RAS inhibitors

Abstract

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

Description

[0001] This application is a divisional application of an invention patent application, the parent application of which was filed on October 7, 2022, with application number 202280080572.8 (PCT / US2022 / 077784) and invention title "RAS Inhibitor". Background Technology

[0002] Most small molecule drugs work by binding to functionally important pockets on target proteins, thereby modulating the activity of those proteins. For example, cholesterol-lowering drugs called statins bind to the active site of HMG-CoA reductase, thus preventing the enzyme from binding to its substrate. Indeed, the existence of many such drug / target interaction pairs may mislead one into believing that small molecule regulators targeting most (if not all) proteins can be discovered, thus justifying the amount of time, effort, and resources required. But this is far from the truth. Currently, it is estimated that only about 10% of all human proteins are suitable targets for small molecules. (Bojadzic and Buchwald, CurrTopMedChem 18:674-699 (2019)). The remaining 90% are currently considered intractable or difficult to treat with the aforementioned small molecule drugs. These targets are often referred to as “undruggable.” These undruggable targets comprise a large and often unexplored reservoir of medically important human proteins. Therefore, there is great interest in discovering novel molecular modalities capable of modulating the function of such undruggable targets.

[0003] The literature has well established that Ras proteins (K-Ras, H-Ras, and N-Ras) play a crucial role in various human cancers, thus making them suitable targets for anticancer therapies. In fact, approximately 30% of all human cancers in the United States are caused by Ras protein mutations, many of which are fatal. Dysregulation of Ras proteins caused by activating mutations, overexpression, or upstream activation is common in human tumors, and activating mutations of Ras are frequently found in human cancers. For example, an activating mutation at codon 12 in the Ras protein significantly biases the Ras mutant protein population towards the "on" (GTP-binding) state (Ras(ON)) by inhibiting GTPase-activating protein (GAP) dependence and intrinsic GTP hydrolysis rate, leading to the action of oncogenic MAPK signaling. Notably, Ras exhibits a picomolar affinity for GTP, allowing it to be activated even in the presence of low concentrations of this nucleotide. Mutations at codons 13 (e.g., G13D) and 61 (e.g., Q61K) in Ras also induce oncogenic activity in some cancers.

[0004] Despite extensive drug discovery efforts targeting Ras over the past decades, only a drug targeting the K-RasG12C mutant has been approved (sotorasib). Further efforts are needed to discover other medicines for cancers driven by other Ras mutations. Summary of the Invention

[0005] This document provides Ras inhibitors. The methods described herein require the formation of a high-affinity three-component complex between a synthetic ligand and two intracellular proteins that do not interact under normal physiological conditions: the target protein of interest (e.g., Ras), and a cytosolic chaperone protein (presenting protein) (e.g., cyclophilin A) that is widely expressed in the cell. More specifically, in some embodiments, the Ras inhibitors described herein induce a novel binding pocket in Ras by driving the formation of a high-affinity triple complex or conjugate between the Ras protein and the widely expressed cytosolic chaperone protein cyclophilin A (CYPA). Without being bound by theory, the inventors believe that one way the compounds of the present invention, and the complexes or conjugates thereto, exert their inhibitory effect on Ras is by spatially blocking the interaction sites between Ras and downstream effector molecules such as RAF and PI3K required for the propagation of oncogenic signals.

[0006] Therefore, in some embodiments, the present invention is characterized by a compound of structural formula I or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0009] X 1 X 2 and X 3 Each is independently selected from CH2, CF2, C=O, or O;

[0010] m is 1 or 2;

[0011] n is 0 or 1;

[0012] R 1 It is hydrogen, optionally substituted C1-C6 heteroalkyl or optionally substituted 3 to 10-membered heterocyclic alkyl;

[0013] R 2 It is an optional substituted C1-C6 alkyl group; and

[0014] R 3It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0015] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0016] In some embodiments, the invention is characterized by a compound having structural formula II or a pharmaceutically acceptable salt thereof:

[0017]

[0018] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0019] R 2 It is an optional substituted C1-C6 alkyl group; and

[0020] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0021] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0022] In some embodiments, the invention is characterized by a compound having structural formula V or a pharmaceutically acceptable salt thereof:

[0023]

[0024] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0025] R 2 It is an optional substituted C1-C6 alkyl group; and

[0026] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0027] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0028] In some embodiments, the invention is characterized by a compound having structural formula VI or a pharmaceutically acceptable salt thereof:

[0029]

[0030] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0031] R 2 It is an optional substituted C1-C6 alkyl group; and

[0032] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0033] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0034] In some embodiments, the invention is characterized by a compound having structural formula VII or a pharmaceutically acceptable salt thereof:

[0035]

[0036] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0037] R 2 It is an optional substituted C1-C6 alkyl group; and

[0038] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0039] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0040] In some embodiments, the invention is further characterized by a compound selected from Table 1 or Table 2 or a pharmaceutically acceptable salt thereof.

