM-W binds to the Ras protein to form a conjugate. For example, the W portion of the RBM-W compound binds, e.g., crosslinks, to an amino acid of the Ras protein to form a conjugate. In some embodiments, the Ras-binding moiety is a K-Ras-binding moiety. In some embodiments, the K-Ras-binding moiety binds to a residue in the K-Ras Switch-II binding pocket of K-Ras. In some embodiments, the Ras-binding moiety is an H-Ras-binding moiety that binds to a residue in the H-Ras Switch-II binding pocket of an H-Ras protein. In some embodiments, the Ras-binding moiety is an N-Ras-binding moiety that binds to a residue in the N-Ras Switch-II binding pocket of an N-Ras protein. The W of the RBM-W compound can include any W described herein. The Ras-binding moiety typically has a molecular weight of less than 1200 Da. See, for example, Johnson et al., 292:12981-12993 (2017), which is incorporated herein by reference for Ras protein domains.

[0250] In some embodiments, the compounds of the invention are or act as prodrugs, for example, for administration to a cell or subject in need thereof. In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0251] Further provided is a conjugate comprising the structure of Formula IV, or a salt thereof: ML 2 -P 1 Formula IV [In the formula, L 2 is the linker, P 1 is a monovalent organic moiety, and M has the structure of Formula V.

[0252] [ka]

[0253] wherein A is an optionally substituted 3- to 6-membered heterocycloalkylene, an optionally substituted 3- to 6-membered cycloalkylene, an optionally substituted 6-membered arylene, or an optionally substituted 5- to 10-membered heteroarylene; X 1 is CH2 or O; m is 1 or 2, n is 0 or 1, R 1 is hydrogen or an optionally substituted 3- to 10-membered heterocycloalkyl; R 2 is an optionally substituted C1-C6 alkyl, and R 3 is an optionally substituted C1-C6 alkyl or an optionally substituted 3- to 6-membered cycloalkyl.

[0254] In some embodiments of the conjugates of the present invention, the monovalent organic moiety is a protein. In some embodiments, the protein is a Ras protein. In some embodiments, the Ras protein is K-Ras G12D or K-Ras G13D. In some embodiments of the conjugates of the present invention, the linker, L 2 is attached to the monovalent organic moiety via a bond to the carboxyl group of the amino acid residue of the monovalent organic moiety.

[0255] Further provided is a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.The cancer can be, for example, pancreatic cancer, colorectal cancer, non-small cell lung cancer, acute myeloid leukemia, multiple myeloma, thyroid cancer, myelodysplastic syndrome, or small squamous cell carcinoma follicular carcinoma.In some embodiments, the cancer contains a Ras mutation, such as K-Ras G12D or K-Ras G13D.Other Ras mutations are described herein.

[0256] Further provided is a method for treating a Ras protein-associated disease in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof.

[0257] Further provided is a method for inhibiting a Ras protein in a cell, comprising contacting the cell with an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. For example, the Ras protein is K-Ras G12D or K-Ras G13D. Other Ras proteins are described herein. The cell can be a cancer cell, such as a pancreatic cancer cell, a colorectal cancer cell, a non-small cell lung cancer cell, an acute myeloid leukemia cell, a multiple myeloma cell, a thyroid cancer cell, a myelodysplastic syndrome cell, or a small squamous cell carcinoma / follicular carcinoma cell. Other cancer types are described herein. The cell can be in vivo or in vitro.

[0258] For compounds of the present invention, one stereoisomer may exhibit better inhibition than another, for example, one atropisomer may exhibit inhibition while another atropisomer may exhibit little or no inhibition.

[0259] In some embodiments, the methods or uses described herein further comprise administering an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy is an EGFR inhibitor, a second Ras inhibitor, an SHP2 inhibitor, an SOS1 inhibitor, a Raf inhibitor, a MEK inhibitor, an ERK inhibitor, a PI3K inhibitor, a PTEN inhibitor, an AKT inhibitor, an mTORC1 inhibitor, a BRAF inhibitor, a PD-L1 inhibitor, a PD-1 inhibitor, a CDK4 / 6 inhibitor, a HER2 inhibitor, or a combination thereof. In some embodiments, the additional anti-cancer therapy is an SHP2 inhibitor. Other additional anti-cancer therapies are described herein.

[0260] Synthesis method The compounds described herein can be made from commercially available starting materials or can be synthesized using known organic, inorganic, or enzymatic processes.

[0261] The compounds of the present invention can be prepared by a number of methods well known to those skilled in the art of organic synthesis. For example, the compounds of the present invention can be synthesized using the methods shown in the schemes below, along with synthetic methods known in the art of organic synthetic chemistry, or variations thereon as would be understood by those skilled in the art. These methods include, but are not limited to, those depicted in the schemes below.

[0262] The compounds in Tables 1, 2, 3, 4, and 6 herein were prepared using the methods disclosed herein or prepared using the methods disclosed herein in combination with the knowledge of those of ordinary skill in the art. The compounds in Table 5 can be prepared using the methods disclosed herein or can be prepared using the methods disclosed herein in combination with the knowledge of those of ordinary skill in the art.

[0263] Scheme 1. General synthesis of aziridine-containing macrocycles

[0264] [ka]

[0265] As shown in Scheme 1, compounds of this type can be prepared by reacting an appropriate amine (1) with a protected amine (2) containing a carboxylic acid in the presence of a standard amide coupling reagent to give 3, followed by deprotection of the amine to give 4. Coupling of an aziridine carboxylate (5) in the presence of a standard amide coupling reagent gives 6. R 1 If is a protecting group, deprotection gives the final compound (7).

[0266] Pharmaceutical Compositions and Methods of Use Pharmaceutical compositions and methods of administration The compounds to which the present invention relates are RAS inhibitors and are useful in the treatment of cancer. Accordingly, one embodiment of the present invention provides pharmaceutical compositions containing a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, as well as methods for preparing such compositions using the compounds of the present invention.

[0267] As used herein, the term "pharmaceutical composition" means a compound, such as a compound of the present invention, or a pharmaceutically acceptable salt thereof, formulated together with a pharmaceutically acceptable excipient.

[0268] In some embodiments, the compound is present in the pharmaceutical composition in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition can be specially formulated for administration in solid or liquid form, including oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, pills, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection as a sterile solution or suspension, or sustained-release formulation; topical application, e.g., as a cream, ointment, or sustained-release patch, or spray applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingual; intraocular; transdermal; or suitable for nasal, pulmonary, and other mucosal surfaces.

[0269] As used herein, "pharmaceutically acceptable excipient" refers to any inert ingredient (e.g., a vehicle capable of suspending or dissolving an active compound) that is toxic and non-inflammatory in a subject. Typical excipients include, for example, anti-adhesives, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, glidants (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or hydration water. Excipients include, but are not limited to, optionally substituted butylated hydroxyl toluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, optionally substituted hydroxyl propyl cellulose, optionally substituted hydroxyl propyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. Those skilled in the art are familiar with the variety of agents and materials useful as excipients.See, e.g., Ansel, et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. In some embodiments, the composition comprises at least two different pharmaceutically acceptable excipients.

[0270] The compounds described herein, whether explicitly stated or not, can be provided or utilized in salt form, for example, in pharmaceutically acceptable salt form, unless explicitly stated to the contrary. As used herein, the term "pharmaceutically acceptable salt" refers to those salts of the compounds described herein that are suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response, etc., within the normal scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base group with a suitable organic acid.

[0271] The compounds of the present invention can have ionic groups, allowing them to be prepared as pharmaceutically acceptable salts. These salts can be acid addition salts with inorganic or organic acids, or in the case of the acidic form of the compounds of the present invention, salts can be prepared from inorganic or organic bases. In some embodiments, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases, such as hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, or tartaric acid for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, etc. for forming base salts, are well known in the art. Methods for preparing suitable salts are well established in the art.

[0272] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-(optionally substituted)hydroxy-ethanesulfonate, and the like. Included are sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.

[0273] As used herein, the term "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human at any stage of development. In some embodiments, "subject" refers to a human patient. In some embodiments, "subject" refers to a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, the subject includes, but is not limited to, a mammal, bird, reptile, amphibian, fish, or worm. In some embodiments, the subject can be a transgenic animal, a genetically modified animal, or a clone.

[0274] As used herein, the term "dosage form" refers to a physically discrete unit of a compound (e.g., a compound of the invention) for administration to a subject. Each unit contains a predetermined amount of the compound. In some embodiments, such amount is a unit dose (or a whole fraction thereof) appropriate for administration according to a dosing regimen (i.e., using a therapeutic dosing regimen) that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population. Those skilled in the art will understand that the total amount of a therapeutic composition or compound administered to a particular subject will be determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0275] As used herein, the term "dosing regimen" refers to a collection of unit doses (usually two or more) administered individually to a subject, usually separated by a period of time. In some embodiments, a given therapeutic compound (e.g., a compound of the present invention) has a recommended dosing regimen, which may have one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated from the other by the same length of time period; in some embodiments, a dosing regimen includes multiple doses and at least two different time periods separating the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dose. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is different from the first dosage amount. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across relevant populations (ie, is a therapeutic dosing regimen).

[0276] By "therapeutic regimen" is meant a dosing regimen in which administration across a relevant population correlates with a desired or beneficial therapeutic outcome. The term "treatment" (plus "treat" or "treating"), in its broadest sense, refers to any administration of a substance (e.g., a compound of the invention) that partially or completely ameliorates, alleviates, reduces, or inhibits a particular disease, disorder, or condition; partially or completely delays the onset of a particular disease, disorder, or condition; partially or completely reduces the severity of a particular disease, disorder, or condition; or partially or completely reduces the occurrence of one or more symptoms, characteristics, or causes of a particular disease, disorder, or condition. In some embodiments, such treatment can be administered to a subject who does not exhibit signs of the associated disease, disorder, or condition, or who exhibits only early signs of a disease, disorder, or condition. Alternatively, or in addition, in some embodiments, such treatment can be administered to a subject who exhibits established signs of one or more of the associated diseases, disorders, or conditions. In some embodiments, treatment can be in a subject who has been diagnosed as suffering from the associated disease, disorder, or condition. In some embodiments, treatment may be in a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the relevant disease, disorder or condition.

[0277] The term "therapeutically effective amount" refers to an amount sufficient to treat a disease, disorder, or condition when administered to a population afflicted with or suspected of having the disease, disorder, or condition in accordance with a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the occurrence or severity of one or more symptoms of the disease, disorder, or condition, or delays the onset of one or more symptoms of the disease, disorder, or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not, in fact, require that successful treatment be achieved in any particular individual. Rather, a therapeutically effective amount can be an amount that, when administered to subjects in need of such treatment, results in a specific, desired pharmacological response in a significant number of subjects. It is specifically understood that certain subjects may, in fact, be "refractory" to a "therapeutically effective amount." In some embodiments, reference to a therapeutically effective amount can be a reference to the amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). Those skilled in the art will appreciate that in some embodiments, a therapeutically effective amount can be formulated or administered in a single dose, hi some embodiments, a therapeutically effective amount can be formulated or administered in multiple doses, for example, as part of a dosing regimen.

[0278] For use as a therapeutic for a subject, the compounds of the present invention, or pharmaceutically acceptable salts thereof, can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired, e.g., prevention, prophylaxis, or therapy, the compounds, or pharmaceutically acceptable salts thereof, will be formulated in a manner consistent with these parameters. A summary of such techniques is provided in Remington: The Science and Practice of Pharmacy, 21 stEdition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0279] The compositions can be prepared according to conventional mixing, granulating, or coating methods, respectively, and the pharmaceutical compositions can contain about 0.1% to about 99%, about 5% to about 90%, or about 1% to about 20% (by weight or volume) of a compound of the present invention or a pharmaceutically acceptable salt thereof. In some embodiments, the compounds described herein or a pharmaceutically acceptable salt thereof can be present in an amount of 1 to 95% in total by weight of the composition, such as a pharmaceutical composition.

[0280] The compositions can be provided in a dosage form suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesical, intraurethral, ​​intrathecal, epidural, otic, or ocular administration, or for injection, inhalation, or direct contact with the nasal, urogenital, reproductive, or oral mucosa. Thus, pharmaceutical compositions can be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels, including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectable solutions, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions can be formulated according to conventional pharmaceutical practice.

[0281] As used herein, the term "administration" refers to the administration of a composition (e.g., a compound or a preparation comprising a compound described herein) to a subject or system. Administration to an animal subject (e.g., a human) can be by any suitable route. For example, in some embodiments, administration can be bronchial (including bronchial infusion), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, intratracheal (including intratracheal infusion), transdermal, intravaginal, or intravitreal.

[0282] The formulation can be prepared in a manner suitable for systemic administration or local or topical administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, or subcutaneous injection), or can be prepared for transdermal, transmucosal, or oral administration. The formulation generally includes a diluent, and may also include an adjuvant, a buffer, a preservative, etc. The compound or its pharmaceutically acceptable salt can also be administered in a liposome composition or as a microemulsion.

[0283] For injection, the preparations can be prepared in conventional forms, either as liquid solutions or suspensions, or as solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol, etc. Such compositions can also contain amounts of nontoxic auxiliary substances (e.g., wetting agents or emulsifying agents), pH buffering agents, etc., such as sodium acetate, sorbitan monolaurate, etc.

[0284] Various sustained release systems for drugs have also been devised, see, for example, U.S. Patent No. 5,624,677. Systemic administration can also include relatively non-invasive methods such as the use of suppositories, transdermal patches, transmucosal delivery, and intranasal administration.Oral administration is also suitable for the compounds of the present invention or their pharmaceutically acceptable salts.Suitable forms include syrups, capsules, and tablets, as understood in the art.

[0285] Each compound described herein or its pharmaceutically acceptable salt can be formulated in various ways known in the art.For example, the first agent and the second agent of combination therapy can be formulated together or separately.Other modalities of combination therapy are described herein.

[0286] Individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, a kit containing two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include any components that aid in administering a unit dose to a subject, such as a vial for reconstituting a powder form, a syringe for injection, a customized IV delivery system, an inhaler, etc. In addition, a unit dose kit can contain instructions for preparing or administering the composition. The kit can be manufactured as a single-use unit dose for a subject, multiple uses for a specific subject (at a fixed concentration or where the potency of the individual compounds, or pharmaceutically acceptable salts thereof, changes as treatment progresses), or the kit can contain multiple doses (bulk packaging) suitable for administration to multiple subjects. The components of the kit can be assembled into a carton, blister pack, bottle, tube, etc.

[0287] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugars, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, optionally substituted hydroxylpropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and smoothing agents, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0288] The two or more compounds can be mixed in a tablet, capsule, or other vehicle, or can be fractionated. In one example, a first compound is contained inside the tablet and a second compound is present on the outside, with a substantial portion of the second compound being released before the release of the first compound.

[0289] Formulations for oral use can be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient can be mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil. Powders, granules, and pellets can be prepared using the ingredients described above under tablets and capsules in a conventional manner, for example, using a mixer, a fluidized bed device, or a spray-drying device.

[0290] Dissolution or diffusion controlled release can be achieved by suitable coating of the compound on tablets, capsules, pellets, or granules, or by incorporating the compound or its pharmaceutically acceptable salt into a suitable matrix. The sustained release coating can include one or more of the above-mentioned coating materials, or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethyl cellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-(optionally substituted) hydroxyl methacrylate, methacrylate hydrogel, 1,3-butylene glycol, ethylene glycol methacrylate, or polyethylene glycol. In sustained-release matrix formulations, the matrix material can also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, Carbopol 934, silicone, glyceryl tristearate, acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, or halogenated fluorocarbons.

[0291] Liquid forms into which the compounds of the present invention, or pharmaceutically acceptable salts thereof, and compositions may be incorporated for oral administration 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.

[0292] Generally, when administered to humans, the oral dose of the compound of the present invention or its pharmaceutically acceptable salt depends on the properties of the compound and can be readily determined by those skilled in the art. The dose may be, for example, about 0.001 mg to about 2000 mg / day, about 1 mg to about 1000 mg / day, about 5 mg to about 500 mg / day, about 100 mg to about 1500 mg / day, about 500 mg to about 1500 mg / day, about 500 mg to about 2000 mg / day, or any range variable thereof.

[0293] In some embodiments, the pharmaceutical composition can further comprise an additional compound with antiproliferative activity. Depending on the administration method, the compound or its pharmaceutically acceptable salt is formulated into a suitable composition that allows easy delivery. Each compound of the combination therapy, or its pharmaceutically acceptable salt, can be formulated in various ways known in the art. For example, the first agent and the second agent of the combination therapy can be formulated together or separately. Preferably, the first agent and the second agent are formulated together for simultaneous or nearly simultaneous administration of the agents.

[0294] It will be understood that the compounds and pharmaceutical compositions of the present invention can be formulated and utilized in combination therapy, i.e., the compounds and pharmaceutical compositions can be formulated or administered simultaneously with, prior to, or after one or more other desired therapeutic agents or medical procedures. The particular combination of therapies (therapeutics or procedures) used in a combination regimen will take into account the compatibility of the desired therapeutic agents or procedures and the desired therapeutic effect to be achieved. Furthermore, it will be understood that the therapies used may achieve the desired effect on the same disease or may achieve different effects (e.g., control of any adverse effects).

[0295] As described herein, administration of each agent in the combination therapy can independently be from one to four times daily for one day to one year, and even for the life of the subject. Chronic long-term administration may be indicated.

[0296] How to use In some embodiments, the present invention discloses methods for treating a disease or disorder characterized by aberrant Ras activity due to Ras mutants. In some embodiments, the disease or disorder is cancer.

[0297] Therefore, also provided is a method for treating cancer in a subject in need of cancer treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising such a compound or salt.In some embodiments, the cancer is colorectal cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, appendiceal cancer, melanoma, acute myeloid leukemia, small intestine cancer, ampullary cancer, germ cell cancer, cervical cancer, cancer of unknown primary origin, endometrial cancer, esophageal cancer, GI neuroendocrine cancer, ovarian cancer, sex cord-stromal tumor cancer, hepatobiliary cancer, or bladder cancer.In some embodiments, the cancer is appendiceal cancer, endometrial cancer, or melanoma.Also provided is a method for treating Ras protein-associated disease in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising such a compound or salt.

[0298] In some embodiments, the compounds of the present invention, or pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising such compounds or salts, and the methods provided herein can be used to treat a wide variety of cancers, including, for example, tumors such as lung cancer, prostate cancer, breast cancer, brain cancer, skin cancer, cervical cancer, and testicular cancer. More specifically, cancers treatable by the compounds or salts thereof, pharmaceutical compositions comprising such compounds or salts, and the methods of the present invention include, but are not limited to, tumor types such as astrocytic, breast, cervical, colorectal, uterine, esophageal, gastric, head and neck, hepatocellular, laryngeal, lung, pharyngeal, ovarian, prostate, and thyroid cancer and sarcoma. Other cancers include, for example: Heart, for example, nonepithelial malignant tumors (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; Lung, e.g., bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchial) carcinoma, bronchial adenoma, non-epithelial malignant tumor, lymphoma, chondroitin hamartoma, mesothelioma; Gastrointestinal, e.g., esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, islet cell tumor, glucagonoma, gastrinoma, carcinoid tumor, VIP tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary organs, e.g., kidney (adenocarcinoma, Wilms' tumor, (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, non-epithelial carcinoma), testicle (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriomas, non-epithelial carcinoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoid tumor, lipoma); Liver, e.g., hepatocarcinoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Biliary tract, e.g., gallbladder cancer, ampullary cancer, cholangiocarcinoma; Bone, 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, chondromyxofibroma, osteoid osteoma, and giant cell tumor; Nervous system, e.g., skull (osteoma, hemangioma, granuloma, xanthomas, osteitis deformans), meninges (meningiomas, meningiosarcomas, gliomatosis), brain (astrocytomas, medulloblastomas, gliomas, ependymomas, pinealomas, glioblastoma multiforme, oligodendrogliomas, schwannomas, retinoblastomas, congenital tumors), spinal cord neurofibromas, neurofibromatosis type I, meningiomas, gliomas, non-epithelial malignant tumors); Gynecological, e.g., uterus (endometrial carcinoma, uterine carcinoma, endometrial carcinoma of the uterine corpus), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovary (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-theca cell tumor, Sertoli-Leydig cell tumor, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma)), fallopian tube (epithelial carcinoma); Hematological, e.g., blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia); myeloproliferative disorders (e.g., myelofibrosis and myeloproliferative neoplasia); multiple myeloma; myelodysplastic syndrome, Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma); Skin, for example, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; Adrenal gland, e.g., neuroblastoma.

[0299] In some embodiments, the Ras protein is wild-type (Ras WT ) Thus, in some embodiments, the compounds of the present invention inhibit Ras WT (e.g., K-Ras WT , H-Ras WT , or N-Ras WT In some embodiments, the Ras protein is used in methods of treating a patient having a cancer comprising Ras amplification (e.g., K-Ras amp ) Thus, in some embodiments, the compounds of the present invention inhibit Ras amp (K-Ras amp , H-Ras amp , or N-Ras amp In some embodiments, the cancer comprises a Ras mutation, such as a Ras mutation described herein. In some embodiments, the mutation is selected from the following: (a) the following K-Ras mutants: G12D, G12V, G12C, G13D, G12R, G12A, Q61H, G12S, A146T, G13C, Q61L, Q61R, K117N, A146V, G12F, Q61K, L19F, Q22K, V14I, A59T, A146P, G13R, G12L, or G13V, and combinations thereof; (b) the following H-Ras mutants: Q61R, G13R, Q61K, G12S, Q61L, G12D, G13V, G13D, G12C, K117N, A59T, G12V, G13C, Q61H, G13S, A18V, D119N, G13N, A146T, A66T, G12A, A146V, G12N, or G12R, and combinations thereof; and (c) the following N-Ras mutants: Q61R, Q61K, G12D, Q61L, Q61H, G13R, G13D, G12S, G12C, G12V, G12A, G13V, G12R, P185S, G13C, A146T, G60E, Q61P, A59D, E132K, E49K, T50I, A146V, or A59T, and combinations thereof; or any combination of the foregoing. In some embodiments, the cancer comprises a K-Ras mutation selected from the group consisting of G12C, G12D, G13C, G12V, G13D, G12R, G12S, Q61H, Q61K, and Q61L. In some embodiments, the cancer comprises an N-Ras mutation selected from the group consisting of G12C, Q61H, Q61K, Q61L, Q61P, and Q61R. In some embodiments, the cancer comprises an H-Ras mutation selected from the group consisting of Q61H and Q61L. In some embodiments, the cancer comprises a Ras mutation selected from the group consisting of G12C, G13C, G12A, G12D, G13D, G12S, G13S, G12V, and G13V. In some embodiments, the cancer contains at least two Ras mutations selected from the group consisting of G12C, G13C, G12A, G12D, G13D, G12S, G13S, G12V, and G13V. In some embodiments, the compounds of the present invention inhibit two or more Ras mutations. For example, the compounds may inhibit both K-Ras G12C and K-Ras G13C. The compounds may inhibit both N-Ras G12C and K-Ras G12C. The compounds may inhibit both N-Ras G12C and K-Ras G12C. In some embodiments, the compounds may inhibit both K-Ras G12C and K-Ras G12D. In some embodiments, the compounds may inhibit both K-Ras G12V and K-Ras G12C. In some embodiments, the compounds may inhibit both K-Ras G12V and K-Ras G12S. In some embodiments, the compounds of the present invention inhibit Ras in addition to one or more additional Ras mutations. WT inhibits (e.g., K, H, or N-Ras WT and K-Ras G12D, G12V, G12C, G13D, G12R, G12A, Q61H, G12S, A146T, G13C, Q61L, Q61R, K117N, A146V, G12F, Q61K, L19F, Q22K, V14I, A59T, A146P, G13R, G12L, or G13V; K, H, or N-Ras WTand H-Ras Q61R, G13R, Q61K, G12S, Q61L, G12D, G13V, G13D, G12C, K117N, A59T, G12V, G13C, Q61H, G13S, A18V, D119N, G13N, A146T, A66T, G12A, A146V, G12N, or G12R; or K, H, or N-Ras WT and N-Ras Q61R, Q61K, G12D, Q61L, Q61H, G13R, G13D, G12S, G12C, G12V, G12A, G13V, G12R, P185S, G13C, A146T, G60E, Q61P, A59D, E132K, E49K, T50I, A146V, or A59T). In some embodiments, the compounds of the invention comprise, in addition to one or more additional Ras mutations, a Ras amp inhibits (e.g., K, H, or N-Ras amp and K-Ras G12D, G12V, G12C, G13D, G12R, G12A, Q61H, G12S, A146T, G13C, Q61L, Q61R, K117N, A146V, G12F, Q61K, L19F, Q22K, V14I, A59T, A146P, G13R, G12L, or G13V; K, H, or N-Ras amp and H-Ras Q61R, G13R, Q61K, G12S, Q61L, G12D, G13V, G13D, G12C, K117N, A59T, G12V, G13C, Q61H, G13S, A18V, D119N, G13N, A146T, A66T, G12A, A146V, G12N, or G12R; or K, H, or N-Ras amp and N-Ras Q61R, Q61K, G12D, Q61L, Q61H, G13R, G13D, G12S, G12C, G12V, G12A, G13V, G12R, P185S, G13C, A146T, G60E, Q61P, A59D, E132K, E49K, T50I, A146V, or A59T).

[0300] The method for detecting Ras mutation is known in the art.Such means include but are not limited to direct sequencing and the use of highly sensitive diagnostic assays (using CE-IVD mark), including TheraScreen PCR; AmoyDx; PNAClamp; RealQuality; EntroGen; LightMix; StripAssay; Hybcell plexA; Devyser; Surveyor; Cobas; and TheraScreen Pyro, as described in, for example, Domagala, et al., Pol J Pathol 3:145-164 (2012), which are incorporated herein by reference in their entirety.See also, for example, WO2020 / 106640.

[0301] In some embodiments, the cancer is non-small cell lung cancer, and the Ras mutation comprises a K-Ras mutation such as K-Ras G12C, K-Ras G12V, or K-Ras G12D. In some embodiments, the cancer is colorectal cancer, and the Ras mutation comprises a K-Ras mutation such as K-Ras G12C, K-Ras G12V, or K-Ras G12D. In some embodiments, the cancer is pancreatic cancer, and the Ras mutation comprises a K-Ras mutation such as K-Ras G12D or K-Ras G12V. In some embodiments, the cancer is pancreatic cancer, and the Ras mutation comprises an N-Ras mutation such as N-Ras G12D. In some embodiments, the cancer is melanoma, and the Ras mutation comprises an N-Ras mutation such as N-RasQ61R or N-RasQ61K. In some embodiments, the cancer is non-small cell lung cancer, and the Ras protein is K-Ras amp The compound also inhibits Ras WT (e.g., K-, H-, or N-Ras WT ), or Ras amp (e.g., K-, H-, or N-Ras amp ) can be inhibited.

[0302] In some embodiments, the cancer is characterized by a Ras mutation and an STK11 LOFIn some embodiments, the cancer is non-small cell lung cancer and comprises a K-Ras G12C mutation. In some embodiments, the cancer is non-small cell lung cancer and comprises a K-Ras G12C mutation and an STK11 LOF In some embodiments, the cancer is non-small cell lung cancer and includes a K-Ras G12C mutation and an STK11 mutation. LOF In some embodiments, the cancer comprises a K-Ras G13C Ras mutation and an STK11 LOF , KEAP1, EPHA5, or NF1 mutations. In some embodiments, the cancer is non-small cell lung cancer and comprises a K-Ras G12D mutation. In some embodiments, the cancer is non-small cell lung cancer and comprises a K-Ras G12V mutation. In some embodiments, the cancer is colorectal cancer and comprises a K-Ras G12C mutation. In some embodiments, the cancer is pancreatic cancer and comprises a K-Ras G12D mutation. In some embodiments, the cancer is pancreatic cancer and comprises a K-Ras G12V mutation. In some embodiments, the cancer is endometrial cancer and comprises a K-Ras G12C mutation. In some embodiments, the cancer is lung cancer, colorectal cancer, or pancreatic cancer and comprises a K-Ras G12D mutation. In some embodiments, the cancer is lung cancer or pancreatic cancer and comprises a K-Ras G12D mutation. In some embodiments, the cancer is lung cancer and comprises a K-Ras G12D mutation. In some embodiments, the cancer is colorectal cancer and comprises a K-Ras G12D mutation. In some embodiments, the cancer is gastric cancer and comprises a K-Ras G12C mutation. In any of the foregoing, the compound also WT (e.g., K-, H-, or N-Ras WT ), or Ras amp (e.g., K-, H-, or N-Ras amp ) can be inhibited.

[0303] Also provided is a method for inhibiting Ras protein in cells, comprising contacting the cells with an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. Also provided is a method for inhibiting RAF-Ras binding, comprising contacting the cells with an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. The cells can be cancer cells. The cancer cells can be from any type of cancer described herein. The cells can be in vivo or in vitro.

[0304] Combination therapy The methods of the present invention may include compounds of the present invention used alone or in combination with one or more additional therapies (e.g., non-drug therapies or therapeutic agents). The dosage of one or more additional therapies (e.g., non-drug therapies or therapeutic agents) may be reduced from the standard dosage when administered alone. For example, dosage may be empirically determined from drug combinations and permutations or estimated by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6 (2005)).

[0305] The compound of the present invention can be administered before, after, or simultaneously with such one or more additional therapies. When combined, the dosage of the compound of the present invention and the dosage of one or more additional therapies (e.g., non-drug treatments or therapeutic agents) provide a therapeutic effect (e.g., a synergistic or additive therapeutic effect). The compound of the present invention and the additional therapy, e.g., an anticancer agent, can be administered together in a single pharmaceutical composition or separately, and when administered separately, can be administered simultaneously or sequentially. Such sequential administration can be closely spaced or spaced apart.

[0306] In some embodiments, the additional therapy is the administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence or severity of side effects of treatment). For example, in some embodiments, the compounds of the present invention can also be used in combination with a therapeutic agent to treat nausea. Examples of agents that can be used to treat nausea include dronabinol, granisetron, metoclopramide, ondansetron, and prochlorperazine, or pharmaceutically acceptable salts thereof.

[0307] In some embodiments, the one or more additional therapies include a non-drug treatment (e.g., surgery or radiation therapy). In some embodiments, the one or more additional therapies include a therapeutic agent (e.g., a compound or biologic that is an anti-angiogenic agent, a signal transduction inhibitor, an anti-proliferative agent, a glycolysis inhibitor, or an autophagy inhibitor). In some embodiments, the one or more additional therapies include a non-drug treatment (e.g., surgery or radiation therapy) and a therapeutic agent (e.g., a compound or biologic that is an anti-angiogenic agent, a signal transduction inhibitor, an anti-proliferative agent, a glycolysis inhibitor, or an autophagy inhibitor). In other embodiments, the one or more additional therapies include two therapeutic agents. In yet other embodiments, the one or more additional therapies include three therapeutic agents. In some embodiments, the one or more additional therapies include four or more therapeutic agents.

[0308] In this Combination Therapy section, all references to the agents mentioned are incorporated by reference, whether or not explicitly stated. Non-drug therapy Examples of non-drug treatments include, but are not limited to, radiation therapy, cryotherapy, hyperthermia, surgery (eg, surgical removal of tumor tissue), and T cell adoptive transfer (ACT) therapy.

[0309] In some embodiments, the compounds of the present invention may be used as an adjuvant therapy after surgery. In some embodiments, the compounds of the present invention may be used as a neoadjuvant therapy before surgery.

[0310] Radiation therapy can be used to inhibit abnormal cell growth or treat hyperproliferative disorders, such as cancer, in a subject (e.g., a mammal (e.g., a human)). Techniques for administering radiation therapy are known in the art. Radiation therapy can be administered by one of several methods or a combination of methods, including, but not limited to, external beam therapy, internal radiation therapy, implant radiation, stereotactic radiosurgery, systemic radiation therapy, radiotherapy, and permanent or temporary interstitial brachytherapy. As used herein, the term "brachytherapy" refers to radiation therapy delivered by spatially confined radioactive materials inserted within the body at or near the site of a tumor or other proliferative tissue disease. The term is intended to include, but is not limited to, exposure to radioactive isotopes (e.g., At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and radioactive isotopes of Lu). Suitable radioactive sources for use as cell conditioning agents of the present invention include both solid and liquid sources. By way of non-limiting example, the radioactive source may be a radionuclide such as I-125, I-131, Yb-169, Ir-192, I-125 as a solid source, or other radionuclide that emits photons, beta particles, gamma rays, or other therapeutic rays. The radioactive material may also be a fluid made from any solution of radionuclide(s), such as a solution of I-125 or I-131, or a radioactive fluid may be produced using a slurry of a suitable fluid containing small particles of a solid radionuclide, such as Au-198 or Y-90. Furthermore, the radionuclide(s) may be embodied in a gel or radioactive microspheres.

[0311] In some embodiments, the compounds of the present invention can make abnormal cells more sensitive to radiation therapy, which is intended to kill or inhibit the proliferation of such cells.Therefore, the present invention also relates to a method for sensitizing abnormal cells in a mammal to radiation therapy, which method comprises administering to the mammal an amount of a compound of the present invention effective to sensitize abnormal cells to radiation therapy.The amount of the compound in this method can be determined according to the means for determining the effective amount of such compounds described herein.In some embodiments, the compounds of the present invention can be used as adjuvant therapy after radiation therapy or as neoadjuvant therapy before radiation therapy.

[0312] In some embodiments, the non-drug treatment is T cell adoptive transfer (ACT) therapy. In some embodiments, the T cells are activated T cells. The T cells can be modified to express a chimeric antigen receptor (CAR). CAR-modified T (CAR-T) cells can be generated by any method known in the art. For example, CAR-T cells can be generated by introducing a suitable expression vector encoding a CAR into T cells. Prior to expansion and genetic modification of the T cells, a source of T cells is obtained from a subject. T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art can be used. In some embodiments, the T cells are autologous T cells. Whether before or after genetic modification of the T cells to express a desired protein (e.g., a CAR), the T cells generally are engineered to express a desired protein (e.g., a CAR), as described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, Activation and proliferation can be achieved using methods described in US Pat. Nos. 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 7,572,631, 5,883,223, 6,905,874, 6,797,514, and 6,867,041.

[0313] therapeutic agent The therapeutic agent can be a compound used in the treatment of cancer or a condition related thereto. For example, the therapeutic agent can be a steroid. Thus, in some embodiments, the one or more additional therapies include a steroid. Suitable steroids include 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, flucortine butyl, flucortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, and flurandrenolide. , fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, and salts or derivatives thereof.

[0314] Further examples of therapeutic agents that can be used in combination therapy with the compounds of the invention include compounds described in the following patents: U.S. Patent Nos. 6,258,812, 6,630,500, 6,515,004, 6,713,485, 5,521,184, 5,770,599, 5,747,498, 5,990,141, 6,235,764, and 8,623,885, and International Patent Application No. WO00 / 064994. Patent Applications WO01 / 37820, WO01 / 32651, WO02 / 68406, WO02 / 66470, WO02 / 55501, WO04 / 05279, WO04 / 07481, WO04 / 07458, WO04 / 09784, WO02 / 59110, WO99 / 45009, WO00 / 59509, WO99 / 61422, WO00 / 12089, and WO00 / 02871.

[0315] The therapeutic agent may be a biologic (e.g., a cytokine (e.g., an interferon or an interleukin such as IL-2)) used to treat cancer or a related condition. In some embodiments, the biologic is an immunoglobulin-based biologic, such as a monoclonal antibody (e.g., a humanized antibody, a fully human antibody, an Fc fusion protein, or a functional fragment thereof), which targets and stimulates an anti-cancer response or antagonizes an antigen important to cancer. Also included are antibody-drug conjugates.

[0316] The therapeutic agent can be a T cell checkpoint inhibitor. In one embodiment, the checkpoint inhibitor is an inhibitory antibody (e.g., a monospecific antibody such as a monoclonal antibody). The antibody can be, for example, a humanized or fully human antibody. In some embodiments, the checkpoint inhibitor is a fusion protein, e.g., an Fc receptor fusion protein. In some embodiments, the checkpoint inhibitor is an agent such as an antibody that interacts with a checkpoint protein. In some embodiments, the checkpoint inhibitor is an agent such as an antibody that interacts with a ligand of a checkpoint protein. In some embodiments, the checkpoint inhibitor is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of CTLA-4 (e.g., an anti-CTLA-4 antibody or fusion protein). In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or small molecule inhibitor) of PD-1. In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or small molecule inhibitor) of PD-L1. In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or Fc fusion or small molecule inhibitor) of PD-L2 (e.g., a PD-L2 / Ig fusion protein). In some embodiments, the checkpoint inhibitor is an inhibitor or antagonist (e.g., an inhibitory antibody or small molecule inhibitor) of B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, a B-7 family ligand, or a combination thereof.In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, PDR001 (NVS), REGN2810 (Sanofi / Regeneron), a PD-L1 antibody such as avelumab, durvalumab, atezolizumab, pidilizumab, JNJ-63723283 (JNJ), BGB-A317 (BeiGene & Celgene), or Preusser, M. et al. al. (2015) Nat. Rev. Neurol., including, but not limited to, ipilimumab, tremelimumab, nivolumab, pembrolizumab, AMP224, AMP514 / MEDI0680, BMS936559, MED14736, MPDL3280A, MSB0010718C, BMS986016, IMP321, lirilumab, IPH2101, 1-7F9, and KW-6002.

[0317] The therapeutic agent can be an anti-TIGIT antibody, such as MBSA43, BMS-986207, MK-7684, COM902, AB154, MTIG7192A, or OMP-313M32 (etigilimab).

[0318] The therapeutic agent can be an agent that treats cancer or a related condition (e.g., a cytotoxic agent, a non-peptide small molecule, or other compound useful in treating cancer or a related condition, collectively an "anti-cancer agent"). The anti-cancer agent can be, for example, a chemotherapeutic agent or a targeted therapy agent.

[0319] Anticancer drugs include mitotic inhibitors, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopirotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione-substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, corticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. Additional anticancer drugs include leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel, and doxetaxel. In some embodiments, the one or more additional therapies comprise two or more anticancer drugs. Two or more anti-cancer drugs can be administered in combination or in a cocktail administered separately. Suitable dosing regimens for combined anti-cancer drugs are known in the art and are described, for example, in Saltz et al., Proc. Am. Soc. Clin. Oncol. 18:233a (1999) and Douillard et al., Lancet 355(9209):1041-1047 (2000).

[0320] Other non-limiting examples of anti-cancer drugs include Gleevec® (imatinib mesylate); Kyprolis® (carfilzomib); Velcade® (bortezomib); Casodex (bicalutamide); Iressa® (gefitinib); alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzamidine, benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines, such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (e.g., the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (e.g., its adolezin, carzelesin, and synthetic analogs of beizeresin; cryptophycins (specifically, cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (e.g., synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin A; spongistatins; nitrogen mustards, e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine, amine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas, e.g., camrustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., enediyne antibiotics (e.g., calicheamicins, e.g., calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Agnew, Chem. Intl. Ed Engl. 33:183-186 (1994)); dynemicins, such as dynemicin A; bisphosphonates, such as clodronate; esperamicin;Neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino - doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; Purine analogues such as ludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid replacement fluids such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid Acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilones such as epothilone B; etoglucide; gallium nitrate; hydroxyurea; lentinan; maytansinoids such as lonidynin, maytansine, and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenameth; pirarubicin; losoxantrone; podophyllic acid;2-Ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes such as T-2 toxin, veraculin A, roridin A, and anguidine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); Cyclophosphamide; thiotepa; taxoids such as Taxol® (paclitaxel), Abraxane® (a chromophore-free, albumin-engineered nanoparticle formulation of paclitaxel), and Taxotere® (doxetaxel); chlorambucil; tamoxifen (Nolvadex™); raloxifene; aromatase-inhibited 4(5)-imidazoles; 4-hydroxytamoxifen; trihydroxyphene; ketoxifene; LY 117018; onapristone; toremifene (Fareston®); flutamide, nilutamide, bicalutamide, leuprolide, goserelin; chlorambucil; Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; esperamicin; capecitabine (e.g., Xeloda®); and pharmaceutically acceptable salts of any of the above.

[0321] Additional non-limiting examples of anti-cancer agents include trastuzumab (Herceptin®), bevacizumab (Avastin®), cetuximab (Erbitux®), rituximab (Rituxan®), Taxol®, Arimidex®, ABVD, avicin, abagovomab, acridine carboxamide, adecatumumab, 17-N-allylamino-17-demethoxygeldanamycin, alpharazine, alvocidib, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, amonafide, anthracenedione, anti-CD22 immunotoxins, antineoplastic agents (e.g., cell cycle non-specific antineoplastic agents, and other antineoplastic agents described herein), antineoplastic herbs, apaziquone, atiprimod, azathioprine, belotecan, bendamustine, BIBW 2992, biricodar, brostallicin, bryostatin, buthionine sulfoximine, CBV (chemotherapy), calyculin, dichloroacetic acid, discodamolide, elsamitol, enocitabine, eribulin, exatecan, exisulind, ferruginol, forodesin, fosfestrol, ICE chemotherapy regimen, IT-101, imexon, imiquimod, indolocarbazole, irofulven, laniquidar, larotaxel, lenalidomide, lucanton, lurtotecan, mafosfamide, mitozolomide, These include nafoxidine, nedaplatin, olaparib, ortataxel, PAC-1, pawpaw, pixantrone, proteasome inhibitors, rebeccamycin, resiquimod, rubitecan, SN-38, salinosporamide A, sapacitabine, Stanford V, swainsonine, talaporfin, tariquidar, tegafur-uracil, temodar, tesetaxel, triplatin tetranitrate, tris(2-chloroethyl)amine, troxacitabine, uramustine, vadimezan, vinflunine, ZD6126, and zosuquidar.

[0322] Further non-limiting examples of anti-cancer drugs include vinca alkaloids (e.g., vinblastine, vincristine, and vinorelbine), epidipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, and idarubicin), anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), mitomycin, enzymes (e.g., that metabolize L-asparagine systemically and synthesize their own asparagine), and steroids (e.g., steroids that metabolize L-asparagine systemically and synthesize their own asparagine). L-asparaginase, which deprives cells of the ability to form tumors; antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide and analogs, melphalan, and chlorambucil); ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa); CDK inhibitors (e.g., CDK4 / 6 inhibitors such as abemaciclib, ribociclib, and palbociclib); seliciclib, UCN-01, and P1446A-05 , PD-0332991, dinaciclib, P27-00, AT-7519, RGB286638, and SCH727965), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine (BCNU) and analogs, and streptozocin), trazeneth-dacarbazine (DTIC), folic acid analogs, pyrimidine analogs (e.g., fluorouracil, floxuridine, and cytarabine), purine analogs, and other antiproliferative / antimitotic metabolites and related inhibitors (e.g., cyclosporine ... For example, mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine), aromatase inhibitors (e.g., anastrozole, exemestane, and letrozole), and platinum coordination complexes (e.g., cisplatin and carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide, histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan, suberoylanilide hydroamic acid, vorinostat, LBH 589, romidepsin, ACY-1215, and panobinostat), mTOR inhibitors (e.g., bistosertib, temsirolimus, everolimus, ridaforolimus, and sirolimus), KSP (Eg5) inhibitors (e.g., Array520), DNA binders (e.g., Zalypsis®), PI3K inhibitors such as PI3K delta inhibitors (e.g., GS-1101 and TGR-1202), PI3K delta and gamma inhibitors (e.g., CAL-130), copanlisib, alpelisib, and idelalisib; multikinase inhibitors (e.g., TG02 and sorafenib), hormones (e.g., estrogen), and hormonal agonists such as leutinizing hormone-releasing hormone (LHRH) agonists (e.g., goserelin, leuprolide, and triptorelin), BAFF neutralizing antibodies (e.g., LY2127399), IKK inhibitors, p38MAPK inhibitors, anti-IL-6 (e.g., CNT0328), telomerase inhibitors (e.g., GRN 163L), Aurora kinase inhibitors (e.g., MLN8237), cell surface monoclonal antibodies (e.g., anti-CD38 (HUMAX-CD38)), anti-CS1 (e.g., elotuzumab), HSP90 inhibitors (e.g., 17AAG and KOS953), P13K / Akt inhibitors (e.g., perifosine), Akt inhibitors (e.g., GSK-2141795), PKC inhibitors (e.g., enzastaurin), FTIs (e.g., Za These include natural products such as rnestra™, anti-CD138 (e.g., BT062), Torcl / 2-specific kinase inhibitors (e.g., INK128), ER / UPR targeting agents (e.g., MKC-3946), cFMS inhibitors (e.g., ARRY-382), JAK1 / 2 inhibitors (e.g., CYT387), PARP inhibitors (e.g., olaparib and veliparib (ABT-888)), and BCL-2 antagonists.

[0323] In some embodiments, the anti-cancer agent is selected from mechlorethamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, Navelbine®, sorafenib, or any analog or derivative variant of the foregoing.

[0324] In some embodiments, the anti-cancer agent is a HER2 inhibitor. Non-limiting examples of HER2 inhibitors include monoclonal antibodies such as trastuzumab (Herceptin®) and pertuzumab (Perjeta®); small molecule tyrosine kinase inhibitors such as gefitinib (Iressa®), erlotinib (Tarceva®), piritinib, CP-654577, CP-724714, canertinib (CI 1033), HKI-272, lapatinib (GW-572016; Tykerb®), PKI-166, AEE788, BMS-599626, HKI-357, BIBW 2992, ARRY-334543, JNJ-26483327, and JNJ-26483327.

[0325] In some embodiments, the anticancer agent is an ALK inhibitor. Non-limiting examples of ALK inhibitors include ceritinib, TAE-684 (NVP-TAE694), PF02341066 (crizotinib or 1066), alectinib, brigatinib, entrectinib, ensartinib (X-396), lorlatinib, ASP3026, CEP-37440, 4SC-203, TL-398, PLB1003, TSR-011, CT-707, TPX-0005, and AP26113. Additional examples of ALK kinase inhibitors are described in Examples 3-39 of WO05 / 016894.

[0326] In some embodiments, the anti-cancer agent is an inhibitor of a downstream member of a receptor tyrosine kinase (RTK) / growth factor receptor (e.g., an SHP2 inhibitor (e.g., SHP099, TNO155, RMC-4550, RMC-4630, JAB-3068, JAB-3312, RLY-1971, ERAS-601, SH3809, PF-07284892, or BBP-398), an SOS1 inhibitor (e.g., BI-1701963, BI-3406, SDR5, MRTX0902, RMC-5845, or BAY-293), a Raf inhibitor, a MEK inhibitor, an ERK inhibitor, a PI3K inhibitor, a PTEN inhibitor, an AKT inhibitor, or an mTOR inhibitor (e.g., an mTORC1 inhibitor or an mTORC2 inhibitor). In some embodiments, the anti-cancer agent is JAB-3312.

[0327] In some embodiments, the anti-cancer agent is an additional Ras inhibitor or a Ras vaccine, or another therapy designed to directly or indirectly reduce the oncogenic activity of Ras. In some embodiments, the anti-cancer agent is an additional Ras inhibitor. In some embodiments, the Ras inhibitor targets Ras in its active or GTP-bound state (Ras(ON)). In some embodiments, the Ras(ON) inhibitor is RMC-6291, RMC-6236, RMC-9805, or RMC-8839. In some embodiments, the Ras inhibitor is a RAS(ON) inhibitor disclosed in WO2021091956, WO2021091967, WO2021091982, WO2022060836, or WO2020132597, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof, which are incorporated by reference herein in their entireties. In some embodiments, the Ras inhibitor targets Ras in its inactive or GDP-bound state. In some embodiments, the Ras inhibitor is an inhibitor of K-Ras G12C, such as, for example, AMG510, MRTX1257, MRTX849, JNJ-74699157 (ARS-3248), LY3499446, or ARS-1620, ARS-853, BPI-421286, LY3537982, JDQ443, ERAS-3490, JAB-21000, BPI-421286, D-1553, JAB-21822, GH-35, ICP-915, IBI351, RMC-6291, or GDC-6036. In some embodiments, the Ras inhibitor is an inhibitor of K-Ras G12D, such as ERAS-4, MRTX1133, RMC-9805, or JAB-22000. In some embodiments, the Ras inhibitor is an inhibitor of K-Ras G12V, such as JAB-23000. In some embodiments, the Ras inhibitor is RMC-6236. Other examples of RAS inhibitors that can be combined with the Ras inhibitors of the present invention are provided below, which are incorporated by reference in their entirety: WO 2022087624, WO 2022087375, WO 2022087371, WO2022083616、WO 2022083569、WO 2022081655、WO 2022078414、WO 2022076917、WO 2022072783、WO 2022066805、WO 2022066646、WO 2022063297、WO 2022061251、WO 2022056307、WO 2022052895、WO 2022047093、WO 2022042630、WO 2022040469、WO 2022037560、WO 2022031678、WO 2022028492、WO 2022028346、WO 2022026726、WO 2022026723、WO 2022015375、WO 2022002102、WO 2022002018、WO 2021259331、WO 2021257828、WO 2021252339、WO 2021248095、WO 2021248090、WO 2021248083、WO 2021248082、WO 2021248079、WO 2021248055、WO 2021245051、WO 2021244603、WO 2021239058、WO 2021231526、WO 2021228161、WO 2021219090、WO 2021219090、WO 2021219072、WO 2021218939、WO 2021217019、WO 2021216770、WO 2021215545、WO 2021215544、WO 2021211864、WO 2021190467、WO 2021185233、WO 2021180181、WO 2021175199、2021173923、WO 2021169990、WO 2021169963、WO 2021168193、WO 2021158071、WO 2021155716、WO 2021152149、WO 2021150613、WO 2021147967、WO 2021147965、WO 2021143693、WO 2021142252、WO 2021141628、WO 2021139748、WO 2021139678、WO 2021129824、WO 2021129820、WO 2021127404、WO 2021126816、WO 2021126799、WO2021124222、WO 2021121371、WO 2021121367、WO 2021121330、WO 2020050890、WO 2020047192、WO 2020035031、WO 2020028706、WO 2019241157、WO 2019232419、WO 2019217691、WO 2019217307、WO 2019215203、WO 2019213526、WO 2019213516、WO 2019155399、WO 2019150305、WO 2019110751、WO 2019099524、WO 2019051291、WO 2018218070、WO 2018217651、WO 2018218071、WO 2018218069、WO 2018206539、WO 2018143315、WO 2018140600、WO 2018140599、WO 2018140598、WO 2018140514、WO 2018140513、WO 2018140512、WO 2018119183、WO 2018112420、WO 2018068017、WO 2018064510、WO 2017201161、WO 2017172979、WO 2017100546、WO 2017087528、WO 2017058807、WO 2017058805、WO 2017058728、WO 2017058902、WO 2017058792、WO 2017058768、WO 2017058915、WO 2017015562、WO 2016168540、WO 2016164675、WO 2016049568、WO 2016049524、WO 2015054572、WO 2014152588、WO 2014143659、WO 2013155223、CN 114195804、CN 114195788、CN 114057776、CN 114057744、CN 114057743、CN 113999226、CN 113980032、CN 113980014、CN 113929676、CN 113754653、CN 113683616、CN 113563323、CN 113527299、CN 113527294、CN 113527293、CN 113493440、CN113429405, CN 113248521, CN 113087700, CN 113024544, CN 113004269, CN 112920183, CN 112778284, CN 112390818, CN 112390788, CN 112300196, CN 112300194, CN 112300173, CN 112225734, CN 112142735, CN 112110918, CN 112094269, CN 112047937, and CN 109574871, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof.