[0041] Pharmaceutical compositions are also provided that comprise a compound of formula I, II, V, VI, or VII, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Pharmaceutical compositions are also provided that comprise a compound of Table 1 or Table 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0042] A method for treating cancer in a subject in need is also provided, the method comprising administering to the subject a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0043] In some embodiments, a method is provided for treating a subject with Ras protein-related conditions, the method comprising administering to the subject a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0044] A method for inhibiting Ras protein in cells is also provided, the method comprising contacting the cells with an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0045] Specifically, upon careful consideration, any limitations discussed regarding one embodiment of the invention may be applied 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 manufacture or utilize any compound or composition of the invention.

[0046] Definitions and chemical terms

[0047] In this application, unless clearly indicated from the context, (i) the term “a (a)” means “one or more”; (ii) the term “or” is used to mean “and / or” unless explicitly indicated that the term refers to an alternative as unique or that the alternatives are mutually exclusive, however, the definition supported by this invention refers to a unique alternative as well as “and / or”; (iii) the terms “comprising” and “including” should be understood to encompass the listed components or steps, whether presenting only the components or steps themselves or presenting the components or steps in combination with one or more additional components or steps; and (iv) when providing a scope, endpoints are included.

[0048] As used herein, the term "about" is used to indicate that a value includes the standard deviation of the error of the means or method used to determine that value. In some embodiments, the term "about" refers to a range of values ​​in any direction (greater or less than) within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lower percentages of the value, unless otherwise specified or otherwise apparent from the context (e.g., when the figure would exceed 100% of the possible value).

[0049] As used herein, in the context of describing adjacent atoms, the term "adjacent" refers to divalent atoms directly connected by covalent bonds.

[0050] As used herein, “compounds of the present invention” and similar terms, whether explicitly indicated or not, refer to the Ras inhibitors described herein, including compounds of any of the formulas I through VII or their subformulas, as well as the compounds listed in Table 1 or Table 2, and their salts (e.g., pharmaceutically acceptable salts), solvates, hydrates, stereoisomers (including transisomers), and tautomers.

[0051] The term "wildtype" refers to an entity that has the structure or activity seen in nature in a "normal" state or condition (as opposed to mutation, disease, alteration, etc.). Those skilled in the art will understand that wildtype genes and polypeptides often exist in many different forms (e.g., alleles).

[0052] Those skilled in the art will understand that some of the compounds described herein may exist in one or more different isomeric forms (e.g., stereoisomers, geometric isomers, transisomers, tautomers) or isotopic forms (e.g., one or more atoms are substituted with different isotopes of that atom, such as hydrogen being substituted with deuterium). Unless otherwise indicated or clearly apparent from the context, the structures depicted are to be understood as representing any such isomeric or isotopic forms, individually or in combination.

[0053] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are covered. 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 are known in the art, such as by resolving racemic mixtures or by stereoselective synthesis. Many geometric isomers, such as alkenes and C=N double bonds, may also be present in the compounds described herein, and all such stable isomers are covered in this invention. Cis and trans geometric isomers of the compounds of the present invention have been described and can be isolated in mixtures of isomers or as separate isomers.

[0054] In some embodiments, one or more compounds described herein may exist in different tautomer forms. As will be clear from the context, unless explicitly excluded, references to such compounds encompass all such tautomer forms. In some embodiments, the tautomer form is obtained by the exchange of a single bond with an adjacent double bond, accompanied by proton migration. In some embodiments, the tautomer form may be a proton-transfer tautomer, which is an isomer protonated state having the same empirical formula and total charge as the reference form. Examples of portions having a proton-transfer tautomer form include keto-enol pairs, amide-imino pairs, lactam-lactamimide pairs, amide-imino pairs, enamine-imide pairs, and cyclic forms in which the proton may occupy two or more positions in the heterocyclic system, such as 1H-imidazole and 3H-imidazole, 1H-triazole, 2H-triazole and 4H-1,2,4-triazole, 1H-isoindole and 2H-isoindole, and 1H-pyrazole and 2H-pyrazole. In some embodiments, the tautomer form may be in equilibrium or spatially locked into one form through appropriate substitution. In some embodiments, the tautomer form is obtained by interconversion of acetals.