[0328] In some embodiments, therapeutic agents that can be combined with compounds of the invention are inhibitors of the MAP kinase (MAPK) pathway (or "MAPK inhibitors"). MAPK inhibitors include, but are not limited to, one or more of the MAPK inhibitors described in Cancers (Basel) 2015 Sep;7(3):1758-1784. For example, MAPK inhibitors include trametinib, binimetinib, selumetinib, cobimetinib, LErafAON (NeoPharm), ISIS 5132, vemurafenib, pimasertib, TAK733, RO4987655 (CH4987655), CI-1040, PD-0325901, CH5126766, MAP855, AZD6244, refametinib (RDEA 119 / BAY 86-9766), GDC-0973 / XL581, AZD8330 (ARRY-424704 / ARRY-704), and RO5126766 (Roche, PLoS One. 2014 Nov. 25;9(11)), and GSK1120212 (or JTP-74057, described in Clin Cancer Res. 2011 Mar 1;17(5):989-1000). The MAPK inhibitor can be PLX8394, LXH254, GDC-5573, LY3009120.

[0329] In some embodiments, the anticancer drug is a disruptor or inhibitor of the RAS-RAF-ERK or PI3K-AKT-TOR or PI3K-AKT signaling pathway. PI3K / AKT inhibitors include, but are not limited to, one or more PI3K / AKT inhibitors described in Cancers (Basel) 2015 Sep;7(3):1758-1784. For example, the PI3K / AKT inhibitor can be selected from one or more of NVP-BEZ235, BGT226, XL765 / SAR245409, SF1126, GDC-0980, PI-103, PF-04691502, PKI-587, and GSK2126458.

[0330] In some embodiments, the anti-cancer agent is a PD-1 or PD-L1 antagonist. In some embodiments, the additional therapeutic agent includes an ALK inhibitor, a HER2 inhibitor, an EGFR inhibitor, an IGF-1R inhibitor, a MEK inhibitor, a PI3K inhibitor, an AKT inhibitor, a TOR inhibitor, an MCL-1 inhibitor, a BCL-2 inhibitor, a SHP2 inhibitor, a proteasome inhibitor, and an immunotherapy. In some embodiments, the therapeutic agent can be a pan-RTK inhibitor, e.g., afatinib.

[0331] IGF-1R inhibitors include linsitinib, or a pharmaceutically acceptable salt thereof. EGFR inhibitors include, but are not limited to, small molecule antagonists, antibody inhibitors, or specific antisense nucleotides or siRNA. Useful antibody inhibitors of EGFR include cetuximab (Erbitux®), panitumumab (Vectibix®), zalutumumab, nimotuzumab, and matuzumab. Additional antibody-based EGFR inhibitors include any anti-EGFR antibody or antibody fragment that can partially or completely block EGFR activation by its natural ligand. Non-limiting examples of antibody-based EGFR inhibitors include those described in Modjtahedi et al., Br. J. Cancer 1993, 67:247-253; Teramoto et al., Cancer 1996, 77:639-645; Goldstein et al., Clin. Cancer Res. 1995, 1:1311-1318; Huang et al., 1999, Cancer Res. 15:59(8):1935-40; and Yang et al., Cancer Res. 1999, 59:1236-1243. The EGFR inhibitor may be the monoclonal antibody Mab E7.6.3 (Yang, supra, 1999), or Mab C225 (ATCC Accession No. HB-8508), or an antibody or antibody fragment having the binding specificity thereof.

[0332] Small molecule antagonists of EGFR include gefitinib (Iressa®), erlotinib (Tarceva®), and lapatinib (TykerB®). See, e.g., Yan et al., Pharmacogenetics and Pharmacogenomics In Oncology Therapeutic Antibody Development, BioTechniques 2005, 39(4):565-8, and Paez et al., EGFR Mutations In Lung Cancer Correlation With Clinical Response To Gefitinib Therapy, Science 2004, 304(5676):1497-500. In some embodiments, the EGFR inhibitor is osimertinib (Tagrisso®). Further non-limiting examples of small molecule EGFR inhibitors include any of the EGFR inhibitors described in the following patent publications, and any pharmaceutically acceptable salts of such EGFR inhibitors: EP 0520722; EP 0566226; WO 96 / 33980; U.S. Pat. No. 5,747,498; WO 96 / 30347; EP 0787772; WO 97 / 30034; WO 97 / 30044; WO 97 / 38994; WO 97 / 49688; EP 837063; WO 98 / 02434; WO 97 / 38983; WO 95 / 19774; WO 95 / 19970; WO 97 / 13771; WO 98 / 02437; WO 9 8 / 02438;WO97 / 32881;DE19629652;WO98 / 33798;WO97 / 32880;WO97 / 32880;EP68202 7;WO97 / 02266;WO97 / 27199;WO98 / 07726;WO97 / 34895;WO96 / 31510;WO98 / 14449;WO 98 / 14450; WO98 / 14451; WO95 / 09847; WO97 / 19065; WO98 / 17662; U.S. Patent Nos. 5,789,427; 5,650,415; 5,656,643; WO99 / 35146; WO99 / 35132; WO99 / 07701; and WO92 / 20642.Additional non-limiting examples of small molecule EGFR inhibitors include any of the EGFR inhibitors described in Traxler et al., Exp. Opin. Ther. Patents 1998, 8(12):1599-1625. In some embodiments, the EGFR inhibitor is an ERBB inhibitor. In humans, the ERBB family contains HER1 (EGFR, ERBB1), HER2 (NEU, ERBB2), HER3 (ERBB3), and HER (ERBB4).

[0333] MEK inhibitors include, but are not limited to, pimasertib, selumetinib, cobimetinib (Cotellic®), trametinib (Mekinist®), and binimetinib (Mektovi®). In some embodiments, the MEK inhibitor targets a MEK mutation that is a Class I MEK1 mutation selected from D67N, P124L, P124S, and L177V. In some embodiments, the MEK mutation is a Class II MEK1 mutation selected from ΔE51-Q58, ΔF53-Q58, E203K, L177M, C121S, F53L, K57E, Q56P, and K57N.

[0334] PI3K inhibitors include wortmannin, 17-hydroxywortmannin analogs described in WO 06 / 044453, 4-[2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as pictilisib or GDC-0941 and described in WO 09 / 036082 and WO 09 / 055730), 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (BEZ 235 or NVP-BEZ), 235 and described in WO 06 / 122806), (S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one (described in WO 08 / 070740), LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (available from Axon Medchem), PI 103 hydrochloride (3-[4-(4-morpholinylpyrido-[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride (available from Axon Medchem), PIK 75 (2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid, monohydrochloride) (available from Axon Medchem), PIK 90 (N-(7,8-dimethoxy-2,3-dihydro-imidazo[1,2-c]quinazolin-5-yl)-nicotinamide (available from Axon Medchem), AS-252424 (5-[1-[5-(4-fluoro-2-hydroxy-phenyl)-furan-2-yl]-meth-(Z)-ylidene]-thiazolidine-2,4-dione (available from Axon Medchem), Examples of suitable anti-inflammatory drugs include, but are not limited to, TGX-221 (7-methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido-[1,2-a]pyrinidin-4-one (available from Axon Medchem), XL-765, and XL-147.Other PI3K inhibitors include demethoxyviridine, perifosine, CAL101, PX-866, BEZ235, SF1126, INK1117, IPI-145, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TGI 00-115, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136.

[0335] AKT inhibitors include Akt-1-1 (inhibits Akt1) (Barnett et al., Biochem. J. 2005, 385(Pt. 2):399-408); Akt-1-1,2 (inhibits Akt1 and 2) (Barnett et al., Biochem. J. 2005, 385(Pt. 2):399-408); API-59CJ-Ome (e.g., Jin et al., Br. J. Cancer 2004, 91:1808-12); 1-H-imidazo[4,5-c]pyridinyl compounds (e.g., WO05 / 011700); indole-3-carbinol and its derivatives (e.g., U.S. Pat. No. 6,656,963; Sarkar and Li J Nutr. 2004, 134(12) Suppl):3493S-3498S); perifosine (e.g., interferes with Akt membrane localization; Dasmahapatra et al. Clin. Cancer Res. 2004, 10(15):5242-52); phosphatidylinositol ether lipid analogs (e.g., Gills and Dennis Expert. Opin. Investig. Drugs 2004, 13:787-97); and triciribine (TCN or API-2 or NCI identifier: NSC 154020; Yang et al., Cancer Res. 2004, 64:4394-9).

[0336] mTOR inhibitors include ATP-competitive mTORC1 / mTORC2 inhibitors, e.g., PI-103, PP242, PP30; 1; FKBP12 potentiators; 4H-1-benzopyran-4-one derivatives; and rapamycin (also known as sirolimus) and its derivatives, such as temsirolimus (Torisel®); everolimus (Afinitor®, WO 94 / 09010); ridaforolimus (also known as deforolimus or AP23573); rapalogs, such as those disclosed in WO 98 / 02441 and WO 01 / 14387, for example, AP23464 and AP23841; 40-(2-hydroxyethyl)rapamycin; 40-[3-hydroxy(hydroxymethyl)methylpropanoate]-rapamycin (also known as CC1779); 40-epi-(tetrazolite)-rapamycin (also referred to as ABT578); 32-deoxo Rapamycin; 16-pentynyloxy-32(S)-dihydrorapamycin; derivatives disclosed in WO 05 / 005434; U.S. Patent Nos. 5,258,389, 5,118,677, 5,118,678, 5,100,883, 5,151,413, 5,120,842, and 5,256,790, and WO 94 / 090101, WO 92 / 051 79, WO93 / 111130, WO94 / 02136, WO94 / 02485, WO95 / 14023, WO94 / 02136, WO95 / 16691, WO96 / 41807, WO96 / 41807, and WO2018 / 204416, as well as phosphorus-containing rapamycin derivatives (e.g., WO05 / 016252). In some embodiments, the mTOR inhibitor is a disteric inhibitor (see, e.g., WO2018 / 204416, WO2019 / 212990, and WO2019 / 212991), e.g., RMC-5552, having the following structure:

[0337] [ka]

[0338] BRAF inhibitors that can be used in combination with the compounds of the present invention include, for example, vemurafenib, dabrafenib, and encorafenib. BRAF can include class 3 BRAF mutations. In some embodiments, class 3 BRAF mutations are selected from one or more of the following amino acid substitutions in human BRAF: D287H; P367R; V459L; G466V; G466E; G466A; S467L; G469E; N581S; N581I; D594N; D594G; D594A; D594H; F595L; G596D; G596R; and A762E.

[0339] MCL-1 inhibitors include, but are not limited to, AMG-176, MIK665, and S63845. Myeloid cell leukemia-1 (MCL-1) protein is one of the major anti-apoptotic members of the B-cell lymphoma-2 (BCL-2) protein family. Overexpression of MCL-1 is closely associated with tumor progression and resistance to not only conventional chemotherapy but also targeted therapeutic agents, including BCL-2 inhibitors such as ABT-263.

[0340] In some embodiments, the additional therapeutic agent is an SHP2 inhibitor. SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene and contributes to multiple cellular functions, such as proliferation, differentiation, cell cycle maintenance, and migration. SHP2 has two N-terminal Src homology 2 domains (N-SH2 and C-SH2), a catalytic domain (PTP), and a C-terminal tail. The two SH2 domains control the intracellular localization and functional regulation of SHP2. This molecule exists in an inactive, autoinhibited conformation stabilized by a binding network involving residues from both the N-SH2 and PTP domains. For example, stimulation by cytokines or growth factors acting through receptor tyrosine kinases (RTKs) leads to exposure of the catalytic site, resulting in enzymatic activation of SHP2.

[0341] SHP2 is involved in signal transduction via the RAS mitogen-activated protein kinase (MAPK), JAK-STAT, or phosphoinositol 3-kinase-AKT pathways. Mutations in the PTPN11 gene and subsequent SHP2 mutations have been identified in several human developmental disorders, such as Noonan syndrome and Leopard syndrome, as well as human cancers such as juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, and breast, lung, and colon cancer. Some of these mutations destabilize the autoinhibitory conformation of SHP2, promoting its self-activation or enhanced growth factor-driven activation. Therefore, SHP2 is a highly attractive target for the development of novel therapies for the treatment of various diseases, including cancer. SHP2 inhibitors (e.g., RMC-4550 or SHP099) in combination with RAS pathway inhibitors (e.g., MEK inhibitors) have been shown to inhibit the growth of multiple cancer cell lines (e.g., pancreatic, lung, ovarian, and breast cancer) in vitro. Therefore, combination therapy using SHP2 inhibitors and RAS pathway inhibitors together may be a general strategy to prevent tumor resistance in a wide range of malignancies.

[0342] Non-limiting examples of such SHP2 inhibitors are known in the art and are described in publications such as Chen et al. Mol Pharmacol. 2006, 70, 562; Sarver et al., J. Med. Chem. 2017, 62, 1793; Xie et al., J. Med. Chem. 2017, 60, 113734; and Igbe et al., Oncotarget, 2017, 8, 113734; and patent applications WO 2022063190, WO 2022043685, WO 2022042331, WO 2022033430, WO 2022033430, WO 2022017444, WO 2022007869, WO 2021259077, WO 2021249449, WO 2021249057, WO 2021244659, WO 2021218755, WO 2021281752, WO 2021197542, WO 2021176072, WO 2021149817, WO 2021148010, WO 2021147879, WO 2021143823, WO 2021143701, WO 2021143680, WO 2021121397, WO 2021119525, WO 2021115286, WO 2021110796, WO 2021088945, WO 2021073439, WO 2021061706, WO 2021061515, WO 2021043077, WO 2021033153, WO 2021028362, WO 2021033153, WO 2021028362, WO 2021018287, WO 2020259679, WO 2020249079, WO 2020210384, WO 2020201991, WO 2020181283, WO 2020177653, WO 2020165734, WO 2020165733, WO 2020165732, WO 2020156243, WO 2020156242, WO 2020108590, WO 2020104635, WO 2020094104, WO 2020094018, WO 2020081848, WO 2020073949, WO 2020073945, WO 2020072656, WO 2020065453, WO2020065452, WO 2020063760, WO 2020061103, WO 2020061101, WO 2020033828, WO 2020033286, WO 2020022323, WO 2019233810, WO 2019213318, WO 2019183367, WO 2019183364, WO 2019182960, WO 2019167000, WO 2019165073, WO 2019158019, WO 2019152454, WO 2019051469, WO 2019051084, WO 2018218133, WO 2018172984, WO 2018160731, WO 2018136265, WO 2018136264, WO 2018130928, WO 2018129402, WO 2018081091, WO 2018057884, WO 2018013597, WO 2017216706, WO 2017211303, WO 2017210134, WO 2017156397, WO 2017100279, WO 2017079723, WO 2017078499, WO 2016203406, WO 2016203405, WO 2016203404, WO 2016196591, WO 2016191328, WO 2015107495, WO 2015107494, WO 2015107493, WO 2014176488, WO 2014113584, US 20210085677, US 10858359, US 10934302, US 10954243, US 10988466, US 11001561, US 11033547, US 11034705, US CN 11044675, CN 114213417, CN 114163457, CN 113896710, CN 113248521, CN 113248449, CN 113135924, CN 113024508, CN 112920131, CN 112823796, CN 112402385, CN 111848599, CN 111704611, CN 111265529, and CN 108113848, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof.

[0343] In some embodiments, the SHP2 inhibitor binds to the active site. In some embodiments, the SHP2 inhibitor is a mixed, irreversible inhibitor. In some embodiments, the SHP2 inhibitor binds to an allosteric site, e.g., a non-covalent allosteric inhibitor. In some embodiments, the SHP2 inhibitor is a covalent SHP2 inhibitor, such as an inhibitor that targets a cysteine ​​residue (C333) outside the active site of the phosphatase. In some embodiments, the SHP2 inhibitor is a reversible inhibitor. In some embodiments, the SHP2 inhibitor is an irreversible inhibitor. In some embodiments, the SHP2 inhibitor is SHP099. In some embodiments, the SHP2 inhibitor is TNO155. In some embodiments, the SHP2 inhibitor is RMC-4550. In some embodiments, the SHP2 inhibitor is RMC-4630. In some embodiments, the SHP2 inhibitor is JAB-3068. In some embodiments, the SHP2 inhibitor is JAB-3312. In some embodiments, the SHP2 inhibitor is RLY-1971. In some embodiments, the SHP2 inhibitor is ERAS-601. In some embodiments, the SHP2 inhibitor is BBP-398.

[0344] In some embodiments, the additional therapeutic agent is selected from the group consisting of a MEK inhibitor, a HER2 inhibitor, a SHP2 inhibitor, a CDK4 / 6 inhibitor, an mTOR inhibitor, a SOS1 inhibitor, and a PD-L1 inhibitor. In some embodiments, the additional therapeutic agent is selected from the group consisting of a MEK inhibitor, a SHP2 inhibitor, and a PD-L1 inhibitor. See, e.g., Hallin et al., Cancer Discovery, DOI: 10.1158 / 2159-8290 (October 28, 2019) and Canon et al., Nature, 575:217 (2019). In some embodiments, a Ras inhibitor of the present invention is used in combination with a MEK inhibitor and a SOS1 inhibitor. In some embodiments, a Ras inhibitor of the present invention is used in combination with a PD-L1 inhibitor and a SOS1 inhibitor. In some embodiments, a Ras inhibitor of the present invention is used in combination with a PD-L1 inhibitor and a SHP2 inhibitor. In some embodiments, a Ras inhibitor of the present invention is used in combination with a MEK inhibitor and a SHP2 inhibitor. In some embodiments, the cancer is colorectal cancer and the treatment comprises administering a Ras inhibitor of the present invention in combination with a second or third therapeutic agent.

[0345] Proteasome inhibitors include, but are not limited to, carfilzomib (Kyprolis®), bortezomib (Velcade®), and oprozomib.

[0346] Immunotherapies include, but are not limited to, monoclonal antibodies, immunomodulatory imides (IMiDs), GITR agonists, engineered T cells (e.g., CAR-T cells), bispecific antibodies (e.g., BiTEs), and anti-PD-1, anti-PD-L1, anti-CTLA4, anti-LAG1, and anti-OX40 agents.

[0347] Immunomodulatory agents (IMiDs) are a class of immunomodulatory drugs (drugs that modify the immune response) that contain an imide group. The IMiD class includes thalidomide and its analogs (lenalidomide, pomalidomide, and apremilast).

[0348] Exemplary anti-PD-1 antibodies, and methods for their use, are described by Goldberg et al., Blood 2007, 110(1):186-192; Thompson et al., Clin. Cancer Res. 2007, 13(6):1757-1761; and WO06 / 121168 A1), and in addition, elsewhere herein.

[0349] GITR agonists include GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as those described in U.S. Pat. Nos. 6,111,090, 8,586,023, WO 2010 / 003118, and WO 2011 / 090754, or those described in, for example, U.S. Pat. No. 7,025,962, EP 1947183, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591, 886, 7,618,632, EP1866339, and the anti-GITR antibodies described in WO2011 / 028683, WO2013 / 039954, WO05 / 007190, WO07 / 133822, WO05 / 055808, WO99 / 40196, WO01 / 03720, WO99 / 20758, WO06 / 083289, WO05 / 115451, and WO2011 / 051726.

[0350] Another example of a therapeutic agent that can be used in combination with the compounds of the present invention is an anti-angiogenic agent.Anti-angiogenic agents include, but are not limited to, in vitro synthetically prepared chemical compositions, antibodies, antigen-binding regions, radionuclides, and combinations and conjugates thereof.Anti-angiogenic agents can be agonists, antagonists, allosteric modulators, toxins, or more generally, they act to inhibit or stimulate their targets (e.g., activate or inhibit receptors or enzymes), thereby promoting cell death or stopping cell proliferation.In some embodiments, one or more additional therapies include an anti-angiogenic agent.

[0351] Antiangiogenic agents can be MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase 11) inhibitors. Non-limiting examples of antiangiogenic agents include rapamycin, temsirolimus (CCI-779), everolimus (RAD001), sorafenib, sunitinib, and bevacizumab. Examples of useful COX-II inhibitors include alecoxib, valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in WO96 / 33172, WO96 / 27583, WO98 / 07697, WO98 / 03516, WO98 / 34918, WO98 / 34915, WO98 / 33768, WO98 / 30566, WO90 / 05719, WO99 / 52910, WO99 / 5288 9, WO99 / 29667, WO99 / 007675, EP0606046, EP0780386, EP1786785, EP1181017, EP0818442, EP1004578, and US2009 / 0012085, as well as U.S. Patent Nos. 5,863,949 and 5,861,510. Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-1. More preferred are those that selectively inhibit MMP-2 or MMP-9 relative to other matrix metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specific examples of MMP inhibitors are AG-3340, RO 32-3555, and RS13-0830.

[0352] Further exemplary anti-angiogenic agents include KDR (kinase domain receptor) inhibitors (e.g., antibodies and antigen-binding regions that specifically bind to kinase domain receptors), anti-VEGF agents (e.g., antibodies or antigen-binding regions that specifically bind to VEGF (e.g., bevacizumab), or a soluble VEGF receptor or ligand-binding region thereof), such as VEGF-TRAP™, and anti-VEGF receptor agents (e.g., antibodies or antigen-binding regions that specifically bind thereto), EGFR inhibitors (e.g., antibodies or antigen-binding regions that specifically bind thereto), such as Vectibix® (panitumumab), erlotinib (Tarceva®), anti-Ang1 and anti-Ang2 agents (e.g., antibodies or antigen-binding regions that specifically bind thereto or their receptors, e.g., Tie2 / Tek), and anti-Tie2 kinase inhibitors (e.g., antibodies or antigen-binding regions that specifically bind thereto). Other anti-angiogenic agents include Cambus, IL-8, B-FGF, Tek antagonists (US2003 / 0162712, US6,413,932), anti-TWEAK agents (e.g., specifically binding antibodies or antigen-binding regions, or soluble TWEAK receptor antagonists, see US6,727,225), ADAM distointegrin domains that antagonize the binding of integrins to their ligands (US2002 / 0042368), specifically binding anti-eph receptor or anti-ephrin antibodies or antigen-binding regions. (U.S. Patent Nos. 5,981,245, 5,728,813, 5,969,110, 6,596,852, 6,232,447, 6,057,124, and patent family members thereof), and anti-PDGF-BB antagonists (e.g., antibodies or antigen-binding regions that specifically bind), as well as antibodies or antigen-binding regions that specifically bind to PDGF-BB ligands, and PDGFR kinase inhibitors (e.g., antibodies or antigen-binding regions that specifically bind thereto). Additional anti-angiogenic agents include SD-7784 (Pfizer, USA), cilengitide (Merck KGaA, Germany, EPO0770622), pegaptanib octasodium (Gilead Sciences, USA), alphastatin (BioActa,UK), M-PGA (Celgene, USA, US5712291), Ilomastat (Arriva, USA, US5892112), Emaxanib (Pfizer, USA, US5792783), Vatalanib (Novartis, Switzerland), 2-Methoxyestradiol (EntreMed, USA), TLC ELL-12 (Elan, Ireland), Anecortave acetate (Alcon, USA), Alpha-D148 Mab (Amgen, USA), CEP-7055 (Cephalon, USA), Anti-Vn Mab (Crucell, Netherlands), DAC Antiangiogenic Agent (ConjuChem, Canada), Angiocidin (InKine Pharmaceutical, USA), KM-2550 (Kyowa Hakko, Japan), SU-0879 (Pfizer, USA), CGP-79787 (Novartis, Switzerland, EP0970070), ARGENT technology (Ariad, USA), YIGSR-Stealth (Johnson & Johnson, USA), fibrinogen-E fragment (BioActa, UK), angiogenesis inhibitors (Trigen, UK), TBC-1635 (Encysive Pharmaceuticals, USA), SC-236 (Pfizer, USA), ABT-567 (Abbott, USA), metastatin (EntreMed, USA), maspin (Sosei, Japan), 2-methoxyestradiol (Oncology Sciences Corporation, USA), ER-68203-00 (IV AX, USA), BeneFin (Lane Labs, USA), Tz-93 (Tsumura, Japan), TAN-1120 (Takeda, Japan), FR-111142 (Fujisawa, Japan, JP02233610), platelet factor 4 (RepliGen, USA, EP407122), vascular endothelial growth factor antagonist (Borean, Denmark), bevacizumab (pINN) (Genentech, USA), angiogenesis inhibitor (SUGEN, USA), XL 784 (Exelixis, USA), XL 647 (Exelixis, USA), MAb, alpha 5 beta 3 integrin, second generation (Applied Molecular Evolution, USA and MediImmune, USA), enzastaurin hydrochloride (Lilly,(USA), CEP 7055 (Cephalon, USA and Sanofi-Synthelabo, France), BC 1 (Genoa Institute of Cancer Research, Italy), rBPI 21 and BPI-derived antiangiogenic agents (XOMA, USA), PI 88 (Progen, Australia), cilengitide (Merck KGaA, Germany, Munich Technical University, Germany, Scripps Clinic and Research Foundation, USA), AVE 8062 (Ajinomoto, Japan), AS 1404 (Cancer Research Laboratory, New Zealand), SG 292 (Telios, USA), endostatin (Boston Children's Hospital, USA), ATN 161 (Attenuon, USA), 2-methoxyestradiol (Boston Children's Hospital, USA), ZD 6474 (AstraZeneca, UK), ZD 6126 (Angiogene Pharmaceuticals, UK), PPI 2458 (Praecis, USA), AZD 9935 (AstraZeneca, UK), AZD 2171 (AstraZeneca, UK), vatalanib (pINN) (Novartis, Switzerland and Schering AG, Germany), tissue factor pathway inhibitor (EntreMed, USA), pegaptanib (Pinn) (Gilead Sciences, USA), xanthorrhizol (Yonsei University, Korea), vaccine, gene-based VEGF-2 (Scripps Clinic and Research Foundation, USA), SPV5.2 (Supratek, Canada), SDX 103 (University of California at San Diego, USA), PX 478 (ProlX, USA), metastatin (EntreMed, USA), troponin I (Harvard University, USA), SU 6668 (SUGEN, USA), OXI 4503 (OXiGENE, USA), o-guanidine (Dimensional Pharmaceuticals,(USA), motuporamine C (University of British Columbia, Canada), CDP 791 (Celltech Group, UK), atiprimod (pINN) (GlaxoSmithKline, UK), E 7820 (Eisai, Japan), CYC 381 (Harvard University, USA), AE 941 (Aeterna, Canada), vaccines, angiogenic agents (EntreMed, USA), urokinase plasminogen activator inhibitor (Dendreon, USA), oglufanide (pINN) (Melmotte, USA), HIF-lalfa inhibitor (Xenova, UK), CEP 5214 (Cephalon, USA), BAY RES 2622 (Bayer, Germany), angiocidin (InKine, USA), A6 (Angstrom, USA), KR 31372 (Korea Research Institute of Chemical Technology, Korea), GW 2286 (GlaxoSmithKline, UK), EHT 0101 (ExonHit, France), CP 868596 (Pfizer, USA), CP 564959 (OSI, USA), CP 547632 (Pfizer, USA), 786034 (GlaxoSmithKline, UK), KRN 633 (Kirin Brewery, Japan), drug delivery system, intraocular, 2-methoxyestradiol, Anginex (Maastricht University, The Netherlands and University of Minnesota, USA), ABT 510 (Abbott, USA), AAL 993 (Novartis, Switzerland), VEGI (ProteomTech, USA), tumor necrosis factor-alpha inhibitor, SU 11248 (Pfizer, USA and SUGEN, USA), ABT 518 (Abbott, USA), YH16 (Yantai Rongchang, China), S-3APG (Boston Children's Hospital, USA and EntreMed, USA), MAb, KDR (ImClone Systems, USA), MAb, alpha5beta (Protein Design, USA), KDR kinase inhibitor (Celltech Group, UK and Johnson & Johnson,USA), GFB 116 (University of South Florida, USA and Yale University, USA), CS 706 (Sankyo, Japan), combretastatin A4 prodrug (Arizona State University, USA), chondroitinase AC (IBEX, Canada), BAY RES 2690 (Bayer, Germany), AGM 1470 (Harvard University, USA, Takeda, Japan, and TAP, USA), AG 13925 (Agouron, USA), tetrathiomolybdate (University of Michigan, USA), GCS 100 (Wayne State University, USA), CV 247 (Ivy Medical, UK), CKD 732 (Chong Kun Dang, Korea), irsogladine (Nippon Shinyaku, Japan), RG 13577 (Aventis, France), WX 360 (Wilex, Germany), squalamine (Genaera, USA), RPI 4610 (Sirna, USA), heparanase inhibitor (InSight, Israel), KL 3106 (Kolon, Korea), honokiol (Emory University, USA), ZK CDK (Schering AG, Germany), ZK Angio (Schering AG, Germany), ZK 229561 (Novartis, Switzerland and Schering AG, Germany), XMP 300 (XOMA, USA), VGA 1102 (Taisho, Japan), VE-cadherin-2 antagonist (ImClone Systems, USA), vasostatin (National Institutes of Health, USA), Flk-1 (ImClone Systems, USA), TZ 93 (Tsumura, Japan), tumstatin (Beth Israel Hospital, USA), truncated soluble FLT 1 (vascular endothelial growth factor receptor 1) (Merck & Co., USA), Tie-2 ligand (Regeneron, USA), and thrombospondin 1 inhibitor (Allegheny Health, Education and Research Foundation, USA).

[0353] Further examples of therapeutic agents that may be used in combination with the compounds of the invention include agents (e.g., antibodies, antigen-binding regions, or soluble receptors) that specifically bind and inhibit the activity of growth factors, such as antagonists of hepatocyte growth factor (HGF, also known as scatter factor), and antibodies or antigen-binding regions that specifically bind to its receptor, c-Met.

[0354] Another example of a therapeutic agent that can be used in combination with the compounds of the present invention is an autophagy inhibitor. Autophagy inhibitors include, but are not limited to, chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil™), bafilomycin A1, 5-amino-4-imidazolecarboxamide riboside (AICAR), okadaic acid, autophagy-inhibiting algal toxins that inhibit type 2A or type 1 protein phosphatases, cAMP analogs, and drugs that increase cAMP levels, such as adenosine, LY204002, N6-mercaptopurine riboside, and vinblastine. In addition, antisense or siRNA inhibitors that inhibit the expression of proteins, including but not limited to ATG5 (related to autophagy), can also be used. In some embodiments, one or more additional therapies include an autophagy inhibitor.

[0355] Another example of a therapeutic agent that can be used in combination with the compound of the present invention is an anti-cancer agent. In some embodiments, one or more additional therapies include an anti-cancer agent. Non-limiting examples of an anti-cancer agent include acemannan, aclarubicin, aldesleukin, alemtuzumab, alitretinoin, altretamine, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, ancer, ancestim, aruglavin, arsenic trioxide, BAM-002 (Novelos), bexarotene, bicalutamide, bromodeoxyuridine, capecitabine, celmoleukin, cetrorelix, cladribine, clotrimazole, cytarabine octophosphate, DA 3030 (Dong-A), daclizumab, denileukin diftitox, deslorelin, dexrazoxane, dilazep, docetaxel, docosanol, doxercalciferol, doxifluridine, doxorubicin, bromocriptine, carmustine, cytarabine, fluorouracil, HIT diclofenac, interferon alpha, daunorubicin, doxorubicin, tretinoin, edelfosine, edrecolomab eflornithine, emiteflu, epirubicin, epoetin beta, etoposide phosphate, exemestane, exisulind, fadrozole, filgrastim, finasteride, fludarabine phosphate, formestane, fotemustine, gallium nitrate, gemcitabine, gemtuzumab zogamicin, gimeracil / oteracil / tegafur combination Combined, glycopin, goserelin, heptaplatin, human chorionic gonadotropin, human fetal alpha-fetoprotein, ibandronic acid, idarubicin (imiquimod, interferon alpha, interferon alpha, natural type, interferon alpha-2, interferon alpha-2a, interferon alpha-2b, interferon alpha-NI, interferon alpha-n3, interferon alphacon-1, interferon alpha, natural type, interferon beta, interferon beta-la, interferon beta-lb, interferon gamma, natural type interferon gamma-la, interferon gamma-lb, interleukin-1 beta, iobenguane, irinotecan, irsogladine,Lanreotide, LC9018 (Yakult), leflunomide, lenograstim, lentinan sulfate, letrozole, leukocyte alpha interferon, leuprorelin, levamisole + fluorouracil, liarozole, lobaplatin, lonidamine, lovastatin, masoprocol, melarsoprol, metoclopramide, mifepristone, miltefosine, millimostim, mispaired double-stranded RNA, mitoguazone, mitolactol, mitoxantrone, molgramostim, nafarelin, naloxone + pentazocine, nartograstim, neda Platin, nilutamide, noscapine, novel erythropoiesis-stimulating protein, NSC631570, octreotide, oprelvekin, osaterone, oxaliplatin, paclitaxel, pamidronate, pegaspargase, peginterferon alfa-2b, pentosan, sodium polysulfate, pentostatin, picibanil, pirarubicin, rabbit antithymocyte polyclonal antibody, polyethylene glycol interferon alfa-2a, porfimer sodium, raloxifene, raltitrexed, rasbuconazole riembodiment), etidronate rhenium Re186, RII retinamide, rituximab, romurtide, samarium (153Sm) lexidronam, sargramostim, sizofiran, sobuzoxane, sonermin, strontium-89 chloride, suramin, tasonermin, tazarotene, tegafur, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, thalidomide, thymalfasin, thyroid-stimulating hormone alpha, topotecan, toremifene, tositumomab-iodine 131, trastuzumab, treosulfan, Tretinoin, trilostane, trimetrexate, triptorelin, tumor necrosis factor alpha, native, ubenimex, bladder cancer vaccine, Maruyama vaccine, melanoma lysate vaccine, valrubicin, verteporfin, vinorelbine, virulence, zinostatin stimalamer or zoledronic acid; abarelix; AE941 (Aeterna), ambamustine, antisense oligonucleotide, bcl-2 (Genta), APC8015 (Dendreon), decitabine, dexaminoglutethimide, diazicon, EL532 (Elan),EM800 (Endorecherche), eniluracil, etanidazole, fenretinide, filgrastim SD01 (Amgen), fulvestrant, galocitabine, gastrin-17 immunogen, HLA-B7 gene therapy (Vical), granulocyte-macrophage colony-stimulating factor, histamine dihydrochloride, ibritumomab tiuxetan, ilomastat, IM862 (Cytran), interleukin-2, iproxifen, LDI200 (Milkhaus), religistim, lintuzumab, CA125 MAb (Biomira), cancer MAb (Japan Pharmaceutical Development), HER-2 and Fc MAb (Medarex), idiotype 105AD7 MAb (CRC Technology), idiotype CEA MAb (Trilex), LYM-1-iodine-131 MAb (Techniclone), polymorphic epithelial mucin-yttrium-90 MAb (Antisoma), marimastat, menogaril, mitumomab, motexafin, gadolinium, MX6 (Galderma), nelarabine, nolatrexed, P30 protein, pegvisomant, pemetrexed, porfiromycin, prinomastat, RL0903 (Shire), rubitecan, satraplatin, sodium phenylacetate, sparfosic acid, SRL172 (SR Pharma), SU5416 (SUGEN), TA077 (Tanabe), tetrathiomolybdate, saliblastin, thrombopoietin, tin ethyl etiopurpurin, tirapazamine, cancer vaccine (Biomira), melanoma vaccine (New York University), melanoma vaccine (Sloan Kettering) Institute), melanoma oncolysate vaccine (New York Medical College), viral melanoma cell lysate vaccine (Royal Newcastle Hospital), or Valspodar.

[0356] Additional examples of therapeutic agents that may be used in combination with the compounds of the invention include ipilimumab (Yervoy®); tremelimumab; galiximab; nivolumab, also known as BMS-936558 (Opdivo®); pembrolizumab (Keytruda®); avelumab (Bavencio®); AMP224; BMS-936559; MPDL3280A, also known as RG7446; MEDI-570; AMG557; MGA271; IMP321; BMS-663513; PF-05082566; CDX-1127; anti-OX40 (Providence Health Services); huMAbOX40L; atacicept; CP-870893; lucatumumab; dacetuzumab; muromonab-CD3; ipilumumab; MEDI4736 (Imfinzi®); MSB0010718C; AMP 224; adalimumab (Humira®); ado-trastuzumab emtansine (Kadcyla®); aflibercept (Eylea®); alemtuzumab (Campath®); basiliximab (Simulect®); belimumab (Benlysta®); basiliximab (Simulect®); belimumab (Benlysta®); brentuximab vedotin (Adcetris®); canakinumab (Ilaris®); certolizumab pegol (Cimzia®); daclizumab (Zenapax®); daratumumab (Darzale x(R)); denosumab (Prolia(R)); eculizumab (Soliris(R)); efalizumab (Raptiva(R)); gemtuzumab ozogamicin (Mylotarg(R)); golimumab (Simponi(R)); ibritumomab tiuxetan (Zevalin(R)); infliximab (Remicade(R)); motavizumab (Numax(R)); natalizumab (Tysabri(R)); obinutuzumab (Gazyva(R)); ofatumumab (Arzerra(R)); omalizumab (Xolair(R)); palivizumab (Synagis(R));Examples of anti-cancer drugs include pertuzumab (Perjeta®); pertuzumab (Perjeta®); ranibizumab (Lucentis®); raxibacumab (Abthrax®); tocilizumab (Actemra®); tositumomab; tositumomab-i-131; tositumomab and tositumomab-i-131 (Bexxar®); ustekinumab (Stelara®); AMG 102; AMG 386; AMG 479; AMG 655; AMG 706; AMG 745; and AMG 951.

[0357] The compounds described herein can be used in combination with the agents disclosed herein or other suitable agents, depending on the condition being treated. Thus, in some embodiments, one or more compounds of the present invention are co-administered with other therapies, such as those described herein. When used in combination therapy, the compounds described herein can be administered simultaneously with the second agent or separately. This combined administration can include simultaneous administration of the two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, the compounds described herein and any of the agents described herein can be formulated together in the same dosage form and administered simultaneously. Alternatively, the compounds of the present invention and any of the therapies described herein can be administered simultaneously, with both agents present in separate formulations. In another alternative, the compounds of the present invention can be administered followed by any of the therapies described herein, or vice versa. In some embodiments of the separate administration protocol, the compounds of the present invention and any of the therapies described herein are administered within minutes, hours, or days of each other.

[0358] In some embodiments of any of the methods described herein, the first therapy (e.g., a compound of the invention) and the one or more additional therapies are administered simultaneously or sequentially in any order. The first therapeutic agent is administered immediately before or immediately after the one or more additional therapies, or up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, up to 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours before or after, or up to 1-7, 1-14, 1-21, or 1-30 days before or after.

[0359] The invention also features kits that include (a) a pharmaceutical composition that includes an agent described herein (e.g., a compound of the invention), and (b) a package insert with instructions for practicing any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition that includes an agent described herein (e.g., a compound of the invention), (b) one or more additional therapies (e.g., non-drug treatments or therapeutic agents), and (c) a package insert with instructions for practicing any of the methods described herein.

[0360] Because one aspect of the present invention contemplates treating a disease or associated symptoms with a combination of pharmaceutically active compounds that can be administered separately, the present invention further relates to combining separate pharmaceutical compositions in the form of a kit. The kit may include two separate pharmaceutical compositions: a compound of the present invention and one or more additional therapies. The kit may include containers for housing the separate compositions, such as divided bottles or divided foil packets. Additional examples of containers include syringes, boxes, and bags. In some embodiments, the kit may include instructions for using the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing healthcare professional.

[0361] Numbered Embodiments Embodiment 1. A compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof:

[0362] [ka]

[0363] wherein A is an optionally substituted 3- to 6-membered heterocycloalkylene, an optionally substituted 3- to 6-membered cycloalkylene, an optionally substituted 6-membered arylene, or an optionally substituted 5- to 10-membered heteroarylene; W is a bridging group comprising an aziridine, epoxide, carbodiimide, oxazoline, thiazoline, chloroethylurea, chloroethylthiourea, chloroethylcarbamate, chloroethylthiocarbamate, trifluoromethylketone, boronic acid, boronic ester, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isoEEDQ or other EEDQ derivatives, oxazolium, or glycal; X 1 is CH2 or O; m is 1 or 2, n is 0 or 1, R 1 is hydrogen or an optionally substituted 3- to 10-membered heterocycloalkyl; R 2 is an optionally substituted C1-C6 alkyl, and R 3 is optionally substituted C1-C6 alkyl or optionally substituted 3- to 6-membered cycloalkyl.

[0364] Embodiment 2. The compound of embodiment 1, wherein A is optionally substituted thiazole, optionally substituted oxazole, optionally substituted morpholino, optionally substituted pyrrolidinyl, optionally substituted piperidinyl, or optionally substituted phenyl, or a pharmaceutically acceptable salt thereof.

[0365] Embodiment 3. A compound of embodiment 1 or 2 having the structure of formula II-1, or a pharmaceutically acceptable salt thereof:

[0366] [ka]

[0367] Embodiment 4. A compound of embodiment 1 or 2, having the structure of formula II-2, or a pharmaceutically acceptable salt thereof:

[0368] [ka]

[0369] [In the formula, R 6 , R 7 , and R 8 are each independently selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted 3- to 6-membered cycloalkyl, and optionally substituted 3- to 6-membered heterocycloalkyl; R 6 and R 7 combine with the atom to which they are attached to form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 8-membered heterocycloalkyl; or R 6 and R 8 combine with the atom to which they are attached to form an optionally substituted 3- to 8-membered cycloalkyl or an optionally substituted 3- to 8-membered heterocycloalkyl.

[0370] Embodiment 5. A compound of embodiment 4 having the structure of formula II-3, or a pharmaceutically acceptable salt thereof:

[0371] [ka]

[0372] Embodiment 6. A compound of embodiment 4 having the structure of formula II-4, or a pharmaceutically acceptable salt thereof:

[0373] [ka]

[0374] [In the formula, X 2 is CH2 or O, and o is 1 or 2.] Embodiment 7. X 2 is CH2, or a pharmaceutically acceptable salt thereof.

[0375] Embodiment 8. The compound of embodiment 7, or a pharmaceutically acceptable salt thereof, wherein o is 1. Embodiment 9. X 2 is O, or a pharmaceutically acceptable salt thereof.

[0376] Embodiment 10. The compound of any one of embodiments 6, 7, and 9, or a pharmaceutically acceptable salt thereof, wherein o is 2. Embodiment 11. R 2 teeth,

[0377] [ka]

[0378] 11. The compound of any one of embodiments 1-10, wherein: Embodiment 12. R 3 The compound of any one of embodiments 1-11, or a pharmaceutically acceptable salt thereof, wherein is optionally substituted C1-C6 alkyl.

[0379] Embodiment 13. R 3 teeth

[0380] [ka]

[0381] or a pharmaceutically acceptable salt thereof. Embodiment 14. R 3 12. The compound of any one of embodiments 1-11, wherein: is or an optionally substituted 3-6 membered cycloalkyl, or a pharmaceutically acceptable salt thereof.

[0382] Embodiment 15. R 3 teeth

[0383] [ka]

[0384] or a pharmaceutically acceptable salt thereof. Embodiment 16. A compound of any one of embodiments 1 to 15, or a pharmaceutically acceptable salt thereof, wherein A is an optionally substituted 5- to 10-membered heteroarylene.

[0385] Embodiment 17. A is

[0386] [ka]

[0387] 17. The compound of embodiment 16, wherein: Embodiment 18. A compound of any one of embodiments 1-15, or a pharmaceutically acceptable salt thereof, wherein A is optionally substituted phenyl.

[0388] Embodiment 19. A is

[0389] [ka]

[0390] 19. The compound of embodiment 18, wherein: Embodiment 20. A compound of any one of embodiments 1-15, or a pharmaceutically acceptable salt thereof, wherein A is an optionally substituted 3-6 membered heterocycloalkylene.

[0391] Embodiment 21. A compound of embodiment 20, wherein A is selected from the following, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0392] [ka]

[0393] Embodiment 22. The compound of any one of embodiments 1 to 21, or a pharmaceutically acceptable salt thereof, wherein m is 1. Embodiment 23. The compound of embodiment 22, or a pharmaceutically acceptable salt thereof, wherein n is 1.

[0394] Embodiment 24. X 1 is CH2, or a pharmaceutically acceptable salt thereof. Embodiment 25. The compound of any one of embodiments 1 to 21, or a pharmaceutically acceptable salt thereof, wherein m is 2.

[0395] Embodiment 26. X 1 is CH2, or a pharmaceutically acceptable salt thereof. Embodiment 27. The compound of embodiment 26, or a pharmaceutically acceptable salt thereof, wherein n is 1.

[0396] Embodiment 28. The compound of embodiment 26, or a pharmaceutically acceptable salt thereof, wherein n is 0. Embodiment 29. X 1 is O, or a pharmaceutically acceptable salt thereof.

[0397] Embodiment 30. The compound of embodiment 29, or a pharmaceutically acceptable salt thereof, wherein n is 1. Embodiment 31. The compound of embodiment 29, or a pharmaceutically acceptable salt thereof, wherein n is 0.

[0398] Embodiment 32. The compound of any one of embodiments 1-31, or a pharmaceutically acceptable salt thereof, wherein W comprises aziridine. Embodiment 33. A compound of embodiment 32, or a pharmaceutically acceptable salt thereof, wherein W comprises an optionally substituted cyclopropyl-aziridinyl moiety.

[0399] Embodiment 34. A compound of embodiment 32 or 33, wherein W is selected from the following, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0400] [ka]

[0401] Embodiment 35. A compound of any one of embodiments 1-31, or a pharmaceutically acceptable salt thereof, wherein W comprises an epoxide. Embodiment 36. A compound of embodiment 35, wherein W is selected from the following, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0402] [ka]

[0403] Embodiment 37. A compound of any one of embodiments 1-36 having the structure of a compound set forth in Table 1 or Table 2, or a pharmaceutically acceptable salt thereof. Embodiment 38. A compound having the structure of Formula III, or a pharmaceutically acceptable salt thereof:

[0404] [ka]

[0405] wherein P is —(CO)R 9 , -(PO)(OH)2, or -Si(R 10 )3, W is a bridging group comprising an aziridine, epoxide, carbodiimide, oxazoline, thiazoline, chloroethylurea, chloroethylthiourea, chloroethylcarbamate, chloroethylthiocarbamate, trifluoromethylketone, boronic acid, boronic ester, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isoEEDQ or other EEDQ derivatives, oxazolium, or glycal; L 1 is an optionally substituted 3- to 9-membered heterocycloalkylene or an optionally substituted C2-C4 heteroalkyl; R 4 is an optionally substituted C1-C6 alkyl; R 5 is optionally substituted C1-C6 alkyl or optionally substituted 3- to 6-membered cycloalkyl.

[0406] R 9 is an optionally substituted C1-C3 alkyl or an optionally substituted C1-C3 heteroalkyl, and Each R 10 are independently optionally substituted C1-C3 alkyl.

[0407] Embodiment 39. R 4 teeth

[0408] [ka]

[0409] or a pharmaceutically acceptable salt thereof. Embodiment 40. R 5is optionally substituted C1-C6 alkyl, or a pharmaceutically acceptable salt thereof.

[0410] Embodiment 41. R 5 teeth

[0411] [ka]

[0412] or a pharmaceutically acceptable salt thereof. Embodiment 42. R 5 The compound of embodiment 38 or 39, or a pharmaceutically acceptable salt thereof, wherein is optionally substituted 3-6 membered cycloalkyl.

[0413] Embodiment 43. R 5 teeth

[0414] [ka]

[0415] or a pharmaceutically acceptable salt thereof. Embodiment 44. P is —(CO)R 9 44. The compound of any one of embodiments 38-43, wherein:

[0416] Embodiment 45. P is

[0417] [ka]

[0418] or a pharmaceutically acceptable salt thereof. Embodiment 46. The compound of any one of embodiments 38-43, or a pharmaceutically acceptable salt thereof, wherein P is —(PO)(OH).

[0419] Embodiment 47. P is —Si(R 10 44. The compound of any one of embodiments 38-43, or a pharmaceutically acceptable salt thereof, wherein: Embodiment 48. P is

[0420] [ka]

[0421] or a pharmaceutically acceptable salt thereof. Embodiment 49. L 1 The compound of any one of embodiments 38-48, or a pharmaceutically acceptable salt thereof, wherein is 3-9 membered heterocycloalkylene.