[0055] Unless otherwise specified, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. Exemplary isotopes that may be incorporated into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as... 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 I. Isotope-labeled compounds (e.g., labeled with...) 3 H and 14 Compounds of C can be used in the determination of the tissue distribution of compounds or substrates. Tritium (i.e., 3 H) and carbon-14 (i.e., ... 14 C) Isotopes are available because they are easy to prepare and detect. Alternatively, heavier isotopes, such as deuterium (i.e.,...), can be used. 2 H) substitution can provide certain therapeutic benefits due to enhanced metabolic stability (e.g., increased half-life in vivo or reduced dose requirement). In some embodiments, one or more hydrogen atoms are... 2H or 3 H substitution, or one or more carbon atoms being... 13 C or 14 Carbon-enriched carbon substitution. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies to examine substrate acceptor 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 a procedure similar to that disclosed for the compounds of the invention described herein, by replacing unlabeled reagents with isotopically labeled reagents.

[0056] As may be used interchangeably herein, “deuterium substitution,” “deuteration,” or “deuterium enrichment” refers to the deuterium (D or) in the compounds of this invention or portions thereof. 2 The H) level has been enriched to exceed the natural abundance of deuterium, which is 0.015%. In some embodiments, the minimum isotopic enrichment factor of the compositions of the present invention is at least 5 (0.075% deuterium inclusion), for example at least 10 (0.15% deuterium inclusion). In other embodiments, the isotope enrichment factor of the composition is at least 50 (0.75% deuterium inclusion), at least 500 (7.5% deuterium inclusion), at least 2000 (30% deuterium inclusion), at least 3000 (45% deuterium inclusion), at least 4000 (60% deuterium inclusion), at least 4500 (67.5% deuterium inclusion), at least 5000 (75% deuterium inclusion), at least 5500 (82.5% deuterium inclusion), at least 6000 (90% deuterium inclusion), or at least 6600 (99% deuterium inclusion).

[0057] Non-limiting examples of compounds of the present invention containing one or more deuterium-substituted portions include the following, wherein "R" at any position can be deuterium (D):

[0058] Other examples include the following:

[0059] And similar to R 1 The deuteration of the type part, where R 1 The definition is found herein (e.g., in compounds of any of formulas I-VII). It also covers the deuteration of portions within crosslinking groups (e.g., optionally substituted aziridine moieties) in the compounds of the invention, wherein said crosslinking groups are as defined herein (see, for example, general formulas I-VII and their sub-formulas, and specific examples of W described herein, such as...).

[0060] Furthermore, it also covers deuteration at any available position in part A of the compounds described herein, for example...

[0061] Furthermore, deuterium substitution can also occur at the linker sites of the compounds of the various forms described herein, for example...

[0062]

[0063] In another embodiment, silylation substitution is also covered, for example, substitution at the following linker positions:

[0064] Other examples of silylation include, for example, the following:

[0065] And similar to R 1 The silanization substitution of the type part, wherein R 1 The definition is found in this document (e.g., in compounds of any of formulas I-VII).

[0066] As is known in the art, many chemical entities can exist in a variety of different solid forms, such as amorphous or crystalline forms (e.g., polymorphs, hydrates, solvates). In some embodiments, the compounds of the present invention can be used in any such form, including any solid form. In some embodiments, the compounds described or depicted herein can be provided in hydrate or solvate form.

[0067] Throughout this specification, substituents in the compounds of the present invention are disclosed by group or by scope. Specifically, the invention is intended to encompass every individual combination of members of each of the said groups and scopes. For example, the term "C1-C6 alkyl" specifically intends to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl. Furthermore, where a compound comprises multiple positions, and where substituents are disclosed by group or by scope at these positions, unless otherwise indicated, the invention is intended to cover individual compounds and groups of compounds (e.g., species and subclasses) containing each individual combination of members at each position.

[0068] The term “optionally substituted X” (e.g., “optionally substituted alkyl”) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein the alkyl group is optionally substituted”). It is not intended that the characteristic “X” (e.g., alkyl) itself is optional. As described herein, some compounds of interest may contain one or more “optionally substituted” moieties. Generally, the term “substituted”, whether or not preceded by the term “optionally”, means that one or more hydrogens of the specified moiety are replaced by a suitable substituent, such as any of the substituents or groups described herein. Unless otherwise indicated, the “optionally substituted” group may have a suitable substituent at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure is substituted by more than one substituent selected from the specified group. For example, in the term “optionally substituted C1-C6 alkyl-C2-C9 heteroaryl,” the alkyl moiety, the heteroaryl moiety, or both may be optionally substituted. The combinations of substituents contemplated in this invention are preferably combinations that form stable or chemically viable compounds. As used herein, the term "stable" means that a compound remains substantially unchanged when subjected to conditions that allow the compound to be produced, detected, and in some embodiments, to be recovered, purified, and used for one or more purposes disclosed herein.

[0069] The suitable monovalent substituent on the substituted carbon atom of the "optionally substituted" group can independently be deuterium; halogen; -(CH2). 0-4 R o ;-(CH2) 0-4 OR o ;-O(CH2) 0-4 R o ;-O-(CH2)0-4C(O)OR o ;-(CH2) 0-4 CH(OR o )2;-(CH2) 0- 4SR o ;-(CH2) 0-4 Ph, this group can be R o Substitution; -(CH2) 0-4 O(CH2) 0-1 Ph, this group can be R o Substitution; -CH=CHPh, this group can be replaced by R o Substitution; -(CH2) 0-4 O(CH2) 0-1 -pyridyl group, this group can be R oSubstitution; 4 to 8-membered saturated or unsaturated heterocyclic alkyl groups (e.g., pyridinyl); 3 to 8-membered saturated or unsaturated cycloalkyl groups (e.g., cyclopropyl, cyclobutyl, or cyclopentyl); -NO2; -CN; -N3; ​​-(CH2)0-4N(R) o )2;-(CH2) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ;-(CH2)0-4N(R o )C(O)NR o 2; -N(R) o )C(S)NR o 2;-(CH2) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2; -N(R) o )N(R o )C(O)OR o ;-(CH2) 0-4 C(O)R o ;-C(S)R o ;-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 -C(O)-N(R o )2;-(CH2) 0-4 -C(O)-N(R o )-S(O)2-R o ;-C(NCN)NR o 2;-(CH2) 0-4 C(O)SR o ;-(CH2) 0-4 C(O)OSiR o 3; -(CH2) 0-4 OC(O)R o ;-OC(O)(CH2) 0-4 SR o ;-SC(S)SR o ;-(CH2) 0-4 SC(O)R o ;-(CH2) 0-4 C(O)NR o 2; -C(S)NRo 2;-C(S)SR o ;-(CH2) 0-4 OC(O)NR o 2; -C(O)N(OR) o )R o ;-C(O)C(O)R o ;-C(O)CH2C(O)R o ;-C(NOR) o )R o ;-(CH2) 0-4 SSR o ;-(CH2) 0-4 S(O)2R o ;-(CH2) 0-4 S(O)2OR o ;-(CH2) 0-4 OS(O)2R o ;-S(O)2NR o 2;-(CH2) 0-4 S(O)R o ;-N(R o )S(O)2NR o 2; -N(R) o )S(O)2R o ;-N(OR) o )R o ;-C(NOR) o )NR o 2;-C(NH)NR o 2; -P(O)2R o ;-P(O)R o 2; -P(O)(OR o )2;-OP(O)R o 2; -OP(O)(OR o )2;-OP(O)(OR o )R v ;-SiR o 3;-(C1-C4 straight-chain or branched-chain alkylene)ON(R) o )2; or -(C1-C4 straight-chain or branched-chain alkylene)C(O)ON(R) o )2, where each R o It can be substituted and independently replaced by hydrogen, -C1-C6 aliphatic groups, -CH2Ph, or -O(CH2) as defined below. 0-1 Ph, -CH2- (5 to 6-membered heteroaryl ring), or 3 to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, or, despite the above definitions, two independently existing R... oTogether with the inserted atoms, they form 3 to 12 saturated, partially unsaturated, or aryl monocyclic or bicyclic rings with 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0070] R o (or two independently existing R) o Suitable monovalent substituents on the ring formed with the inserted atom can independently be halogens, -(CH2). 0-2 R ● -(halogenated R) ● -(CH2) 0-2 OH, -(CH2) 0-2 OR ● -(CH2) 0-2 CH(OR ● )2、-O(halogenated R ● ), -CN, -N3, -(CH2)0-2C(O)R ● -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● -(CH2) 0-2 SR ● -(CH2)0-2SH, -(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-chain or branched-chain alkylene)C(O)OR ● or -SSR ● , where each R ● It is either unsubstituted or, if preceded by "halogenated", substituted with only one or more halogens, and is independently selected from C1-C4 aliphatic groups, -CH2Ph, and -O(CH2). 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur. R o Suitable divalent substituents on saturated carbon atoms include =O and =S.

[0071] Suitable divalent substituents on the saturated carbon atom of the "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-, where R * When occurring independently, it is selected from hydrogen; a C1-C6 aliphatic group, which may be substituted as defined below; or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to the adjacent substituted carbon of the "optionally substituted" group include: -O(CR * 2) 2- 3O-, where R * It is selected from hydrogen each time it appears independently; a C1-C6 aliphatic group, which may be substituted as defined below; or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0072] R * Suitable substituents on aliphatic groups include halogens, -R ● -(halogenated R) ● -OH, -OR ● -O (halogenated R) ● -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● -NR ● 2 or -NO2, where each R ● It is either unsubstituted or, if preceded by "halogenated", substituted with only one or more halogens, and is independently a C1-C4 aliphatic group, -CH2Ph, -O(CH2)0-1Ph, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0073] Suitable substituents on the substituted nitrogen of the "optionally substituted" group include or Among them each Independently hydrogen; a C1-C6 aliphatic group, which may be substituted as defined below; an unsubstituted -OPh; or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, despite the above definitions, two independently existing... Together with the inserted atoms, they form 3 to 12 saturated, partially unsaturated, or aryl monocyclic or bicyclic rings with 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur.