[0422] Embodiment 50. L 1 or its stereoisomers:

[0423] [ka]

[0424] 50. The compound of embodiment 49, wherein: Embodiment 51. L 1 The compound of any one of embodiments 38-48, or a pharmaceutically acceptable salt thereof, wherein is optionally substituted C2-C4 heteroalkyl.

[0425] Embodiment 52. L 1 teeth

[0426] [ka]

[0427] or a pharmaceutically acceptable salt thereof. Embodiment 53. A compound according to any one of embodiments 38-52, or a pharmaceutically acceptable salt thereof, wherein W is an optionally substituted cyclopropyl-aziridinyl moiety.

[0428] Embodiment 54. W is or a stereoisomer thereof:

[0429] [ka]

[0430] 54. The compound of any one of embodiments 38-53, wherein: Embodiment 55. A compound of any one of embodiments 38-54 having the structure of a compound set forth in Table 3 or Table 4, or a pharmaceutically acceptable salt thereof.

[0431] Embodiment 56. A compound having the structure of a compound set forth in Table 5, or a pharmaceutically acceptable salt thereof. Embodiment 57. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 56, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0432] Embodiment 58. A conjugate comprising the structure of Formula IV, or a salt thereof: ML 2 -P 1 Formula IV [In the formula, L 2 is the linker, P 1 is a monovalent organic moiety, and M has the structure of Formula V.

[0433] [ka]

[0434] wherein A is an optionally substituted 3- to 6-membered heterocycloalkylene, an optionally substituted 3- to 6-membered cycloalkylene, an optionally substituted 6-membered arylene, or an optionally substituted 5- to 10-membered heteroarylene; X 1 is CH2 or O; m is 1 or 2, n is 0 or 1, R 1 is hydrogen or an optionally substituted 3- to 10-membered heterocycloalkyl; R 2 is an optionally substituted C1-C6 alkyl, and R 3 is optionally substituted C1-C6 alkyl or optionally substituted 3- to 6-membered cycloalkyl.

[0435] Embodiment 59. The conjugate of embodiment 58, or a salt thereof, wherein the monovalent organic moiety is a protein. Embodiment 60. The conjugate of embodiment 59, or a salt thereof, wherein the protein is a Ras protein.

[0436] Embodiment 61. The conjugate of embodiment 60, or a salt thereof, wherein the Ras protein is K-Ras G12D or K-Ras G13D. Embodiment 62. The linker L 2 is attached to the monovalent organic moiety via a bond to a carboxyl group of an amino acid residue of the monovalent organic moiety.

[0437] Embodiment 63. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of any one of embodiments 1 to 56, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of embodiment 57.

[0438] Embodiment 64. The method of embodiment 63, wherein the cancer is pancreatic cancer, non-small cell lung cancer, colorectal cancer, or endometrial cancer. Embodiment 65. The method of embodiment 63 or 64, wherein the cancer comprises a Ras mutation.

[0439] Embodiment 66. The method of embodiment 65, wherein the Ras mutation is K-Ras G12D or K-Ras G13D. Embodiment 67. A method of treating a Ras protein-associated disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of any one of embodiments 1 to 56, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of embodiment 57.

[0440] Embodiment 68. A method of inhibiting a Ras protein in a cell, comprising contacting the cell with a compound of any one of embodiments 1 to 56, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of embodiment 57.

[0441] Embodiment 69. The method of embodiment 67 or 68, wherein the Ras protein is K-Ras G12D or K-Ras G13D. Embodiment 70. The method of embodiment 68 or 69, wherein the cell is a cancer cell.

[0442] Embodiment 71. The method of embodiment 70, wherein the cancer cells are pancreatic cancer cells, non-small cell lung cancer cells, colorectal cancer cells, or endometrial cancer cells. Embodiment 72. The method or use according to any one of embodiments 63 to 71, wherein the method comprises administering an additional anti-cancer therapy.

[0443] Embodiment 73. The method of embodiment 72, wherein the additional anticancer therapy is an EGFR inhibitor, a second Ras inhibitor, a SHP2 inhibitor, a SOS1 inhibitor, a Raf inhibitor, a MEK inhibitor, an ERK inhibitor, a PI3K inhibitor, a PTEN inhibitor, an AKT inhibitor, an mTORC1 inhibitor, a BRAF inhibitor, a PD-L1 inhibitor, a PD-1 inhibitor, a CDK4 / 6 inhibitor, a HER2 inhibitor, or a combination thereof.

[0444] Embodiment 74. The method of embodiment 72 or 73, wherein the additional anticancer therapy is an SHP2 inhibitor. Example The present invention is further illustrated by the following examples and synthetic examples, which should not be construed as limiting the scope or spirit of the present invention to the specific procedures described herein. It should be understood that the examples are provided to illustrate particular embodiments and that no limitation on the scope of the present invention is intended thereby. It should be further understood that resort can be made to various other embodiments, modifications, and equivalents thereof, which may suggest themselves to those skilled in the art, without departing from the spirit of the present invention or the scope of the appended claims.

[0445] chemical synthesis Definitions used in the examples below and elsewhere in this specification are as follows:

[0446] CH2Cl2, DCM methylene chloride, dichloromethane CH3CN, MeCN, Acetonitrile CuI Copper(I) iodide DIPEA Diisopropylethylamine DMF N,N-dimethylformamide EtOAc ethyl acetate hS time H2O Water HCl Hydrochloric acid K3PO2 Potassium phosphate (tribasic acid) MeOH Methanol Na2SO4 Sodium Sulfate NMP N-methylpyrrolidone Pd(dppf)Cl2 [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) Synthesis of intermediates Intermediate 1. Synthesis of 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylpropan-1-ol

[0447] [ka]

[0448] Step 1: Synthesis of 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropan-1-one To a mixture of 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropanoyl chloride (65 g, 137 mmol, crude) in DCM (120 mL) was slowly added 1 M SnCl in DCM (137 mL, 137 mmol) under N2 atmosphere at 0 °C. After the mixture was stirred at 0 °C for 30 min, a solution of 5-bromo-1H-indole (26.8 g, 137 mmol) in DCM (40 mL) was added dropwise. After the mixture was stirred at 0 °C for 45 min, it was diluted with EtOAc (300 mL), washed with brine (4 × 100 mL), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (55 g, 75% yield). LCMS (ESI) m / z: [M+H]C 29 H 32 Calculated for BrNO2SiNa 556.1; found 556.3.

[0449] Step 2: Synthesis of 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropan-1-one To a mixture of 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropan-1-one (50 g, 93.6 mmol) and THF (100 mL) was added LiBH (6.1 g, 281 mmol) at 0 °C under an atmosphere of N. The mixture was heated to 60 °C and stirred for 20 h. After that, MeOH (10 mL) and EtOAc (100 mL) were added, the mixture was washed with brine (50 mL), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with DCM (50 mL), cooled to 10 °C, and dirudin (9.5 g, 37.4 mmol) and TsOH·HO (890 mg, 4.7 mmol) were added. The mixture was stirred at 10° C. for 2 hours, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (41 g, 84% yield). LCMS (ESI) m / z: [M+H]C 29 H 34 Calculated for BrNOSi: 519.2; Found 520.1.

[0450] Step 3: Synthesis of 5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-2-iodo-1H-indole To a mixture of 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropan-1-one (1.5 g, 2.9 mmol) and I (731 mg, 2.9 mmol) in THF (15 mL) was added AgOTf (888 mg, 3.5 mmol) at room temperature. The mixture was stirred at room temperature for 2 hours, then diluted with EtOAc (200 mL), washed with saturated aqueous NaSO (100 mL), dried over anhydrous NaSO, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (900 mg, 72% yield) as a solid.

[0451] Step 4: Synthesis of (1S)-1-(3-bromopyridin-2-yl)ethanol To a stirred mixture of HCOH (66.3 g, 1.44 mol) and EtN (728 g, 7.2 mol) under an Ar atmosphere at 0 °C, (4S,5S)-2-chloro-2-methyl-1-(4-methylbenzenesulfonyl)-4,5-diphenyl-1,3-diaza-2-ruthenacyclopentanecimene (3.9 g, 6.0 mmol) was added in several portions. The mixture was heated to 40 °C and stirred for 15 min, then cooled to room temperature, and 1-(3-bromopyridin-2-yl)ethanone (120 g, 600 mmol) was added in small portions. The mixture was heated to 40 °C and stirred for an additional 2 h, after which the solvent was concentrated under reduced pressure. Brine (2 L) was added to the residue, and the mixture was extracted with EtOAc (4 × 700 mL), dried over anhydrous NaSO, and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give the product (100 g, 74% yield) as an oil. LCMS (ESI) m / z calculated for [M+H]C7H8BrNO: 201.98; found: 201.9.

[0452] Step 5: Synthesis of 3-bromo-2-[(1S)-1-methoxyethyl]pyridine To a stirred mixture of (1S)-1-(3-bromopyridin-2-yl)ethanol (100 g, 495 mmol) in DMF (1 L) at 0 °C, NaH (60% dispersion in oil) (14.25 g, 594 mmol) was added in small portions. The mixture was stirred at 0 °C for 1 h. MeI (140.5 g, 990 mmol) was added dropwise at 0 °C, and the mixture was warmed to room temperature and stirred for 2 h. The mixture was cooled to 0 °C, and saturated aqueous NH4Cl (5 L) was added. The mixture was extracted with EtOAc (3 × 1.5 L), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (90 g, 75% yield) as an oil. LCMS (ESI) m / z [M+H]C8H 10 Calculated for BrNO: 215.99; Found: 215.9.

[0453] Step 6: Synthesis of 2-[(1S)-1-methoxyethyl]-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine To a stirred mixture of 3-bromo-2-[(1S)-1-methoxyethyl]pyridine (90 g, 417 mmol) in toluene (900 mL) at room temperature under an Ar atmosphere, bis(pinacolato)diboron (127 g, 500 mmol), KOAc (81.8 g, 833 mmol), and Pd(dppf)Cl (30.5 g, 41.7 mmol) were added. The mixture was heated to 100 °C and stirred for 3 h. The filtrate was concentrated under reduced pressure, and the residue was purified by AlO column chromatography to give the product (100 mg, 63% yield) as a semi-solid. LCMS (ESI) m / z [M+H] C 14 H 22 Calculated value for BNO3: 264.17; Found value: 264.1.

[0454] Step 7: Synthesis of 5-bromo-3-[3-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylpropyl]-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-1H-indole To a stirred mixture of 5-bromo-3-[3-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylpropyl]-2-iodo-1H-indole (140 g, 217 mmol) and 2-[(1S)-1-methoxyethyl]-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (100 g, 380 mmol) in dioxane (1.4 L) was added KCO (74.8 g, 541 mmol), Pd(dppf)Cl (15.9 g, 21.7 mmol), and HO (280 mL) in small portions under an Ar atmosphere at room temperature. The mixture was heated to 85 °C and stirred for 4 h. After cooling, HO (5 L) was added, and the mixture was extracted with EtOAc (3 × 2 L). The combined organic layers were washed with brine (2 x 1 L), dried over anhydrous NaSO, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (71 g, 45% yield) as a solid. LCMS (ESI) m / z [M+H] C 37 H 43 Calculated for BrN2O2Si: 655.23; Found: 655.1.

[0455] Step 8: Synthesis of 5-bromo-3-[3-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylpropyl]-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indole To a stirred mixture of 5-bromo-3-[3-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylpropyl]-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-1H-indole (71 g, 108 mmol) in DMF (0.8 L) was added CsCO (70.6 g, 217 mmol) and EtI (33.8 g, 217 mmol) in small portions at 0 °C under an atmosphere of N. The mixture was allowed to warm to room temperature and stirred for 16 h. After that, HO (4 L) was added and the mixture was extracted with EtOAc (3 × 1.5 L). The combined organic layers were washed with brine (2 × 1 L), dried over anhydrous NaSO, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (66 g, 80% yield) as an oil. LCMS(ESI) m / z[M+H]C 39 H 47 Calculated for BrN2O2Si: 683.26; Found: 683.3.

[0456] Step 9: Synthesis of 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylpropan-1-ol To a stirred mixture of TBAF (172.6 g, 660 mmol) in THF (660 mL) at room temperature under an atmosphere of N was added 5-bromo-3-[3-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylpropyl]-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indole (66 g, 97 mmol) in small portions. The mixture was heated to 50 °C and stirred for 16 h, cooled, diluted with HO (5 L), and extracted with EtOAc (3 × 1.5 L). The combined organic layers were washed with brine (2 × 1 L), dried over anhydrous NaSO, and filtered. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (30 g, 62% yield) as a solid. LCMS (ESI) m / z [M+H]C 23 H 29 Calculated for BrN2O2: 445.14; Found: 445.1.

[0457] Intermediate 2. Alternative synthesis via the Fischer indole route.

[0458] [ka]

[0459] Step 1: Synthesis of 5-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-2,2-dimethyl-5-oxopentanoic acid To a mixture of i-PrMgCl (2 M in THF, 0.5 L) was added n-BuLi (2.5 M in hexane, 333 mL, 833 mmol) dropwise over 15 min at −10° C. under an atmosphere of N. After the mixture was stirred for 30 min at −10° C., a solution of 3-bromo-2-[(1S)-1-methoxyethyl]pyridine (180 g, 833 mmol) in THF (0.5 L) was added dropwise over 30 min at −10° C. The resulting mixture was warmed to −5° C. and stirred for 1 h, after which a solution of 3,3-dimethyloxane-2,6-dione (118 g, 833 mmol) in THF (1.2 L) was added dropwise over 30 min at −5° C. The mixture was warmed to 0° C. and stirred for 1.5 h before being quenched by the addition of pre-pooled 4 M HCl in dioxane (0.6 L) at 0° C. to adjust the pH to approximately 5. The mixture was diluted with HO (3 L) at 0° C. and extracted with EtOAc (3×2.5 L). The combined organic layers were dried over anhydrous NaSO, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (87 g, 34% yield) as a solid. LCMS (ESI) m / z [M+H]C 15 H 21 Calculated for NO4: 280.15; Found: 280.1.

[0460] Step 2: Synthesis of 3-(5-bromo-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-1H-indol-3-yl)-2,2-dimethylpropanoic acid and ethyl (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropanoate To a mixture of 5-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-2,2-dimethyl-5-oxopentanoic acid (78 g, 279 mmol) and EtOH (0.78 L) was added (4-bromophenyl)hydrazine hydrochloride (68.7 g, 307 mmol) in small portions at room temperature under an atmosphere of N. The mixture was heated to 85° C. and stirred for 2 hours. After cooling to room temperature, 4 M HCl (in dioxane, 69.8 mL, 279 mmol) was added dropwise. The mixture was heated to 85° C. and stirred for an additional 3 hours. It was then concentrated under reduced pressure, and the residue was dissolved in TFA (0.78 L). The mixture was heated to 60° C., stirred for 1.5 hours, concentrated under reduced pressure, and the residue was basified with saturated NaHCO to pH 8 and extracted with EtOAc (3×1.5 L). The combined organic layers were dried over anhydrous Na2SO4 and filtered, the filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give the product (78 g, crude). LCMS (ESI) m / z [M+H]C 21 H 23 Calculated for BrN2O3: 430.1 and C 23 H 27 Calculated for BrN2O3: 459.12; Found: 431.1 (carboxylic acid) and 459.1.

[0461] Step 3: Synthesis of ethyl 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylpropanoate To a mixture of 3-(5-bromo-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-1H-indol-3-yl)-2,2-dimethylpropanoic acid and ethyl (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropanoate (198 g, 459 mmol) in DMF (1.8 L) was added CsCO (449 g, 1.38 mol) in small portions at 0 °C under an atmosphere of N. Then, a solution of EtI (215 g, 1.38 mmol) in DMF (200 mL) was added dropwise at 0 °C. The mixture was allowed to warm to room temperature and stirred for 4 h, then diluted with brine (5 L) and extracted with EtOAc (3 × 2.5 L). The combined organic layers were washed with brine (2 x 1.5 L), dried over anhydrous NaSO, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (160 g, 57% yield) as a solid. LCMS (ESI) m / z [M+H]C 25 H 31 Calculated for BrN2O3: 487.17; Found: 487.2.

[0462] Step 4: Synthesis of 3-(5-bromo-1-ethyl-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol To a mixture of ethyl 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylpropanoate (160 g, 328 mmol) and THF (1.6 L) was added LiBH (28.6 g, 1.3 mol) under an atmosphere of N at 0 °C. The mixture was heated to 60 °C for 16 h, cooled, and quenched with pre-pooled (0 °C) saturated aqueous NH Cl (5 L). The mixture was extracted with EtOAc (3 × 2 L), and the combined organic layers were washed with brine (2 × 1 L), dried over anhydrous Na SO , and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give two atropisomers (as single atropisomers) of 3-(5-bromo-1-ethyl-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol (60 g, 38% yield and 40 g, 26% yield), both as solids. LCMS (ESI) m / z [M+H]C 23 H 29 Calculated for BrN2O2: 445.14; Found: 445.2.

[0463] Intermediate 3. Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((triisopropylsilyl)oxy)phenyl)propanoyl)hexahydropyridazine-3-carboxylate

[0464] [ka]

[0465] Step 1: Synthesis of (S)-methyl 2-(tert-butoxycarbonylamino)-3-(3-(triisopropylsilyloxy)phenyl)-propanoate To a mixture of (S)-methyl 2-(tert-butoxycarbonylamino)-3-(3-hydroxyphenyl)propanoate (10.0 g, 33.9 mmol) in DCM (100 mL) was added imidazole (4.6 g, 67.8 mmol) and TIPSCl (7.8 g, 40.7 mmol). The mixture was stirred overnight at room temperature, then diluted with DCM (200 mL) and washed with HO (3 x 150 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the product (15.0 g, 98% yield) as an oil. LCMS (ESI) m / z [M+Na]C 24 H 41 Calculated for NO5SiNa: 474.22; Found: 474.2.

[0466] Step 2: Synthesis of (S)-methyl 2-(tert-butoxycarbonylamino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(triisopropylsilyloxy)phenyl)-propanoate A mixture of (S)-methyl 2-(tert-butoxycarbonylamino)-3-(3-(triisopropylsilyloxy)phenyl)-propanoate (7.5 g, 16.6 mmol), PinB2 (6.3 g, 24.9 mmol), [Ir(OMe)(COD)]2 (1.1 g, 1.7 mmol), and 4-tert-butyl-2-(4-tert-butyl-2-pyridyl)pyridine (1.3 g, 5.0 mmol) was purged with Ar, and then THF (75 mL) was added. The mixture was placed under an atmosphere of Ar and sealed. The mixture was heated to 80 °C and stirred for 16 h. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (7.5 g, 78% yield) as a solid. LCMS (ESI) m / z [M+Na]C 30 H 52 Calculated for BNO7SiNa: 600.35; Found: 600.4.

[0467] Step 3: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((triisopropylsilyl)oxy)phenyl)propanoic acid At 0° C., to a mixture of triisopropylsilyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((triisopropylsilyl)oxy)phenyl)propanoate (4.95 g, 6.9 mmol) in MeOH (53 mL) was added a solution of LiOH (840 mg, 34.4 mmol) in water (35 mL). The mixture was stirred at 0° C. for 2 h, then acidified with 1 M HCl to pH ∼5 and extracted with EtOAc (2 × 250 mL). The combined organic layers were washed with brine (3 × 100 mL), dried over anhydrous NaSO, filtered, and the filtrate was concentrated under reduced pressure to give the product (3.7 g, 95% yield), which was used directly in the next step without further purification. LCMS (ESI) m / z [M+NH]C 29 H 50 Calculated for BNO7SiNH4: 581.38; Found: 581.4.

[0468] Step 4: Synthesis of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((triisopropylsilyl)oxy)phenyl)propanoyl)hexahydropyridazine-3-carboxylate To a mixture of methyl (S)-hexahydropyridazine-3-carboxylate (6.48 g, 45.0 mmol) in DCM (200 mL) at 0° C. was added NMM (41.0 g, 405 mmol), a solution of (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-((triisopropylsilyl)oxy)phenyl)propanoic acid (24 g, 42.6 mmol) in DCM (50 mL), followed by HOBt (1.21 g, 9.0 mmol), and EDCI HCl salt (12.9 g, 67.6 mmol). The mixture was allowed to warm to room temperature and stirred for 16 h, then diluted with DCM (200 mL) and washed with HO (3 × 150 mL). The organic layer was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (22, 71% yield) as an oil. LCMS (ESI) m / z [M+H]C 35 H 60 Calculated for BN3O8Si: 690.42; Found: 690.5.

[0469] Intermediate 4. tert-Butyl ((6 3 S,4S)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-2 5 -((triisopropylsilyl)oxy)-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate

[0470] [ka]

[0471] Step 1: Synthesis of methyl (3S)-1-[(2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-[1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-5-yl]-5-[(triisopropylsilyl)oxy]phenyl]propanoyl]-1,2-diazinane-3-carboxylate 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylpropan-1-ol (30 g, 67 mmol) and methyl (3S)-1-[(2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborola To a stirred mixture of [(triisopropylsilyl)oxy]phenyl]propanoyl]-1,2-diazinan-3-carboxylate (55.8 g, 80.8 mmol) in dioxane (750 mL), Na2CO3 (17.9 g, 168.4 mmol), Pd(DtBPF)Cl2 (4.39 g, 6.7 mmol), and HO (150 mL) were added in small portions. The mixture was heated to 85 °C and stirred for 3 h, cooled, diluted with HO (2 L), and extracted with EtOAc (3 × 1 L). The combined organic layers were washed with brine (2 × 500 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (50 g, 72% yield) as a solid. LCMS (ESI) m / z [M+H]C 52 H 77 Calculated for N5O8Si: 928.56; Found: 928.8.

[0472] Step 2: Synthesis of (3S)-1-[(2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-[1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-5-yl]-5-[(triisopropylsilyl)oxy]phenyl]propanoyl]-1,2-diazinane-3-carboxylic acid To a stirred mixture of methyl (3S)-1-[(2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-[1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-5-yl]-5-[(triisopropylsilyl)oxy]phenyl]propanoyl]-1,2-diazinan-3-carboxylate (50 g, 54 mmol) and DCE (500 mL) was added trimethyltin hydroxide (48.7 g, 269 mmol) in small portions at room temperature. The mixture was heated to 65 °C and stirred for 16 h, then filtered, and the filter cake was washed with DCM (3 × 150 mL). The filtrate was concentrated under reduced pressure to give the product (70 g, crude), which was used directly in the next step without further purification. LCMS(ESI) m / z[M+H]C 51 H 75 Calculated for N5O8Si: 914.55; Found: 914.6.

[0473] Step 3: tert-Butyl ((6 3 S,4S)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-2 5 -((triisopropylsilyl)oxy)-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate To a stirred mixture of (3S)-1-[(2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-[1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-5-yl]-5-[(triisopropylsilyl)oxy]phenyl]propanoyl]-1,2-diazinan-3-carboxylic acid (70 g) in DCM (5 L) was added DIPEA (297 g, 2.3 mol), HOBT (51.7 g, 383 mmol), and EDCI (411 g, 2.1 mol) in small portions under an atmosphere of N at 0° C. The mixture was allowed to warm to room temperature and stirred for 16 h, then diluted with DCM (1 L), washed with brine (3 × 1 L), dried over anhydrous NaSO, and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give the product (36 g, 42% yield) as a solid. LCMS (ESI) m / z [M+H]C 51 H 73 Calculated for N5O7Si: 896.54; Found: 896.5.

[0474] Intermediate 5. (6 3 S,4S)-4-amino-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphane-5,7-dione

[0475] [ka]

[0476] Step 1: Synthesis of methyl (S)-3-(3-bromophenyl)-2-((tert-butoxycarbonyl)amino)propanoate To a solution of (2S)-3-(3-bromophenyl)-2-[(tert-butoxycarbonyl)amino]propanoic acid (100 g, 290 mmol) in DMF (1 L) at room temperature, NaHCO (48.8 g, 581.1 mmol) and MeI (61.9 g, 435.8 mmol) were added. The reaction mixture was stirred for 16 h, then quenched with HO (1 L) and extracted with EtOAc (3 × 1 L). The combined organic layers were washed with brine (3 × 500 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (13% EtOAc / petroleum ether) to give the final product (109 g, crude). LCMS (ESI) m / z [M+Na]C 15 H 20 Calculated for BrNO4 380.05; Found: 380.0.

[0477] Step 2: Synthesis of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate To a stirred solution of methyl (2S)-3-(3-bromophenyl)-2-[(tert-butoxycarbonyl)amino]propanoate (108 g, 301.5 mmol) and bis(pinacolato)diboron (99.53 g, 391.93 mmol) in dioxane (3.2 L) was added KOAc (73.97 g, 753.70 mmol) and Pd(dppf)Cl (22.06 g, 30.15 mmol). The reaction mixture was heated to 90 °C for 3 h, cooled to room temperature, and extracted with EtOAc (2 × 3 L). The combined organic layers were washed with brine (3 × 800 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% EtOAc / petroleum ether) to give the product (96 g, 78.6% yield). LCMS(ESI) m / z[M+Na]C 21 H 32 Calculated value for BNO6: 428.22; Found: 428.1.