[0074] Suitable substituents on the aliphatic group are independently halogens, -R ● -(halogenated R) ● -OH, -OR ● -O (halogenated R) ● -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● -NR ● 2 or -NO2, where each R ● It is either unsubstituted or, if preceded by "halogenated", substituted with one or more halogens, and is independently a C1-C4 aliphatic group, -CH2Ph, or -O(CH2). 0-1 Ph or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on saturated carbon atoms include =O and =S.

[0075] As used herein, the term "acetyl" refers to the group -C(O)CH3.

[0076] As used herein, the term "alkoxy" refers to -O-Cl-C 20 Alkyl group, wherein the alkoxy group is attached to the rest of the compound via an oxygen atom.

[0077] As used herein, the term "alkyl" refers to a saturated, straight-chain or branched monovalent hydrocarbon group containing 1 to 20 carbon atoms (e.g., 1 to 10 or 1 to 6). In some embodiments, the alkyl group is non-branched (i.e., linear); in some embodiments, the alkyl group is branched. Alkyl groups are, for example, but not limited to, methyl, ethyl, n-propyl and isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl, and neopentyl.

[0078] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a straight-chain or branched saturated hydrocarbon, and examples include methylene, ethylene, isopropylene, etc. The term "C" x -C y "Alkylene" indicates an alkylene having between x and y carbons. Indicative 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-C...). 10 C2-C 20C2-C6, C2-C 10 Or C2-C 20 Alkylene). In some embodiments, the alkylene may be further substituted with 1, 2, 3 or 4 substituents as defined herein.

[0079] Unless otherwise specified, as used herein, the term "alkenyl" refers to a monovalent straight-chain or branched group having 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds, and examples are vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl. Alkenyl groups include both cis and trans isomers. Unless otherwise specified, as used herein, the term "alkenylene" refers to a divalent straight-chain or branched group having 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds.

[0080] As used herein, the term "alkynyl" refers to a monovalent straight-chain or branched chain group containing a carbon-carbon triple bond and having 2 to 20 carbons (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons), and examples of such groups include ethynyl and 1-propynyl.

[0081] As used herein, the term "amino" indicates For example, -NH2 and -N(CH3)2.

[0082] As used herein, the term "aminoalkyl" refers to an alkyl moiety in which one or more carbon atoms are replaced by one or more amino moieties.

[0083] As described herein, the term "amino acid" refers to a molecule having a side chain, an amino group, and an acid group (e.g., -CO2H or -SO3H), wherein the amino acid is linked to a parent molecule group via said side chain, amino group, or acid group (e.g., 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 forming one or more peptide bonds. In some embodiments, the amino acid has the universal structure H2N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any one of the twenty 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 hydroxyl valine, isoleucine, leucine, lysine, methionine, valine, ornithine, phenylalanine, proline, pyrrolidone, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine.

[0084] As used herein, the term "aryl" refers to a monovalent monocyclic, bicyclic, or polycyclic system formed of carbon atoms, wherein the ring attached to a side group is an aromatic ring. Examples of aryl groups include phenyl, naphthyl, phenanthryl, and anthracene. An aromatic ring may be attached to its side group at any heteroatom or carbocyclic atom that produces a stable structure, and unless otherwise specified, any ring atom may optionally be substituted.

[0085] As used herein, the term “C0” signifies a bond. For example, a portion of the term -N(C(O)-(C0-C5 alkylene-H)- includes -N(C(O)-(C0 alkylene-H)-, also represented as -N(C(O)-H)-.

[0086] As used herein, the terms "carbocyclic" and "carbocyclic group" refer to a monovalent, optionally substituted 3- to 12-membered monocyclic, bicyclic, or tricyclic structure, which may be a bridging ring, a fused ring, or a spirocyclic ring, wherein all rings are formed of carbon atoms, and at least one ring is a non-aromatic ring. Carbocyclic structures include cycloalkyl, cycloalkenyl, and cycloynyl groups. Examples of carbocyclic groups include cyclohexyl, cyclohexenyl, cyclooctynyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indenyl, dihydroindenyl, decahydronaphthyl, etc. The carbocyclic ring may be attached to its side group at any ring atom that produces a stable structure, and unless otherwise specifically stated, any ring atom may be optionally substituted.

[0087] As used herein, the term "carbonyl" refers to a C(O) group, which can also be represented as C=O.

[0088] As used herein, the term "carboxyl" means -CO2H, (C=O)(OH), COOH or C(O)OH, or its unprotonated counterpart.

[0089] As used herein, the term "cyano" refers to the -CN group.

[0090] As used herein, the term "cycloalkyl" refers to a monovalent saturated cyclic hydrocarbon group, which, unless otherwise specified, can be a bridging ring, fused ring, or spiro ring having three to eight cyclic carbons, and examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cycloheptyl.

[0091] As used herein, the term "cycloalkenyl" refers to a monovalent, non-aromatic saturated cyclic hydrocarbon group, which, unless otherwise specified, can be a bridging ring, fused ring, or spiro ring having three to eight cyclic carbons and containing one or more carbon-carbon double bonds.

[0092] As used in this article, the term "diastereomer" means a stereoisomer that is not a mirror image of another and cannot be superimposed on another.

[0093] As used herein, the term "enantiomer" means each individual optically active form of the compound of the invention having at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98% optical purity or enantiomer excess (as determined by standard methods in the art).

[0094] As used herein, the term "haloacetyl" refers to an acetyl group in which at least one hydrogen atom is replaced by a halogen.

[0095] As used herein, the term "haloalkyl" refers to an alkyl moiety in which one or more carbon atoms are replaced by one or more identical or different halogen moieties.

[0096] As used herein, the term "halogen" refers to a halogen selected from bromine, chlorine, iodine, or fluorine.

[0097] As used herein, the term "heteroalkyl" refers to an alkyl group as defined herein in which at least one carbon atom is replaced by a heteroatom (e.g., an O, N, or S atom). The heteroatom may be present in the middle or at the end of the group.

[0098] As used herein, the term "heteroaryl" refers to a monovalent, monocyclic, or polycyclic structure containing at least one fully aromatic ring: that is, it contains 4n+2 π electrons within the monocyclic or polycyclic system and contains at least one heteroatom selected from N, O, or S in the aromatic ring. An illustrative unsubstituted heteroaryl has 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 fused any of the aforementioned heteroaryl rings with one or more aromatic 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. The heteroaryl ring can be attached to its side group at any ring atom that produces a stable structure, and any ring atom may optionally be substituted unless otherwise specified. In one embodiment, the heteroaryl group is substituted with 1, 2, 3, or 4 substituents.

[0099] As used herein, the term "heterocyclic alkyl" means at least one ring is a non-aromatic ring and wherein the non-aromatic ring contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, in a monovalent, monocyclic, bicyclic, or polycyclic system, which may be a bridging ring, a fused ring, or a spirocyclic ring. Five-membered rings have zero to two double bonds, and six-membered and seven-membered rings have zero to three double bonds. Illustrative unsubstituted heterocyclic alkyl groups have 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 "heterocyclic alkyl" also means a heterocyclic compound having a bridging polycyclic structure, wherein one or more carbons or heteroatoms bridge a non-adjacent member of a monocyclic ring, such as a quinine cycloyl group. The term "heterocyclic alkyl" includes bicyclic, tricyclic, and tetracyclic groups fused with any of the aforementioned heterocycles and one or more aromatic, carbocyclic, heteroaromatic, or heterocyclic rings, such as aryl, cyclohexane, cyclohexene, cyclopentane, cyclopentene, pyridine, or pyrrolidine rings. Examples of heterocyclic alkyl groups include pyrrolidinyl, piperidinyl, 1,2,3,4-tetrahydroquinolinyl, decahydroquinolinyl, dihydropyrrolopyridinyl, and decahydronaphthidyl. Heterocyclic alkyl rings may be attached to their side groups at any ring atom that produces a stable structure, and unless otherwise specified, any ring atom may optionally be substituted.

[0100] As used herein, the term "hydroxyl group" refers to the -OH group.

[0101] As used herein, the term "hydroxyalkyl" refers to an alkyl moiety in which one or more carbon atoms are replaced by one or more -OH moieties.

[0102] As used herein, the term "isomer" means any tautomer, stereoisomer, transisomer, enantiomer, or diastereomer of any compound of the present invention. It should be understood that compounds of the present invention may have one or more chiral centers or double bonds, and thus exist in stereoisomeric form, such as as double-bonded isomers (i.e., E / Z geometric isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). According to the present invention, the chemical structures described herein and therefore the compounds of the present invention cover all corresponding stereoisomers, i.e., stereoisomerically pure (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) forms, as well as mixtures of enantiomers and stereoisomers, such as racemates. The enantiomers and stereoisomers of the compounds of this invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral gas chromatography, chiral high-performance liquid chromatography, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. The enantiomers and stereoisomers can also be obtained from stereoisomerically pure or enantiomerically pure intermediates, reagents, and catalysts via well-known asymmetric synthetic methods.