[0478] Step 3: Synthesis of methyl (S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate To a mixture of methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propanoate (94 g, 231.9 mmol) and 3-(5-bromo-1H-indol-3-yl)-2,2-dimethylpropyl acetate (75.19 g, 231.93 mmol) in dioxane (1.5 L) and HO (300 mL) was added KCO (64.11 g, 463.85 mmol) and Pd(DtBPF)Cl (15.12 g, 23.19 mmol). The reaction mixture was heated to 70 °C and stirred for 4 h. The reaction mixture was extracted with EtOAc (2 x 2 L), and the combined organic layers were washed with brine (3 x 600 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20% EtOAc / petroleum ether) to give the product (130 g, crude). LCMS (ESI) m / z: [M+H]C 30 H 38 Calculated for N2O6: 523.28; Found 523.1.

[0479] Step 4: Synthesis of methyl (S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate To a solution of methyl (2S)-3-(3-[3-[3-(acetoxy)-2,2-dimethylpropyl]-1H-indol-5-yl]phenyl)-2-[(tert-butoxycarbonyl)amino]propanoate (95.0 g, 181.8 mmol) and iodine (36.91 g, 145.41 mmol) in THF (1 L) was added AgOTf (70.0 g, 272.7 mmol) and NaHCO (22.9 g, 272.65 mmol) at −10° C. The reaction mixture was stirred for 30 min and then quenched by the addition of saturated NaSO (100 mL) at 0° C. The resulting mixture was extracted with EtOAc (3 × 1 L), and the combined organic layers were washed with brine (3 × 500 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give methyl (S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate (49.3 g, 41.8% yield). LCMS (ESI) m / z [M+H]C 30 H 37 Calculated for IN2O6: 649.18; Found: 649.1.

[0480] Step 5: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)propanoic acid To a solution of methyl (2S)-3-(3-[3-[3-(acetyloxy)-2,2-dimethylpropyl]-2-iodo-1H-indol-5-yl]phenyl)-2-[(tert-butoxycarbonyl)amino]propanoate (60 g, 92.5 mmol) in THF (600 mL) was added a solution of LiOH·HO (19.41 g, 462.5 mmol) in water (460 mL). The resulting solution was stirred overnight, and then the pH was adjusted to 6 with HCl (1 M). The resulting solution was extracted with EtOAc (2 × 500 mL), and the combined organic layers were washed with brine (2 × 500 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give the product (45 g, 82.1% yield). LCMS (ESI) m / z [M+Na]C 27 H 33 Calculated for IN2O6 615.13; Found: 615.1.

[0481] Step 6: Synthesis of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylate To a solution of (2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-[3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl]phenyl]propanoic acid (30 g, 50.6 mmol) and methyl (3S)-1,2-diazinane-3-carboxylate (10.9 g, 75.9 mmol) in DCM (400 mL) was added NMM (40.97 g, 405.08 mmol), HOBt (2.05 g, 15.19 mmol), and EDCI (19.41 g, 101.27 mmol). After stirring the reaction mixture overnight, the mixture was washed with saturated aqueous NH4Cl (2 x 200 mL) and brine (2 x 200 mL), the mixture was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the desired product (14 g, 38.5% yield). LCMS (ESI) m / z: [M+H]C 33 H 43 Calculated for IN4O6: 718.23; Found 719.4.

[0482] Step 7: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylic acid To a solution of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylate (92 g, 128.0 mmol) in THF (920 mL) was added a solution of LiOH·HO (26.86 g, 640.10 mmol) in water (640 mL) at 0 °C. The reaction mixture was stirred for 2 h and then concentrated under reduced pressure to give the product (90 g, crude). LCMS (ESI) m / z: [M+H]C 32 H 41 Calculated for IN4O6: 705.22; Found 705.1.

[0483] Step 8: tert-Butyl ((6 3 S,4S)-1 2 -Iodo-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate To a solution of (3S)-1-[(2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-[3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl]phenyl]propanoyl]-1,2-diazinan-3-carboxylic acid (90 g, 127.73 mmol) in DCM (10 L) was added HOBt (34.52 g, 255.46 mmol), DIPEA (330.17 g, 2554.62 mmol), and EDCI (367.29 g, 1915.96 mmol) at 0 °C. The reaction mixture was stirred for 16 h and then concentrated under reduced pressure. The mixture was extracted with DCM (2 × 2 L), and the combined organic layers were washed with brine (3 × 1 L), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give the product (70 g, 79.8% yield). LCMS (ESI) m / z: [M+H]C 32 H 39 Calculated for IN4O5: 687.21; Found 687.1.

[0484] Step 9: tert-Butyl ((6 3 S,4S)-10,10-dimethyl-5,7-dioxo-12-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate In a 1 L round bottom flask at room temperature, tert-butyl ((6 3 S,4S)-1 2 -Iodo-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate (22.0 g, 32.042 mmol), toluene (300.0 mL), Pd2(dba)3 (3.52 g, 3.845 mmol), S-Phos (3.95 g, 9.613 mmol), and KOAc (9.43 g, 96.127 mmol) were charged. 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (26.66 g, 208.275 mmol) was added dropwise to the mixture at room temperature with stirring. The resulting solution was stirred for 3 hours at 60 °C. The resulting mixture was filtered, and the filter cake was washed with EtOAc. The filtrate was concentrated under reduced pressure and the remaining residue was purified by silica gel column chromatography to give the product (22 g, 90% yield) as a solid. LCMS (ESI) m / z: [M+H]C 38 H 51 Calculated for BN4O7: 687.3; found 687.4.

[0485] Step 10: tert-Butyl ((6 3 S,4S)-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate tert-Butyl ((6 3 S,4S)-10,10-dimethyl-5,7-dioxo-1 2 -(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1A mixture of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate (2.0 g, 2.8 mmol), 3-bromo-2-[(1S)-1-methoxyethyl]pyridine (0.60 g, 2.8 mmol), Pd(dppf)Cl (0.39 g, 0.5 mmol), and KPO (1.2 g, 6.0 mmol) in dioxane (50 mL) and HO (10 mL) was heated to 70 °C under an atmosphere of N and stirred for 2 h. The mixture was diluted with HO (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (3 × 50 mL), dried over anhydrous NaSO, and filtered. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the desired product (1.5 g, 74% yield). LCMS (ESI) m / z: [M+H]C 40 H 49 Calculated for N5O6: 695.4; Found 696.5.

[0486] Step 11: tert-Butyl ((6 3 S,4S)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate At 0°C, tert-butyl ((6 3 S,4S)-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-11 To a solution of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate (20 g, 28.7 mmol) and CsCO (18.7 g, 57.5 mmol) in DMF (150 mL) was added a solution of EtI (13.45 g, 86.22 mmol) in DMF (50 mL). The resulting mixture was stirred overnight at 35 °C and then diluted with HO (500 mL). The mixture was extracted with EtOAc (2 × 300 mL), and the combined organic layers were washed with brine (3 × 100 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the product (4.23 g, 18.8% yield) and the atropisomer (5.78 g, 25.7%) as solids. LCMS(ESI) m / z:[M+H]C 42 H 53 Calculated for N5O6: 724.4; Found 724.6.

[0487] Step 12:(6 3 S,4S)-4-amino-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphane-5,7-dione tert-Butyl ((6 3 S,4S)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1A mixture of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate (1.3 g, 1.7 mmol) with TFA (10 mL) and DCM (20 mL) was stirred at 0° C. for 2 hours. The mixture was concentrated under reduced pressure to give the product (1.30 g, crude) as a solid. LCMS (ESI) m / z: [M+H]C 37 H 45 Calculated for N5O4: 623.3; Found 624.4.

[0488] Intermediate 6. (6 3 S,4S)-4-amino-1 1 -ethyl-2 5 -(fluoromethyl)-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphane-5,7-dione

[0489] [ka]

[0490] Step 1: Synthesis of 1-bromo-3-(fluoromethyl)-5-iodobenzene BAST (247.45 g, 1118.45 mmol) was added dropwise to a solution of (3-bromo-5-iodophenyl)methanol (175.0 g, 559.23 mmol) in DCM (2 L) at 0 °C. The resulting mixture was stirred at room temperature for 16 h. The reaction was quenched with saturated aqueous NaHCO3. The organic layer was washed with H2O (3 × 700 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (3% EtOAc / petroleum ether) to give the desired product (120 g, 68% yield).

[0491] Step 2: Synthesis of methyl (2S)-3-[3-bromo-5-(fluoromethyl)phenyl]-2-[(tert-butoxycarbonyl)amino]propanoate To a 1000 mL three-necked round-bottom flask were added Zn powder (32.40 g, 495.358 mmol) in DMF (350.0 mL) and I2 (967.12 mg, 3.810 mmol). To the mixture was added a solution of methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-iodopropanoate (27.0 g, 82.03 mmol) in DMF (10 mL). The mixture was heated to 30 °C for 10 minutes. Next, to the mixture was added a solution of methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-iodopropanoate (54.0 g, 164.07 mmol) in DMF (20 mL). The resulting mixture was stirred at room temperature for 30 minutes and filtered. A mixture of 1-bromo-3-(fluoromethyl)-5-iodobenzene (60 g, 190.522 mmol), tris(furan-2-yl)phosphane (2.65 g, 11.431 mmol), and Pd2(dba)3 (3.49 g, 3.810 mmol) in DMF (400 mL) was added to the resulting solution at room temperature under an argon atmosphere. The reaction mixture was heated to 60 °C for 10 min, and then the oil bath was removed. The resulting mixture was stirred for approximately 1 h until the temperature cooled to 50 °C. The reaction was quenched with saturated aqueous NH4Cl (3000 mL), and the resulting mixture was extracted with EtOAc (3 × 1000 mL). The combined organic layers were washed with brine (2 × 1000 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (9% EtOAc / petroleum ether) to give the desired product (45 g, 60% yield).

[0492] Step 3: Synthesis of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-5-(fluoromethyl)phenyl)propanoate A mixture of methyl (2S)-3-[3-bromo-5-(fluoromethyl)phenyl]-2-[(tert-butoxycarbonyl)amino]propanoate (75.28 g, 192.905 mmol), (S)-3-(1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol (95 g, 192.905 mmol), Pd(dppf)Cl (14.11 g, 19.291 mmol), and KCO (53.32 g, 385.810 mmol) in dioxane (900 mL) and HO (180 mL) was stirred at 80 °C for 2 h. The resulting mixture was concentrated under reduced pressure and then diluted with H2O. The resulting mixture was extracted with EtOAc (3 x 1200 mL), and the combined organic layers were washed with H2O (3 x 500 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give the desired product (105 g, 80% yield). LCMS (ESI) m / z [M+H]C 39 H 50 Calculated for FN3O6: 676.38; Found: 676.1.

[0493] Step 4: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-5-(fluoromethyl)phenyl)propanoic acid To a stirred solution of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-5-(fluoromethyl)phenyl)propanoate (108 g, 159.801 mmol) in THF (500 mL) was added a solution of LiOH·HO (11.48 g, 479.403 mmol) in water (500 mL) at 0 °C. The resulting mixture was stirred for 2 h at 0 °C and then acidified to pH 6 with 1 M HCl (aq). The mixture was extracted with EtOAc (3 × 800 mL), and the combined organic layers were washed with brine (2 × 200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure to give the desired product (101 g, crude). LCMS (ESI) m / z [M+H]C 38 H 48 Calculated for FN3O6: 662.36; Found: 662.1.

[0494] Step 5: Synthesis of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-5-(fluoroethyl)phenyl)propanoyl)hexahydropyridazine-3-carboxylate To a stirred solution of (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-5-(fluoromethyl)phenyl)propanoic acid (103 g, 155.633 mmol) and NMM (157.42 g, 1556.330 mmol) in DCM (1200 mL) at 0° C., methyl (3S)-1,2-diazinane-3-carboxylate (33.66 g, 233.449 mmol), HOBt (10.51 g, 77.816 mmol), and EDCI (59.67 g, 311.265 mmol) were added in small portions. The resulting mixture was stirred at room temperature for 16 hours. The organic layer was then washed with 0.5 M HCl (2 x 1000 mL) and brine (2 x 800 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give the desired product (103 g, 83% yield). LCMS (ESI) m / z [M+H]C 44 H 58 Calculated for FN5O7: 788.44; Found: 788.1.

[0495] Step 6: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-5-(fluoromethyl)phenyl)propanoyl)hexahydropyridazine-3-carboxylic acid To a stirred solution of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-5-(fluoroethyl)phenyl)propanoyl)hexahydropyridazine-3-carboxylate (103 g, 130.715 mmol) in THF (700 mL) was added a solution of LiOH·HO (27.43 g, 653.575 mmol) in water (700 mL) at 0° C. The resulting mixture was stirred for 2 h at 0° C. and then neutralized to pH 6 with 1 M HCl. The resulting mixture was extracted with EtOAc (3 x 800 mL) and the combined organic layers were washed with brine (2 x 600 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the desired product (101 g, crude). LCMS (ESI) m / z [M+H]C 43 H 56 Calculated for FN5O7: 774.43; Found: 774.1.

[0496] Step 7: tert-Butyl ((6 3 S,4S)-1 1 -ethyl-2 5 -(fluoromethyl)-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate To a stirred solution of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-5-(fluoromethyl)phenyl)propanoyl)hexahydropyridazine-3-carboxylic acid (101 g, 130.50 mmol) in DCM (5500 mL) was added DIPEA (227.31 mL, 1305.0 mmol), HOBt (88.17 g, 652.499 mmol), and EDCI (375.26 g, 1957.498 mmol) at 0° C. The resulting mixture was stirred at room temperature overnight. The mixture was then washed with 0.5 M HCl (2 x 2000 mL), brine (2 x 2000 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give the desired product (68 g, 65% yield). LCMS (ESI) m / z [M+H]C 43 H 54 Calculated for FN5O6: 756.42; Found: 756.4.

[0497] Step 8:(2S)-N-((6 3 S,4S)-1 1 -ethyl-2 5 -fluoromethyl)-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)-3-methyl-2-(methylamino)butanamide At 0°C, tert-butyl ((6 3 S,4S)-1 1 -ethyl-2 5 -(fluoromethyl)-1 2-(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 To a stirred solution of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate (350 mg, 0.403 mmol) in DCM (4 mL) was added TFA (1.5 mL). The resulting mixture was stirred at room temperature for 1.5 hours and then concentrated under reduced pressure to give the desired product (600 mg, crude). LCMS (ESI) m / z [M+H]C 38 H 46 Calculated for FN5O4: 656.36; Found: 656.4.

[0498] Intermediate 7. (6 3 S,4S)-4-amino-2 5 -(difluoromethyl)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphane-5,7-dione

[0499] [ka]

[0500] Step 1: Synthesis of methyl (S)-3-(3-bromo-5-(difluoromethyl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate To a 1000 mL three-necked round-bottom flask, Zn powder (43.42 g, 663.835 mmol) and I2 (1.30 g, 5.106 mmol) in DMF (400 mL) were added at room temperature. To the above mixture, a solution of methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-iodopropanoate (36.42 g, 110.64 mmol) in DMF (10 mL) was added. The mixture was heated to 30 °C for 10 minutes. Next, to the mixture at room temperature, a solution of methyl (2R)-2-[(tert-butoxycarbonyl)amino]-3-iodopropanoate (72.83 g, 221.28 mmol) in DMF (20 mL) was added dropwise. The resulting mixture was stirred for 30 minutes. The mixture was filtered, and the solution was added to a mixture of 1-bromo-3-(difluoromethyl)-5-iodobenzene (85.0 g, 255.321 mmol), tris(furan-2-yl)phosphate (3.56 g, 15.319 mmol), and Pd(dba) (4.68 g, 5.106 mmol) in DMF (400 mL) at room temperature under an argon atmosphere. The reaction mixture was heated to 60 °C for 10 minutes, then removed from the oil bath, and stirred for 1 hour until the temperature of the resulting mixture cooled to 50 °C. The reaction was quenched with saturated aqueous NHCl (3000 mL), and the aqueous layer was extracted with EtOAc (3 × 1000 mL). The combined organic layers were washed with brine (2 × 1000 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (9% EtOAc / petroleum ether) to give the desired product (59 g, 56.6% yield).

[0501] Step 2: Synthesis of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(difluoromethyl)-5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)propanoate Methyl (2S)-3-[3-bromo-5-(difluoromethyl)phenyl]-2-[(tert-butoxycarbonyl)amino]propanoate (90.0 g, 220.459 mmol), (S)-3-(1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1 A mixture of H-indol-3-yl)-2,2-dimethylpropan-1-ol (1.50 g, 3.046 mmol), Pd(dppf)Cl (16.13 g, 22.046 mmol), and KPO (116.99 g, 551.148 mmol) in dioxane (600 mL), HO (200 mL), and toluene (200 mL) was stirred at 70 °C for 2 h. The resulting mixture was concentrated under reduced pressure and then diluted with HO (300 mL). The mixture was extracted with EtOAc (3 × 500 mL). The combined organic layers were washed with HO (3 × 500 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give the desired product (128 g, 83.7% yield). LCMS(ESI) m / z[M+H]C 39 H 49 Calculated for F2N3O6: 694.37; Found: 694.2.

[0502] Step 3: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(difluoromethyl)-5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)propanoic acid To a stirred solution of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(difluoromethyl)-5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)propanoate (125.0 g, 180.159 mmol) in THF (800 mL) was added dropwise a solution of LiOH·HO (11.48 g, 479.403 mmol) in water (200 mL) at 0° C. The resulting mixture was stirred for 2 h at 0° C. The mixture was acidified to pH 6 with 1 M HCl (aq) and then extracted with EtOAc (3 × 800 mL). The combined organic layers were washed with brine (2 × 200 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure to give the desired product (125 g, crude). LCMS (ESI) m / z [M+H]C 38 H 47 Calculated for F2N3O6: 680.37; Found: 680.2.

[0503] Step 4: Synthesis of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(difluoromethyl)-5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylate To a stirred solution of methyl (3S)-1,2-diazinan-3-carboxylate (39.77 g, 275.814 mmol) and NMM (185.98 g, 1838.760 mmol) in DCM (1500 mL) at 0° C. was added (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(difluoromethyl)-5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)propanoic acid (125.0 g, 183.876 mmol), HOBt (12.42 g, 91.938 mmol), and EDCI (70.50 g, 367.752 mmol) in small portions. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was then washed with 0.5 M HCl (2 x 1000 mL) and brine (2 x 800 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give the desired product (110 g, 74.2% yield). LCMS (ESI) m / z [M+H]C 44 H 57 Calculated for F2N5O7: 806.43; Found: 806.2.

[0504] Step 5: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(difluoromethyl)-5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylic acid To a stirred solution of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(difluoromethyl)-5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylate (110.0 g, 136.482 mmol) in THF (800 mL) was added a solution of LiOH·HO (17.18 g, 409.446 mmol) in water (200 mL) in small portions at 0 °C. The resulting mixture was stirred for 2 h at 0 °C and then neutralized to pH 6 with 0.5 M HCl. The resulting mixture was extracted with EtOAc (3 x 800 mL) and the combined organic layers were washed with brine (2 x 600 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the desired product (100 g, crude). LCMS (ESI) m / z [M+H]C 43 H 55 Calculated for F2N5O7: 792.42; Found: 792.4.

[0505] Step 6: tert-Butyl ((6 3 S,4S)-2 5 -(difluoromethyl)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate To a stirred solution of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(difluoromethyl)-5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylic acid (100.0 g, 126.273 mmol) in DCM (6000 mL) was added DIPEA (163.20 g, 1262.730 mmol), HOBt (85.31 g, 631.365 mmol), and EDCI (363.10 g, 1894.095 mmol) dropwise at 0° C. The resulting mixture was stirred at room temperature overnight. The mixture was then washed with 0.5 M HCl (2 x 2000 mL) and brine (2 x 2000 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give the desired product (70 g, 71.6% yield). LCMS (ESI) m / z [M+H]C 43 H 53 Calculated for F2N5O6: 774.41; Found: 774.0.

[0506] Step 7:(6 3 S,4S)-4-amino-2 5 -(difluoromethyl)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphane-5,7-dione At 0°C, tert-butyl ((6 3 S,4S)-2 5 -(difluoromethyl)-1 1 -ethyl-1 2-(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 To a stirred solution of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate (202.0 mg, 0.261 mmol) in DCM (2 mL) was added TFA (1.0 mL) dropwise. The resulting mixture was stirred for 1.5 hours at 0°C and then concentrated under reduced pressure to give the desired product. LCMS (ESI) m / z [M+H]C 38 H 45 Calculated for F2N5O4: 674.35; Found: 674.5.

[0507] Intermediate 8. tert-Butyl ((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate

[0508] [ka]

[0509] Step 1: Synthesis of (S)-3-(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid To a solution of methyl (2S)-3-(4-bromo-1,3-thiazol-2-yl)-2-[(tert-butoxycarbonyl)amino]propanoate (110 g, 301.2 mmol) in THF (500 mL) and HO (200 mL) at room temperature was added LiOH (21.64 g, 903.6 mmol). The resulting solution was stirred for 1 hour and then concentrated under reduced pressure. The resulting residue was adjusted to pH 6 with 1 M HCl and extracted with DCM (3 x 500 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give the desired product (108 g, crude). LCMS (ESI) m / z [M+H]C 11 H 15 Calculated for BrN2O4S: 351.00; Found: 351.0.

[0510] Step 2: Synthesis of methyl (S)-1-((S)-3-(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate To a solution of (S)-3-(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid (70 g, 199.3 mmol) in DCM (500 mL) at 0 °C, methyl (3S)-1,2-diazinan-3-carboxylate bis(trifluoroacetate) salt (111.28 g, 298.96 mmol), NMM (219.12 mL, 1993.0 mmol), EDCI (76.41 g, 398.6 mmol), and HOBt (5.39 g, 39.89 mmol) were added. The resulting solution was warmed to room temperature and stirred for 1 h. The reaction was then quenched with HO (500 mL) and extracted with EtOAc (3 × 500 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0->50% EtOAc / petroleum ether) to give the desired product (88.1 g, 93% yield). LCMS (ESI) m / z [M+H]C 17 H 25 Calculated for BrN4O5S: 477.08; Found: 477.1.

[0511] Step 3: Synthesis of (S)-3-(1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol To a solution of 3-(5-bromo-1-ethyl-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol (60 g, 134.7 mmol) in toluene (500 mL) at room temperature, bis(pinacolato)diboron (51.31 g, 202.1 mmol), Pd(dppf)Cl (9.86 g, 13.48 mmol), and KOAc (26.44 g, 269.4 mmol) were added. The reaction mixture was then heated to 90 °C and stirred for 2 h. The reaction solution was then cooled to room temperature and concentrated under reduced pressure. Purification by silica gel column chromatography (0 → 50% EtOAc / petroleum ether) afforded the desired product (60.6 g, 94% yield). LCMS (ESI) m / z [M+H]C 29 H 41 Calculated for BN2O4: 493.32; Found: 493.3.

[0512] Step 4: Synthesis of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)propanoyl)hexahydropyridazine-3-carboxylate (S)-3-(1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol (30 g, 60.9 mmol) in toluene (600 mL), dioxane (200 mL), and HO (200 mL) at room temperature. To the solution was added methyl (S)-1-((S)-3-(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (43.62 g, 91.4 mmol), KPO (32.23 g, 152.3 mmol), and Pd(dppf)Cl (8.91 g, 12.18 mmol). The resulting solution was heated to 70 °C and stirred overnight. The reaction mixture was then cooled to room temperature and quenched with HO (200 mL). The resulting mixture was extracted with EtOAc (3 × 1000 mL), and the combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0 → 90% EtOAc / petroleum ether) to give the desired product (39.7 g, 85% yield). LCMS(ESI) m / z[M+H]C 40 H 54 Calculated for N6O7S: 763.39; Found: 763.3.

[0513] Step 5: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid To a solution of methyl (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)propanoyl)hexahydropyridazine-3-carboxylate (39.7 g, 52.0 mmol) in THF (400 mL) and HO (100 mL) was added LiOH·HO (3.74 g, 156.2 mmol) at room temperature. The resulting mixture was stirred for 1.5 h and then concentrated under reduced pressure. The residue was acidified to pH 6 with 1 M HCl and extracted with DCM (3 × 1000 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give the desired product (37.9 g, crude). LCMS (ESI) m / z [M+H]C 39 H 52 Calculated for N6O7S: 749.37; Found: 749.4.

[0514] Step 6: tert-Butyl ((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate To a solution of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid (37.9 g, 50.6 mmol), HOBt (34.19 g, 253.0 mmol), and DIPEA (264.4 mL, 1518 mmol) in DCM (4 L) was added EDCI (271.63 g, 1416.9 mmol) at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was then quenched with HO and washed with 1 M HCl (4 × 1 L). The organic layer was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-70% EtOAc / petroleum ether) to give the desired product (30 g, 81% yield). LCMS (ESI) m / z [M+H]C 39 H 50 Calculated for N6O6S: 731.36; Found: 731.3.