[0103] As used herein, the "monovalent organic fraction" is less than 500 kDa. In some embodiments, the "monovalent organic fraction" is less than 400 kDa. In some embodiments, the "monovalent organic fraction" is less than 300 kDa. In some embodiments, the "monovalent organic fraction" is less than 200 kDa. In some embodiments, the "monovalent organic fraction" is less than 100 kDa. In some embodiments, the "monovalent organic fraction" is less than 50 kDa. In some embodiments, the "monovalent organic fraction" is less than 25 kDa. In some embodiments, the "monovalent organic fraction" is less than 20 kDa. In some embodiments, the "monovalent organic fraction" is less than 15 kDa. In some embodiments, the "monovalent organic fraction" is less than 10 kDa. In some embodiments, the "monovalent organic fraction" is less than 1 kDa. In some embodiments, the "monovalent organic fraction" is less than 500 g / mol. In some embodiments, the "monovalent organic fraction" is in the range between 500 g / mol and 500 kDa.

[0104] As used herein, the term "stereoisomer" refers to all possible different isomers and configurations that a compound (e.g., any compound of the formula described herein) may have, particularly all possible stereochemical and configurational isomers of the basic molecular structure, all diastereomers, enantiomers, or configurational isomers, including transisomers. Some compounds of the present invention may exist in different tautomer forms, all of which are included within the scope of the present invention.

[0105] As used herein, the term "sulfonyl" refers to the -S(O)2- group.

[0106] As used herein, the term "thiocarbonyl" refers to the -C(S)- group.

[0107] Those skilled in the art will understand upon reading this invention that certain compounds described herein may be provided or utilized in any of a variety of forms, such as salt form, protected form, prodrug form, ester form, isomer form (e.g., optical or structural isomers), isotopic form, etc. In some embodiments, a specific compound mentioned may refer to a specific form of that compound. In some embodiments, a specific compound mentioned may refer to the compound in any form. In some embodiments, for example, a formulation of a single stereoisomer of a compound may be considered as a different form of the compound rather than a racemic mixture of the compound; a specific salt of a compound may be considered as a different form from another salt of the compound; a formulation of a configurational isomer ((Z) or (E)) containing a double bond may be considered as a different form from a formulation of another configurational isomer ((E) or (Z)) containing the double bond; a formulation in which one or more isotopes differ from the isotopes present in a reference formulation may be considered as a different form. Brief description of the attached diagram

[0108] Figure 1 The results show that compounds A and B, representative covalent KRAS G12D inhibitors of the present invention, are effective in human pancreatic adenocarcinoma HPACKRAS. G12D / wt The compounds A and B exhibited strong and durable regulatory effects on the RAS pathway in a mouse xenograft model. Regulation of the RAS / ERK signaling pathway was assessed by measuring human DUSP6 mRNA levels in a qPCR assay. Both compounds A and B induced inhibition of DUSP6 mRNA levels in HPAC xenograft tumors 4 hours after administration, indicating strong RAS pathway regulation.

[0109] Figure 2 The results showed that compounds A and B exhibited strong tumor cross-linking at 4 hours and up to 24 hours, consistent with significant DUSP6 inhibition. (From...) Figure 1 Tumor samples collected for assay were homogenized to extract proteins. Western blots were performed on the protein lysate using Ras rabbit mAb (Abcam ab108602) and β-actin mAb (CST-4967). The presence of higher molecular weight (MW) bands (crosslinked KRAS G12D bands) in the tumor samples was detected, in which compounds are covalently bound to the KRAS G12D protein.

[0110] Figure 3AThe results showed that in the HPAC CDX mouse xenograft model with heterozygous KRAS G12D, single-drug compounds A and B, administered orally at 100 mg / kg daily, caused regression of all tumors at the end of treatment (day 28 after the start of treatment) (regression was defined as >10% tumor regression relative to baseline).

[0111] Figure 3B The results showed that no weight loss was observed with compound A (100 mg / kg po qd) or compound B (100 mg / kg po qd), indicating that in human pancreatic adenocarcinoma HPAC KRAS G12D / wt The two compounds were well tolerated at 100 mg / kg in a mouse xenograft model.