[0515] Intermediate 9. (6 3 S)-4-amino-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-2 1 ,2 2 ,2 3 ,2 6 ,6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Decahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacyclodecaphane-5,7-dione

[0516] [ka]

[0517] Step 1: Synthesis of methyl 2-((tert-butoxycarbonyl)amino)acrylate To a solution of methyl (tert-butoxycarbonyl)-L-serinate (10 g, 45 mmol) in anhydrous MeCN (150 mL) was added DIPEA (17 g, 137 mmol). The reaction mixture was stirred at 45° C. for 2 hours, after which the product was obtained in solution. LCMS (ESI): m / z [M+Na]C9H 15 Calculated value for NO4 201.1; measured value 224.1.

[0518] Step 2: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)acrylate To a solution of methyl 2-((tert-butoxycarbonyl)amino)acrylate (12 g, 60 mmol) in anhydrous MeCN (150 mL) was added DMAP (13 g, 90 mmol) and (Boc)O (26 g, 120 mmol) at 0 °C. The reaction was stirred for 6 h, then quenched with HO (100 mL) and extracted with DCM (3 × 200 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the product (12.5 g, 65% yield) as a solid. LCMS (ESI): m / z [M+Na]C 14 H 23 Calculated value for NO6: 301.2; measured value: 324.1.

[0519] Step 3: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)-3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)propanoate To a mixture of 5-bromo-1,2,3,6-tetrahydropyridine (8.0 g, 49 mmol) and MeOH (120 mL) under an Ar atmosphere, methyl 2-{bis[(tert-butoxy)carbonyl]amino}prop-2-enoate (22 g, 74 mmol) was added. After stirring for 16 hours, the mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (12 g, 47% yield) as an oil. LCMS (ESI) m / z: [M+H]C19 H 31 Calculated for BrN2O6: 462.1; found 463.1.

[0520] Step 4: Synthesis of 3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid To a mixture of methyl 2-(bis(tert-butoxycarbonyl)amino)-3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)propanoate (14 g, 30 mmol) in dioxane (30 mL) and HO (12 mL) was added LiOH (3.6 g, 151 mmol). The mixture was heated to 35 °C and stirred for 12 h, after which 1 M HCl was added to adjust the pH to approximately 3-4. The mixture was extracted with DCM (2 × 300 mL), and the combined organic layers were dried over anhydrous NaSO and filtered. The filtrate was concentrated under reduced pressure to give the product (10 g, 85% yield) as a solid. LCMS (ESI) m / z [M+H]C 13 H 21 Calculated for BrN2O4: 348.1; Found: 349.0.

[0521] Step 5: Synthesis of methyl (3S)-1-(3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate To a mixture of 3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid (10 g, 30 mmol), DIPEA (12 g, 93 mmol), and methyl (3S)-1,2-diazinan-3-carboxylate (5.4 g, 37 mmol) in DMF (100 mL) was added HATU (13 g, 34 mmol) under an atmosphere of Ar at 0 °C. After the mixture was stirred at 0 °C for 2 h, HO was added and the mixture was extracted with EtOAc (2 × 300 mL). The combined organic layers were dried over anhydrous NaSO and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase chromatography to give the product (9.0 g, 55% yield) as a solid. LCMS (ESI) m / z [M+H]C 31 Calculated for BrN4O5 474.1; Found: 475.1.

[0522] Step 6: Synthesis of methyl (3S)-1-(2-((tert-butoxycarbonyl)amino)-3-(5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-3,6-dihydropyridin-1(2H)-yl)propanoyl)hexahydropyridazine-3-carboxylate Under an Ar atmosphere, methyl (3S)-1-(3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (9.0 g, 18 mmol), K2CO3 (4.5 g, 32 mmol), Pd(dppf)Cl2·DCM (1.4 g, 2 mmol), 3-( A mixture of 1-ethyl-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indol-3-yl)-2,2-dimethylpropan-1-ol (9.8 g, 20 mmol) in dioxane (90 mL) and HO (10 mL) was heated to 75 °C and stirred for 2 h. HO was added, and the mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were dried over NaSO, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 5-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-2,2-dimethyl-5-oxopentanoic acid (4.0 g, 25% yield) as a solid. LCMS (ESI) m / z [M+H]C 42 H 60 Calculated for N6O7: 760.5; Found: 761.4.

[0523] Step 7: Synthesis of (3S)-1-(2-((tert-butoxycarbonyl)amino)-3-(5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-3,6-dihydropyridin-1(2H)-yl)propanoyl)hexahydropyridazine-3-carboxylic acid To a mixture of methyl (3S)-1-(2-((tert-butoxycarbonyl)amino)-3-(5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-3,6-dihydropyridin-1(2H)-yl)propanoyl)hexahydropyridazine-3-carboxylate (4.1 g, 5.0 mmol) and THF (35 mL) was added LiOH (0.60 g, 27 mmol) at 0 °C. After the mixture was stirred at 0 °C for 1.5 h, 1 M HCl was added to adjust the pH to approximately 6–7, and the mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were dried over NaSO and filtered, and the filtrate was concentrated under reduced pressure to give the product (3.6 g, 80% yield) as a solid. LCMS(ESI) m / z [M+H]C 41 H 58 Calculated for N6O7: 746.4; Found: 747.4.

[0524] Step 8: tert-Butyl ((6 3 S)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-2 1 ,2 2 ,2 3 ,2 6 ,6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Decahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacyclodecaphan-4-yl)carbamate To a mixture of (3S)-1-(2-((tert-butoxycarbonyl)amino)-3-(5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)-3,6-dihydropyridin-1(2H)-yl)propanoyl)hexahydropyridazine-3-carboxylic acid (3.6 g, 5.0 mmol) and DIPEA (24 g, 190 mmol) in DCM (700 mL) was added EDCI·HCl (28 g, 140 mmol) and HOBt (6.5 g, 50 mmol) under an Ar atmosphere. The mixture was heated to 30°C and stirred at 30°C for 16 hours, then concentrated under reduced pressure. The residue was diluted with EtOAc (200 mL) and washed with HO (2 x 200 mL), brine (200 mL), dried over NaSO, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (1.45 g, 40% yield) as a solid. LCMS (ESI) m / z: [M+H]C 41 H 56 Calculated for N6O6: 728.4; Found 729.4.

[0525] Step 9:(6 3 S)-4-amino-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-2 1 ,2 2 ,2 3 ,2 6 ,6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Decahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacyclodecaphane-5,7-dione At 0°C, tert-butyl ((6 3 S)-1 1 -ethyl-1 2-(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-2 1 ,2 2 ,2 3 ,2 6 ,6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Decahydro-1 1 To a mixture of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(5,1)-pyridinacyclodecaphan-4-yl)carbamate (130 mg, 0.20 mmol) in DCM (1.0 mL) was added TFA (0.3 mL). The mixture was warmed to room temperature and stirred for 2 hours, then concentrated under reduced pressure to give the product, which was used directly in the next step without further purification. LCMS (ESI) m / z [M+H]C 36 H 48 Calculated for N6O4 628.4; Found: 629.4.

[0526] Intermediate 10. (2 2 S,6 3 S,4S)-4-amino-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-morpholina-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-5,7-dione

[0527] [ka]

[0528] Step 1: Synthesis of tert-butyl (2R)-2-formylmorpholin-4-ylformate To a solution of tert-butyl (2R)-2-(hydroxymethyl)morpholin-4-ylformate (50 g, 230 mmol) in EtOAc (1 L) was added TEMPO (715 mg, 4.6 mmol) and NaHCO3 (58 g, 690 mmol) at room temperature. After the mixture was cooled to -50 °C, a solution of TCCA (56 g, 241 mmol) in EtOAc (100 mL) was added dropwise over 30 min. The reaction mixture was warmed to 5 °C for 2 h, then quenched with 10% Na2SO3 (200 mL) and stirred for 20 min. The resulting mixture was filtered, and the organic phase was separated. The aqueous phase was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with HO (100 mL) and brine (100 mL) and then dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure to give the product (50 g, crude) as an oil.

[0529] Step 2: Synthesis of tert-butyl (S,Z)-2-(2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxoprop-1-en-1-yl)morpholine-4-carboxylate To a solution of tert-butyl (2R)-2-formylmorpholin-4-ylformate (49 g, 153 mmol) and methyl 2-{[(benzyloxy)carbonyl]amino}-2-(dimethoxyphosphoryl)acetate (60 g, 183 mmol) in MeCN (300 mL) was added tetramethylguanidine (35 g, 306 mmol) at 0-10°C. The reaction mixture was stirred at 10°C for 30 minutes and then warmed to room temperature for 2 hours. The reaction mixture was diluted with DCM (200 mL) and washed with 10% citric acid (200 mL) and 10% aqueous NaHCO3 (200 mL). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography to give the product (36 g, 90% yield) as a solid. LCMS (ESI): m / z [M+Na]C 21 H 28 Calculated value for N2O4: 420.2; Measured value: 443.1.

[0530] Step 3: Synthesis of tert-butyl (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxopropyl)morpholine-4-carboxylate To a solution of tert-butyl (S,Z)-2-(2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxoprop-1-en-1-yl)morpholine-4-carboxylate (49 g, 0.12 mol) in MeOH (500 mL) was added (S,S)-Et-DUPHOS-Rh (500 mg, 0.7 mmol). The mixture was stirred under an atmosphere of H2 (60 psi) for 48 h at room temperature. The reaction was concentrated and purified by silica gel column chromatography to give the product (44 g, 90% yield) as a solid. LCMS (ESI): m / z [M+Na]C 21 H 30 Calculated for N2O7 422.2; Found 445.2.

[0531] Step 4: Synthesis of methyl (S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-morpholin-2-yl)propanoate To a stirred solution of tert-butyl (S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxopropyl)morpholine-4-carboxylate (2.2 g, 5.2 mmol) in EtOAc (2 mL) at 15° C. was added HCl / EtOAc (25 mL). The reaction was stirred at 15° C. for 2 h and then concentrated under reduced pressure to give the product (1.51 g, 90.4% yield) as an oil. LCMS (ESI) m / z [M+H]C 16 Calculated for H22N2O5: 322.1; Found: 323.2.

[0532] Step 5: Synthesis of (S)-5-bromo-3-(3-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpropyl)-1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indole To a solution of 3-(5-bromo-1-ethyl-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-3-yl)-2,2-dimethylpropan-1-ol (100 g, 0.22 mol) and imidazole (30.6 g, 0.45 mol) in DCM (800 mL) was added a solution of TBSCl (50.7 g, 0.34 mol) in DCM (200 mL) at 0 °C. The reaction was stirred at room temperature for 2 h. The resulting solution was washed with HO (3 × 300 mL) and brine (2 × 200 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the product (138 g, 90% yield) as a solid. LCMS (ESI) m / z: [M+H]C 29 H 43 Calculated for BrN2O2Si 558.2; found 559.2.

[0533] Step 6: Synthesis of methyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-[(2S)-4-(3-{3-[(tert-butyldimethylsilyl)oxy]-2,2-dimethylpropyl}-1-ethyl-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-5-yl)morpholin-2-yl]propanoate To a stirred solution of (S)-5-bromo-3-(3-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpropyl)-1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indole (50 g, 89.3 mmol) in dioxane (500 mL) was added methyl(2S)-2-{[(benzyloxy)carbonyl]amino}-3-[(2 S)-morpholin-2-yl]propanoate (31.7 g, 98.2 mmol), RuPhos (16.7 g, 35.7 mmol), di-μ-chlorobis(2-amino-1,1-biphenyl-2-yl-C,N)dipalladium(II) (2.8 g, 4.4 mmol), and cesium carbonate (96 g, 295 mmol) were added, followed by RuPhos-Pd-G2 (3.5 g, 4.4 mmol). The reaction mixture was stirred for 6 hours at 105 °C under a N2 atmosphere. The resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC chromatography to give the desired product (55 g, 73% yield) as a solid. LCMS (ESI) m / z: [M+H]C 45 H 64 Calculated for N4O7Si: 800.5; Found 801.5.

[0534] Step 7: Synthesis of (2S)-2-{[(benzyloxy)carbonyl]amino}-3-[(2S)-4-(3-{3-[(tert-butyldimethylsilyl)oxy]-2,2-dimethylpropyl}-1-ethyl-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-5-yl)morpholin-2-yl]propanoic acid To a solution of methyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-[(2S)-4-(3-{3-[(tert-butyldimethylsilyl)oxy]-2,2-dimethylpropyl}-1-ethyl-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-5-yl)morpholin-2-yl]propanoate (10 g, 12 mmol) in THF (270 mL) was added a solution of LiOH (1.3 g, 31 mmol) in water (45 mL) at room temperature. The reaction was stirred at room temperature for 2 h and then treated with 1N HCl at 0-5 °C to adjust the pH to 4-5. The resulting mixture was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine and dried over anhydrous NaSO. The organic phase was then concentrated under reduced pressure to give the product (9.5 g, 97% yield) as a solid. LCMS(ESI) m / z:[M+H]C 44 H 62 Calculated for N4O7Si: 786.4; Found 787.4.

[0535] Step 8: Synthesis of methyl (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(3-(3-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpropyl)-1-ethyl-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylate To a stirred solution of (2S)-2-{[(benzyloxy)carbonyl]amino}-3-[(2S)-4-(3-{3-[(tert-butyldimethylsilyl)oxy]-2,2-dimethylpropyl}-1-ethyl-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-5-yl)morpholin-2-yl]propanoic acid (10 g, 12.7 mmol) in DMF (150 mL) was added methyl (S)-hexahydropyridazine-3-carboxylate (2 g, 14 mmol), cooled to 0 °C, and added DIPEA (32.8 g, 254 mmol) followed by HATU (9.7 g, 25.4 mmol) at 0-5 °C. The reaction mixture was stirred at 0-5 °C for 1 h. The resulting mixture was diluted with EtOAc (500 mL) and HO (200 mL). The organic layer was separated, washed with HO (2 x 100 mL) and brine (100 mL), and dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product. LCMS (ESI) m / z: [M+H]C 50 H 72 Calculated value for N6O8Si: 912.5; Found: 913.4.

[0536] Step 9: Synthesis of methyl (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylate Methyl (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(3-(3-((tert-butyldimethylsilyl)oxy)-2,2-dimethylpropyl)-1-ethyl-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylate (8.5 g) at room temperature To a solution of (4, 9 mmol) in THF (8 mL) was added tetrabutylammonium fluoride (1 M in THF, 180 mL, 180 mmol) and AcOH (11 g, 200 mmol). The reaction mixture was stirred at 75 °C for 3 h. The resulting mixture was diluted with EtOAc (150 mL) and washed with HO (6 x 20 mL). The organic phase was concentrated under reduced pressure to give the product (7.4 g, 100% yield) as a solid. LCMS (ESI) m / z: [M+H]C 44 H 58 Calculated for N6O8 799.4; Found: 798.4.

[0537] Step 10: Synthesis of (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid To a solution of methyl (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylate (8 g, 10 mmol) in THF (200 mL) was added a solution of LiOH (600 mg, 25 mm) in water (30 mL). The reaction mixture was stirred at room temperature for 1 h, then treated with 1N HCl at 0-5 °C to adjust the pH to 4-5 and extracted with EtOAc (2 x 500 mL). The organic phase was washed with brine and concentrated under reduced pressure to give the product (8 g, crude) as a solid. LCMS (ESI) m / z [M+H]C 43 H 56 Calculated for N6O8 784.4; Found: 785.4.

[0538] Step 11: Benzyl ((2 2 S,6 3 S,4S)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis to obtain H-8-oxa-2(4,2)-morpholina-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate Under an argon atmosphere, EDCI (88 g, 458 mmol) and HOBt (27.6 g, 204 mmol) were added to a mixture of (S)-1-((S)-2-(((benzyloxy)carbonyl)amino)-3-((S)-4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)morpholin-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid (8 g, 10.2 mmol) and DIPEA (59 g, 459 mmol) in DCM (800 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product (5 g, 66% yield) as a solid. LCMS(ESI) m / z:[M+H]C 43 H 54 Calculated for N6O7 766.4; found 767.4.

[0539] Step 12:(2 2 S,6 3 S,4S)-4-amino-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-morpholina-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-5,7-dione Under H2 atmosphere at room temperature, benzyl ((2 2 S,63S,4S)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6-Hexahydro-1 1 To a solution of H-8-oxa-2(4,2)-morpholina-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate (400 mg, 0.5 mmol) in MeOH (20 mL) was added Pd / C (200 mg) and ammonium acetate (834 mg, 16 mmol), and the mixture was stirred for 2 h. The resulting mixture was filtered and concentrated under reduced pressure. The residue was redissolved in DCM (20 mL) and washed with HO (5 mL × 2), then concentrated under reduced pressure to give the product (320 mg, 97% yield) as a solid. LCMS (ESI) m / z: M+H]C 35 H 48 Calculated value for N6O5: 632.4; Found: 633.3.

[0540] Intermediate 11. tert-Butyl ((6 3 S,4S,Z)-11-ethyl-1 2 -(2-((S)-1-Methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate

[0541] [ka]

[0542] Step 1: Synthesis of benzyl (S)-4-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate A 3 L three-necked round-bottom flask, purged and maintained with an inert atmosphere of argon, was charged with 3-bromo-5-iodo-2-[(1S)-1-methoxymethyl]pyridine (147 g, 429.8 mmol), benzylpiperazine-1-carboxylate (94.69 g, 429.8 mmol), Pd(OAc) (4.83 g, 21.4 mmol), BINAP (5.35 g, 8.6 mmol), CsCO (350.14 g, 1074.6 mmol), and toluene (1 L). The resulting solution was stirred overnight at 100 °C in an oil bath. The reaction mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The MeOH / DCM residue was purified by silica gel column chromatography (50% EtOAc / hexanes) to give the product (135 g, 65% yield) as a solid. LCMS(ESI) m / z:[M+H]C 20 H 24 Calculated for BrN3O3 433.1; found 434.1.

[0543] Step 2: Synthesis of benzyl (S)-4-(6-(1-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)piperazine-1-carboxylate A 3 L three-necked round-bottom flask purged and maintained with an inert atmosphere of argon was charged with benzyl 4-[5-bromo-6-[(1S)-1-methoxyethyl]pyridin-3-yl]piperazine-1-carboxylate (135 g, 310.8 mmol), bis(pinacolato)diboron (86.82 g, 341.9 mmol), Pd(dppf)Cl2 (22.74 g, 31.0 mmol), KOAc (76.26 g, 777.5 mmol), and toluene (1 L). The resulting solution was stirred at 90 °C in an oil bath for 2 days. The reaction mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by neutral alumina column chromatography (30% EtOAc / hexanes) to give the product (167 g, crude) as a solid. LCMS (ESI) m / z: [M+H]C 26 H 36 Calculated for BN3O5 481.3; found 482.1.

[0544] Step 3: Synthesis of benzyl (S)-4-(5-(5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate A 3 L, three-necked round-bottom flask, purged with and maintained under an inert atmosphere of argon, was charged with (S)-4-(6-(1-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)piperazine-1-carboxylate (167 g, 346.9 mmol), 5-bromo-3-[3-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylpropyl]-2-iodo-1H-indole (224.27 g, 346.9 mmol), Pd(dppf)Cl (25.38 g, 34.6 mmol), dioxane (600 mL), HO (200 mL), KPO (184.09 g, 867.2 mmol), and toluene (200 mL). The resulting solution was stirred overnight at 70° C. in an oil bath. The reaction mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The MeOH / DCM residue was purified by silica gel column chromatography (50% EtOAc / hexanes) to give the product (146 g, 48% yield) as a solid. LCMS (ESI) m / z: [M+H]C 49 H 57 Calculated for BrN4O4Si 872.3; found 873.3.

[0545] Step 4: Synthesis of benzyl (S)-4-(5-(5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-1-ethyl-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate To a stirred mixture of benzyl (S)-4-(5-(5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate (146 g, 167.0 mmol) and CsCO (163.28 g, 501.1 mmol) in DMF (1200 mL) under a N atmosphere at 0 °C, ethyl iodide (52.11 g, 334.0 mmol) was added in small portions. The final reaction mixture was stirred at room temperature for 12 h. The resulting mixture was diluted with EtOAc (1 L) and washed with brine (3 × 1.5 L). The organic layer was dried over anhydrous NaSO and filtered. After filtration, the filtrate was concentrated under reduced pressure to give the product (143 g, crude) as a solid, which was used directly in the next step without further purification. LCMS (ESI) m / z: [M+H]C 51 H 61 Calculated for BrN4O4Si 900.4; found 901.4.

[0546] Step 5: Synthesis of benzyl (S)-4-(5-(5-bromo-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate To a stirred mixture of benzyl (S)-4-(5-(5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-1-ethyl-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate (143 g, 158.5 mmol) and DMF (1250 mL) was added CsF (72.24 g, 475.5 mmol). The reaction mixture was then stirred at 60 °C for 2 days under a N atmosphere. The resulting mixture was diluted with EtOAc (1 L) and washed with brine (3 × 1 L). The organic phase was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (30% EtOAc / petroleum ether) to give two atropisomers A (38 g, 36% yield) and B (34 g, 34% yield), both as solids. LCMS(ESI) m / z:[M+H]C35 H 43 Calculated for BrN4O4 663.2; found 662.2.

[0547] Step 6: Synthesis of benzyl (S)-4-(5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate In a 500 mL three-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, benzyl (S)-4-(5-(5-bromo-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate (14 g, 21.1 mmol), bis(pinacolato)diboron (5.89 g, 23.21 mmol), Pd(dppf)Cl (1.54 g, 2.1 mmol), KOAc (5.18 g, 52.7 mmol), and toluene (150 mL) were placed. The resulting solution was stirred at 90 °C in an oil bath for 5 hours. The reaction mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (30% EtOAc / petroleum ether) to give the product (12 g, 76% yield) as a solid. LCMS (ESI) m / z: [M+H]C 41 H 55 Calculated for BN4O6 710.4; found 711.3.

[0548] Step 7: Synthesis of methyl (S)-1-((S)-3-(4-(2-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-5-yl)thiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate Into a 250 mL round-bottom flask purged and maintained with an inert atmosphere of argon was added benzyl (S)-4-(5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate (10.8 g, 15.2 mmol), methyl (3S)-1-[(2S)-3-(4-bromo-1,3-thiazol-2-yl)-2-[(tert-butoxycarbonyl)amino]propanoyl]-1,2-diazinan-3-carboxylate (7.98 g, 16.7 mmol), Pd(dtbpf)Cl (0.99 g, 1.52 mmol), KPO (8.06 g, 37.9 mmol), toluene (60 mL), dioxane (20 mL), and HO (20 mL) were added. The resulting solution was stirred at 70 °C in an oil bath for 3 hours. The reaction mixture was cooled to room temperature. The resulting solution was extracted with EtOAc (2 × 50 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (10% EtOAc / hexane). The solvent was removed under reduced pressure to give the product (8 g, 51% yield) as a solid. LCMS(ESI)m / z [M+H] C52H 68 Calculated value for N8O9S: 980.5; Found: 980.9.

[0549] Step 8: Synthesis of (S)-1-((S)-3-(4-(2-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-5-yl)thiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylic acid To a stirred mixture of methyl (S)-1-((S)-3-(4-(2-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-5-yl)thiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (12 g, 12.23 mmol) in THF (100 mL) / HO (100 mL) was added LiOH (2.45 g, 61.1 mmol) under a N atmosphere, and the resulting mixture was stirred for 2 hours at room temperature. The THF was removed under reduced pressure. At 0 °C, the pH of the aqueous phase was acidified to 5 with 1 N HCl. The aqueous layer was extracted with DCM (3 x 100 mL). The organic phase was concentrated under reduced pressure to give the product (10 g, 85% yield) as a solid. LCMS (ESI) m / z [M+H] C 51 H 66 Calculated value for N8O9S: 966.5; Found: 967.0.

[0550] Step 9: Benzyl 4-(5-((6 3 S,4S,Z)-4-((tert-butoxycarbonyl)amino)-1 1 -ethyl-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-1 2 Synthesis of (S)-1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate Into a 3 L round-bottom flask, purged with and maintained under an inert atmosphere of nitrogen, was placed (S)-1-((S)-3-(4-(2-(5-(4-((benzyloxy)carbonyl)piperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-5-yl)thiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylic acid (18 g, 18.61 mmol), MeCN (1.8 L), DIPEA (96.21 g, 744.4 mmol), EDCI (107.03 g, 558.3 ​​mmol), and HOBt (25.15 g, 186.1 mmol). The resulting solution was stirred at room temperature and then concentrated under reduced pressure. The resulting solution was diluted with DCM (1 L) and washed with 1 M HCl (3 x 1 L) and HO (3 x 1 L). The organic layer was then concentrated under reduced pressure and purified by silica gel column chromatography (50% EtOAc / hexanes) to give the product (10.4 g, 55% yield) as a solid. LCMS (ESI) m / z: [M+H]C 51 H 64 Calculated value for N8O8S: 948.5; Found: 949.3.

[0551] Step 10: tert-Butyl ((6 3 S,4S,Z)-11-ethyl-1 2 -(2-((S)-1-Methoxyethyl)-5-(piperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate In a 250 mL round bottom flask purged and maintained with a nitrogen reducing atmosphere, benzyl 4-(5-((6 3S,4S,Z)-4-((tert-butoxycarbonyl)amino)-1 1 -ethyl-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-1 2 A mixture of (S)-1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylate (10.40 g, 10.9 mmol), Pd(OH) / C (5 g, 46.9 mmol), and MeOH (100 mL) was added. The resulting solution was stirred at room temperature under 2 atmospheres of H for 3 hours. The solid was filtered, and the filter cake was washed with MeOH (3 x 100 mL). The combined organic phase was concentrated under reduced pressure to give the product (8.5 g, 90% yield) as a solid. LCMS (ESI) m / z [M+H] C 43 H 58 Calculated value for N8O6S: 814.4; Found: 815.3.

[0552] Step 11: tert-Butyl ((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate In a 1000 mL round-bottom flask purged and maintained with a nitrogen reducing atmosphere, tert-butyl ((6 3 S,4S,Z)-1 1 -ethyl-12 -(2-((S)-1-Methoxyethyl)-5-(piperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate (8.5 g, 10.4 mmol), MeOH (100 mL), and AcOH (1.88 g, 31.2 mmol) were placed in a flask. After stirring for 15 minutes, HCHO (1.88 g, 23.15 mmol, 37% aqueous solution) and NaBHCN (788 mg, 12.5 mmol) were added at room temperature. The resulting solution was stirred for 3 hours. The mixture was then quenched with HO (100 mL) and concentrated under reduced pressure to remove MeOH. The resulting solution was diluted with DCM (300 mL) and washed with HO (3 × 100 mL). The solution was concentrated under reduced pressure to give the product (8.2 g, 90% yield) as a solid. LCMS(ESI) m / z [M+H] C 44 H 60 Calculated value for N8O6S: 828.4; Found: 829.3.

[0553] Intermediate 12: (6 3 S,4S,Z)-4-amino-1 1 -ethyl-1 2 -(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-5,7-dione

[0554] [ka]

[0555] Step 1: Synthesis of (S)-3-(5-bromo-1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate To a stirred solution of (S)-3-(5-bromo-1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol (100 g, 224.517 mmol) and EtN (45.44 g, 449.034 mmol) in DCM (1 L) at 0 °C under an argon atmosphere, DMAP (2.74 g, 22.452 mmol) and AcO (27.50 g, 269.420 mmol) were added in small portions. The resulting mixture was stirred for 3 h at room temperature. The resulting mixture was concentrated under reduced pressure and then diluted with 1000 mL of EtOAc. The resulting mixture was washed with 1 M HCl (500 mL), followed by saturated NaHCO (500 mL) and brine (500 mL), and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by trituration with petroleum ether (500 mL) to give the product (93.3 g, 85% yield) as a white solid. LCMS (ESI) m / z [M+H]C 25 H 31 Calculated for BrN2O3: 487.16; Found: 489.2.

[0556] Step 2: Synthesis of (S)-(5-(3-(3-acetoxy-2,2-dimethylpropyl)-5-bromo-1-ethyl-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)boronic acid To a stirred solution of (S)-3-(5-bromo-1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (93.3 g, 191.409 mmol) and B2PIN2 (72.91 g, 287.113 mmol) in THF (370 mL) at room temperature under an argon atmosphere, dtbpy (7.71 g, 28.711 mmol) and chloro(1,5-cyclooctadiene)iridium(I) dimer (6.43 g, 9.570 mmol) were added in small portions. The resulting mixture was stirred overnight at 75 °C. The resulting mixture was concentrated under reduced pressure to give the product (190 g, crude) as an oil. LCMS (ESI) m / z: [M+H]C 25 H 32 Calculated for BBrN2O5: 531.17; Found: 533.3.

[0557] Step 3: Synthesis of (S)-3-(5-bromo-1-ethyl-2-(5-iodo-2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate To a stirred solution of (S)-(5-(3-(3-acetoxy-2,2-dimethylpropyl)-5-bromo-1-ethyl-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)boronic acid (110 g, 207.059 mmol) and chloramine-T trihydrate (349.96 g, 1242.354 mmol) in THF (550 mL) was added a solution of NaI (186.22 g, 1242.354 mmol) in water (225 mL) in small portions at 0°C under an air atmosphere. The resulting mixture was stirred overnight at 50°C under an argon atmosphere. The resulting mixture was concentrated under reduced pressure and washed with CHCl (500 mL). The resulting mixture was filtered, and the filter cake was washed with CHCl (3 x 250 mL). The filtrate was extracted with CHCl (3 x 500 mL). The combined organic layers were washed with NaSO (500 mL), washed with brine (2 x 200 mL), and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (18% EtOAc / petroleum ether) to give the product (24 g, 18% yield) as a solid. LCMS (ESI) m / z: [M+H]C 25 H 30 Calculated for BrIN2O3: 613.06; Found: 614.7.

[0558] Step 4: Synthesis of 3-(5-bromo-1-ethyl-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate To a stirred solution of 3-(5-bromo-1-ethyl-2-{5-iodo-2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-3-yl)-2,2-dimethylpropyl acetate (9 g, 14.674 mmol), (S)-octahydropyrazino[2,1-c][1,4]oxazine (2.469 g, 17.609 mmol), CsCO (11.953 g, 36.685 mmol), and BINAP (456.9 mg, 0.734 mmol) at room temperature under an argon atmosphere, Pd(OAc) (329.44 mg, 1.467 mmol) was added. The resulting mixture was stirred at 6 °C for 100 h. After filtration, the filter cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (8% MeOH / DCM) to give the product (6.9 g, 75% yield) as a solid. LCMS (ESI) m / z: [M+H]C 32 H 43 Calculated for BrN4O4: 627.25; found 627.4.

[0559] Step 5: Synthesis of 3-(1-ethyl-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate To a stirred solution of 3-(5-bromo-1-ethyl-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (3.2 g, 5.115 mmol), KOAc (1.51 g, 15.345 mmol), and 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (2.60 g, 10.230 mmol) in toluene (48 mL) at room temperature under an argon atmosphere, Pd(dppf)Cl (0.37 g, 0.512 mmol) was added in small portions. The resulting mixture was stirred for 1.5 hours at 90°C. The resulting mixture was filtered, and the filter cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (8% MeOH / DCM) to give the product (3.0 g, 88% yield) as a solid. LCMS (ESI) m / z: [M+H]C 38 H 55 Calculated for BN4O6: 675.43; Found: 675.1.

[0560] Step 6: Synthesis of methyl (S)-1-((S)-3-(4-(3-(3-acetoxy-2,2-dimethylpropyl)-1-ethyl-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate To a stirred mixture of 3-(1-ethyl-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (5 g, 7.433 mmol) and KPO (4.26 g, 20.067 mmol) in toluene (54 mL) was added dioxane (17.82 mL, 210.307 mmol) and HO (17.82 mL) at room temperature under an argon atmosphere. The resulting mixture was stirred for 2 hours at 70° C. The resulting mixture was filtered, and the filter cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure. The resulting mixture was extracted with EtOAc (200 mL). The combined organic layers were washed with brine (100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (8% MeOH / DCM) to give the product (4.6 g, 66% yield) as a solid. LCMS (ESI) m / z: [M+H]C 49 H 68 Calculated for N8O9S: 945.49; Found: 945.7.

[0561] Step 7: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-5-yl)azol-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid To a stirred solution of methyl (S)-1-((S)-3-(4-(3-(3-acetoxy-2,2-dimethylpropyl)-1-ethyl-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (6 g, 6.361 mmol) in THF (43 mL) was added LiOH·HO (573.92 mg, 13.677 mmol) at 0 °C. The resulting mixture was stirred for 16 h at room temperature. The mixture was acidified to pH 6 with hydrochloric acid. The resulting mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give the product (4 g, crude) as a solid. LCMS (ESI) m / z: [M+H]C 45 H 60 Calculated for N8O9S: 889.43; Found: 889.7.

[0562] Step 8: tert-Butyl ((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate At 0°C under an argon atmosphere, (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-3-(3-hydroxy-2,2-dimethylpropyl)-1H-in To a stirred solution of (azol-5-yl) and (azol-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid (4 g, 4.51 mmol), HOBt (6.09 g, 45.09 mmol), and DIPEA (23.31 g, 180.36 mmol) in DCM (200 mL) was added dropwise a solution of EDCI (25.93 g, 135.27 mmol) in DCM (200 mL). The resulting mixture was stirred for 16 h at room temperature and then concentrated under reduced pressure. At 0 °C, the reaction was quenched with HO and extracted with EtOAc (500 mL). The combined organic layers were washed with brine and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (8% MeOH / DCM) to give the product (2.0 g, 52% yield) as a solid. LCMS(ESI) m / z:[M+H]C 46 H 62 Calculated for N8O7S: 870.4; Found: 871.8.

[0563] Step 9:(6 3 S,4S,Z)-4-amino-1 1 -ethyl-1 2 -(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-5,7-dione At 0°C under an argon atmosphere, tert-butyl ((6 3S,4S,Z)-1 1 -ethyl-1 2 -(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 To a stirred solution of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate (316 mg, 0.345 mmol) in DCM (3 mL) was added TFA (1 mL) dropwise. The resulting mixture was stirred for 2 hours at room temperature. The mixture was neutralized to pH 8 with saturated NaHCO. The resulting mixture was extracted with EtOAc (3 x 80 mL). The combined organic layers were washed with brine (3 x 40 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The crude product mixture was used directly in the next step without further purification. LCMS (ESI) m / z: [M+H]C 41 H 54 Calculated for N8O5S: 771.4; Found 771.6.

[0564] Intermediate 13: (6 3 S,4S,Z)-4-amino-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-5,7-dione

[0565] [ka]

[0566] Step 1: Synthesis of 3-(5-bromo-1-ethyl-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate To a stirred solution of 3-(5-bromo-1-ethyl-2-{5-iodo-2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-3-yl)-2,2-dimethylpropyl acetate (9 g, 14.674 mmol), (R)-octahydro-2H-pyrido[1,2-a]pyrazine (2.469 g, 17.609 mmol), CsCO (11.9523 g, 36.685 mmol), and BINAP (456.85 mg, 0.734 mmol) at room temperature under an argon atmosphere, Pd(OAc) (329.44 mg, 1.467 mmol) was added in small portions. The resulting mixture was stirred for 6 hours at 100 °C, after which the mixture was filtered and the filter cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (8% MeOH / DCM) to give the product (6 g, 65% yield) as a solid. LCMS (ESI) m / z: [M+H]C 33 H 45 Calculated for BrN4O3: 625.28; Found: 627.4.

[0567] Step 2: Synthesis of 3-(1-ethyl-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate To a stirred solution of 3-(5-bromo-1-ethyl-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (3.2 g, 5.115 mmol), KOAc (1.51 g, 15.345 mmol), and 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (2.60 g, 10.230 mmol) in toluene (48 mL) at room temperature under an argon atmosphere, Pd(dppf)Cl (0.37 g, 0.512 mmol) was added in small portions. The resulting mixture was stirred for 1.5 hours at 90°C. The resulting mixture was filtered, and the filter cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure and purified by preparative TLC (8% MeOH / DCM) to give the product (3.1 g, 81% yield) as a solid. LCMS (ESI) m / z: [M+H]C 39 H 57 Calculated for BN4O5: 673.45; Found: 673.4.

[0568] Step 3: Synthesis of methyl (S)-1-((S)-3-(4-(3-(3-acetoxy-2,2-dimethylpropyl)-1-ethyl-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate At room temperature under an argon atmosphere, 3-(1-ethyl-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (5 g, 7.433 mmol), methyl (S)-1-((S)-3 To a stirred mixture of -(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (3.89 g, 8.176 mmol) and KPO (4.26 g, 20.067 mmol) in toluene (54 mL), dioxane (18 mL), and HO (18 mL) was added Pd(dtbpf)Cl (969 mg, 1.486 mmol). The resulting mixture was stirred at 70° C. for 2 hours. The mixture was filtered, and the filter cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure, and the resulting mixture was extracted with EtOAc (200 mL). The combined organic layers were washed with brine (100 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (8% MeOH / DCM) to give the product (6.8 g, 83% yield) as a solid. LCMS (ESI) m / z: [M+H]C 50 H 70 Calculated for N8O8S: 943.51; Found: 943.4.

[0569] Step 4: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)propanoyl)hexahydropyridazine-3-carboxylic acid To a stirred solution of methyl (S)-1-((S)-3-(4-(3-(3-acetoxy-2,2-dimethylpropyl)-1-ethyl-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1H-indol-5-yl)thiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (6 g, 6.361 mmol) in THF (43 mL) at 0 °C under an argon atmosphere, LiOH·HO (573.92 mg, 13.677 mmol) was added. The resulting mixture was stirred for 16 h at room temperature. The mixture was acidified to pH 6 with hydrochloric acid. The resulting mixture was extracted with EtOAc (3 × 150 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give the product (4 g, crude) as a solid. LCMS (ESI) m / z: [M+H]C 47 H 66 Calculated for N8O7S: 887.49; Found: 887.6.

[0570] Step 5: tert-Butyl ((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate At 0°C under an argon atmosphere, (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1H-indole-5- To a stirred solution of (2-( ... LCMS(ESI) m / z:[M+H]C 47 H 64 Calculated for N8O6S: 869.47; Found: 869.8.

[0571] Step 6:(6 3 S,4S,Z)-4-amino-1 1 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-10,10-dimethyl-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-5,7-dione At 0°C, tert-butyl ((6 3 S,4S,Z)-11 -ethyl-1 2 -(2-((S)-1-Methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 To a stirred solution of H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphan-4-yl)carbamate (900 mg, 1.035 mmol) in DCM (9 mL) was added TFA (3 mL) dropwise. The resulting mixture was stirred for 2 hours at room temperature. The mixture was basified with saturated aqueous NaHCO to pH = 8 and extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (3 x 50 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure to give the product (800 mg), which was used directly in the next step without further purification. LCMS (ESI) m / z: [M+H]C 42 H 56 Calculated for N8O4S: 769.42; Found 769.5.

[0572] Intermediate 14: (6 3 S,4S)-4-amino-1 2 -(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-1 1 -(2,2,2-trifluoroethyl)-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphane-5,7-dione

[0573] [ka]

[0574] Step 1: Synthesis of (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate To a stirred solution of (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropan-1-ol (60 g, 0.12 mol) and EtN (24.33 g, 0.24 mol) in DCM (600 mL) was added DMAP (1.46 g, 0.012 mol) and acetic anhydride (14.7 g, 144 mmol) dropwise at 0 °C under an argon atmosphere. The resulting mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under reduced pressure and washed with HCl (500 mL). The resulting mixture was washed with saturated aqueous NaHCO (500 mL). The combined organic layers were washed with brine (500 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure to give the product (59.6 g, 92% yield) as an oil. LCMS (ESI) m / z: [M+H]C 25 H 28 Calculated for BrF3N2O3: 541.13; Found: 543.2.

[0575] Step 2: Synthesis of (S)-(5-(3-(3-acetoxy-2,2-dimethylpropyl)-5-bromo-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)boronic acid To a stirred mixture of (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (55.1 g, 101.771 mmol) and 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (38.77 g, 152.656 mmol) in THF (40 mL) at room temperature under an argon atmosphere, dtbpy (4.10 g, 15.266 mmol) and chloro(1,5-cyclooctadiene)iridium(I) dimer (3.42 g, 5.089 mmol) were added in small portions. The resulting mixture was stirred at 75 °C for 5 hours. The resulting mixture was concentrated under reduced pressure to give the product (102.4 g, crude) as an oil. LCMS (ESI) m / z: [M+H]C 25 H 29 Calculated for BBrF3N2O5: 585.14; Found: 585.2.

[0576] Step 3: (S)-3-(5-bromo-2-(5-iodo-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate At 0° C. under an argon atmosphere, to a stirred solution of (S)-(5-(3-(3-acetoxy-2,2-dimethylpropyl)-5-bromo-1-(2,2,2-trifluoroethyl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)boronic acid (51.2 g, 87.487 mmol) and sodium chloro[(4-methylbenzene)sulfonyl]azanid (197 g, 699.896 mmol) in THF (258 mL) was added dropwise a solution of NaI (104.91 g, 699.896 mmol) in water (129 mL). The resulting mixture was stirred at 55° C. for 16 h. The resulting mixture was concentrated under reduced pressure and extracted with CHCl (2 × 200 mL). The combined organic layers were washed with brine (2 × 20 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20% EtOAc / petroleum ether) to give the product (15.3 g, 26% yield) as a solid. LCMS (ESI) m / z: [M+H]C 32 H 40 Calculated for BrF3N4O4: 666.0; Found: 667.3.

[0577] Step 4: Synthesis of methyl (S)-1-((S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate To a stirred mixture of (S)-3-(5-bromo-2-(5-iodo-2-(1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (2.70 g, 4.046 mmol) and (S)-octahydropyrazino[2,1-c][1,4]oxazine dihydrochloride (1.044 g, 4.855 mmol) in toluene (18.9 mL) at room temperature under an argon atmosphere, CsCO (5932.38 mg, 18.207 mmol) and BINAP (125.97 mg, 0.202 mmol) were added in small portions. To the mixture was added Pd(OAc) (90.84 mg, 0.405 mmol) in small portions. The resulting mixture was stirred at 90° C. for an additional 16 hours. After the mixture was cooled to room temperature, it was filtered and the filter cake was washed with EtOAc (2×20 mL). The filtrate was concentrated under reduced pressure. The MeOH / DCM residue was purified by silica gel column chromatography (10% MeOH / DCM) to give the product (2.3 g, 83% yield) as a solid. LCMS (ESI) m / z: [M+H]C 32 H 40 Calculated for BrF3N4O4: 681.23; Found: 681.4.

[0578] Step 5: Synthesis of methyl (S)-1-((S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate Methyl (S)-1-((S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (2.33 g, 4.512 mmol) and KPO (1.59 g, 7.490 mmol) were added to a 250 mL three-necked round-bottom flask at room temperature under an air atmosphere. To a stirred mixture of HO (8.20 mL) and dioxane (8.20 mL) with toluene at room temperature, Pd(dtbpf)Cl (0.29 g, 0.451 mmol) was added in small portions. The resulting mixture was stirred at 65 °C for 3 h. The resulting mixture was filtered, and the filter cake was washed with EtOAc (2 × 100 mL). The filtrate was concentrated under reduced pressure. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with brine (2 × 150 mL) and dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The MeOH / DCM residue was purified by silica gel column chromatography (3 → 4% MeOH / DCM) to give the product (2.7 g, 90% yield) as a solid. LCMS (ESI) m / z: [M+H]C 52 H 68 Calculated for F3N7O9: 991.5; Found: 992.7.

[0579] Step 6: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-3-(3-hydroxy-2,2-dimethylpropyl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylic acid To a 100 mL three-necked round-bottom flask was added methyl (S)-1-((S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propanoyl)hexahydropyridazine-3-carboxylate (3 g, 3.024 mmol) and THF (30 mL) at room temperature. A solution of LiOH (0.30 g, 12.701 mmol) in water (12.7 mL) followed. The resulting mixture was stirred for 16 hours at room temperature. The mixture was acidified to pH 5 with 1 N HCl. The resulting mixture was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with brine (2 x 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give the product (2.7 g, crude) as a solid. LCMS (ESI) m / z: [M+H]C 49 H 64 Calculated for F3N7O8: 936.48; Found: 936.7.

[0580] Step 7: tert-Butyl ((6 3 S,4S)-12-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1 1 -(2,2,2-trifluoroethyl)-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphan-4-yl)carbamate To a 2 L three-necked round-bottom flask was added (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(2-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-3-(3-hydroxy-2,2-dimethylpropyl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)propanoyl)hexahydropyridazine-3-carboxylic acid (3.12 g, 3.333 mmol) and DCM (624 mL) at room temperature. To the above mixture at 0°C, DIPEA (17.23 g, 133.320 mmol) and HOBt (4.50 g, 33.330 mmol) were added in small portions. The resulting mixture was stirred for an additional 30 minutes. To the above mixture at room temperature for 16 hours, EDCI (19.17 g, 99.990 mmol) was added in small portions. The resulting mixture was concentrated under reduced pressure. The reaction was quenched with H2O at 0°C. The resulting mixture was extracted with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (3 x 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The MeOH / DCM residue was purified by silica gel column chromatography (3->4% MeOH / DCM) to give the product (3 g, 98% yield) as a solid. LCMS (ESI) m / z: [M+H]C 49 H 62 Calculated for F3N7O7: 918.47; Found: 918.8.

[0581] Step 8:(6 3 S,4S)-4-amino-1 2 -(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-1 1 -(2,2,2-trifluoroethyl)-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1Synthesis of H-8-oxa-1(5,3)-indola-6(1,3)-pyridazina-2(1,3)-benzenacycloundecaphane-5,7-dione At 0°C under an argon atmosphere, tert-butyl ((6 3 S,4S)-12-(5-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1 1 -(2,2,2-trifluoroethyl)-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -Hexahydro-1 1 To a stirred solution of H-8-oxa-1(5,3)-indola-6(1,3)-pyrid...

Claims

1. A compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 wherein A is an optionally substituted 3- to 6-membered heterocycloalkylene, an optionally substituted 3- to 6-membered cycloalkylene, an optionally substituted 6-membered arylene, or an optionally substituted 5- to 10-membered heteroarylene; W is a bridging group comprising an aziridine, epoxide, carbodiimide, oxazoline, thiazoline, chloroethylurea, chloroethylthiourea, chloroethylcarbamate, chloroethylthiocarbamate, trifluoromethylketone, boronic acid, boronic ester, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isoEEDQ or other EEDQ derivatives, oxazolium, or glycal; X １ is CH ２ or O; m is 1 or 2; n is 0 or 1; R １ is hydrogen or an optionally substituted 3- to 10-membered heterocycloalkyl; R ２ is an optionally substituted C １ -C ６ alkyl, and R ３ is an optionally substituted C １ -C ６ alkyl, or optionally substituted 3- to 6-membered cycloalkyl.

2. 2. The compound of claim 1, wherein A is optionally substituted thiazole, optionally substituted oxazole, optionally substituted morpholino, optionally substituted pyrrolidinyl, optionally substituted piperidinyl, or optionally substituted phenyl, or a pharmaceutically acceptable salt thereof.

3. R ２ teeth, 【Chemistry 2】 2. The compound of claim 1, wherein:

4. R ３ is an optionally substituted C １ -C ６ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

5. R ３ 2. The compound of claim 1, wherein: is or an optionally substituted 3- to 6-membered cycloalkyl, or a pharmaceutically acceptable salt thereof.

6. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein A is an optionally substituted 5- to 10-membered heteroarylene.

7. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein A is optionally substituted phenyl.

8. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein A is an optionally substituted 3- to 6-membered heterocycloalkylene.

9. 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein W comprises aziridine.

10. 10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein W comprises an optionally substituted cyclopropyl-aziridinyl moiety.

11. 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein W comprises an epoxide.

12. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21

13. A compound having the structure of Formula III, or a pharmaceutically acceptable salt thereof: 【Transformation 3】 wherein P is —(CO)R ９ , -(PO)(OH) ２ , or -Si(R １０ ) ３ and W is a bridging group comprising an aziridine, epoxide, carbodiimide, oxazoline, thiazoline, chloroethylurea, chloroethylthiourea, chloroethylcarbamate, chloroethylthiocarbamate, trifluoromethylketone, boronic acid, boronic ester, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), isoEEDQ or other EEDQ derivatives, oxazolium, or glycal; L １ is an optionally substituted 3- to 9-membered heterocycloalkylene or an optionally substituted C ２ -C ４ is heteroalkylene, R ４ is an optionally substituted C １ -C ６ is alkyl, R ５ is an optionally substituted C １ -C ６ alkyl or optionally substituted 3- to 6-membered cycloalkyl; R ９ is an optionally substituted C １ -C ３ Alkyl or optionally substituted C １ -C ３ is heteroalkyl, and Each R １０ are independently optionally substituted C １ -C ３ alkyl].

14. The compound of claim 13, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following: Table 2-1 Table 2-2 15. A compound, or a pharmaceutically acceptable salt thereof, having a structure selected from the following compounds: Table 3-1 Table 3-2

16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

17. A conjugate comprising the structure of Formula IV, or a salt thereof: M-L ２ -P １ Formula IV [In the formula, L ２ is the linker, P １ is a monovalent organic moiety, and M has the structure of Formula V 【Chemistry 4】 wherein A is an optionally substituted 3- to 6-membered heterocycloalkylene, an optionally substituted 3- to 6-membered cycloalkylene, an optionally substituted 6-membered arylene, or an optionally substituted 5- to 10-membered heteroarylene; X １ is CH ２ or O; m is 1 or 2; n is 0 or 1; R １ is hydrogen or an optionally substituted 3- to 10-membered heterocycloalkyl; R ２ is an optionally substituted C １ -C ６ alkyl, and R ３ is an optionally substituted C １ -C ６ alkyl, or optionally substituted 3- to 6-membered cycloalkyl.

18. A pharmaceutical composition for treating cancer, comprising a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof.

19. A pharmaceutical composition for treating a Ras protein-associated disease, comprising a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof.

20. A pharmaceutical composition for inhibiting Ras protein in a cell, comprising the compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof.

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