[0112] Figure 3C The results showed that in human pancreatic adenocarcinoma HPACKRAS G12D / wt In the mouse xenograft model, 8 / 10 of the tumors in compound A group (100 mg / kg po qd) and 9 / 10 of the tumors in compound B group (100 mg / kg po qd) achieved complete regression by day 28 (complete regression was defined as >85% tumor regression relative to baseline). Detailed Implementation

[0113] compound

[0114] This document provides Ras inhibitors. The methods described herein require the formation of a high-affinity three-component complex or conjugate between a synthetic ligand and two intracellular proteins that do not interact under normal physiological conditions: the target protein of interest (e.g., Ras), and a cytosolic chaperone protein (presenting protein) (e.g., cyclophilin A) that is widely expressed in the cell. More specifically, in some embodiments, the Ras inhibitors described herein induce a new binding pocket in Ras by driving the formation of a high-affinity triple complex or conjugate between the Ras protein and the widely expressed cytosolic chaperone protein cyclophilin A (CYPA). Without being bound by theory, the inventors believe that one way the compounds of the present invention, and the complexes or conjugates thereto, exert their inhibitory effect on Ras is by spatially blocking the interaction sites between Ras and downstream effector molecules such as RAF required for the propagation of oncogenic signals.

[0115] Not bound by theory, the inventors hypothesize that the covalent and non-covalent interactions of the compounds of this invention with Ras and chaperone proteins (e.g., cyclic protein A) can contribute to the inhibition of Ras activity. In some embodiments, the compounds of this invention form covalent adducts with the side chains of Ras proteins (e.g., the -CH2-COOH or -CH2-COO- side chains of aspartic acid at positions 12 or 13 of mutant Ras proteins). Covalent adducts may also be formed with other side chains of Ras. Alternatively or additionally, non-covalent interactions may play a role: for example, van der Waals interactions, hydrophobic interactions, hydrophilic interactions, and hydrogen bonding interactions, and combinations thereof, can contribute to the ability of the compounds of this invention to form complexes and act as Ras inhibitors. Therefore, the compounds of the present invention can inhibit a variety of Ras proteins (e.g., K-Ras, N-Ras, H-Ras and their mutants with mutations at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D and Q61L, and other mutants described herein).

[0116] Methods for determining the formation of covalent adducts are known in the art. One method for determining the formation of covalent adducts is to perform a "crosslinking" determination, as described below:

[0117] Note: The following procedure describes a method for monitoring the crosslinking of K-Ras G12D (GMP-PNP) with the compounds of this invention. This procedure can also be implemented using other Ras proteins or nucleotides.

[0118] The purpose of this biochemical assay was to measure the ability of the test compound to covalently label a nucleotide-loaded K-Ras isoform. A stock solution of 5 μM K-Ras(1-169)G12D loaded with GMP-PNP was diluted 10-fold to a final concentration of 0.5 μM in an assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 5 μM cyclic protein A, and 2 μM of the test compound; the final sample volume was 100 μL.

[0119] Samples were incubated at 25°C for up to 24 hours, followed by quenching with 10 μL of 5% formic acid. The quenched samples were centrifuged at 15,000 rpm for 15 minutes in a benchtop centrifuge, and then 10 μL aliquots were injected into a reversed-phase C4 column and eluted to a mass spectrometer using an increasing acetonitrile gradient in the mobile phase. Analysis of the raw data was performed using Waters MassLynxMS software, where the binding percentage was calculated from the deconvolution of labeled and unlabeled K-Ras protein peaks.

[0120] Therefore, this article provides a compound having the structure of formula 0 or a pharmaceutically acceptable salt thereof:

[0121]

[0122] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0123] X 1 X 2 and X 3 Each is independently selected from CH2, CF2, C=O, or O;

[0124] m is 1 or 2;

[0125] n is 0 or 1;

[0126] R 1 It is hydrogen, optionally substituted C1-C6 heteroalkyl or optionally substituted 3 to 10-membered heterocyclic alkyl;

[0127] R 2 It is an optional substituted C1-C6 alkyl group; and

[0128] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0129] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0130] In some embodiments, the compounds of the present invention have the structure of Formula I, or a pharmaceutically acceptable salt thereof:

[0131]

[0132] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0133] X 1 X 2 and X 3 Each is independently selected from CH2, CF2, C=O, or O;

[0134] m is 1 or 2;

[0135] n is 0 or 1;

[0136] R 1 It is hydrogen, optionally substituted C1-C6 heteroalkyl or optionally substituted 3 to 10-membered heterocyclic alkyl;

[0137] R 2It is an optional substituted C1-C6 alkyl group; and

[0138] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0139] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0140] In some embodiments, the compounds of the present invention have the structure of formula Ia, formula Ib, formula Ic, or a pharmaceutically acceptable salt thereof:

[0141]

[0142]

[0143] Each D indicates that the isotopic enrichment factor of deuterium is at least 5 hydrogens.

[0144] In some embodiments, the compounds of the present invention have the structure of Formula II, or a pharmaceutically acceptable salt thereof:

[0145]

[0146] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0147] R 2 It is an optional substituted C1-C6 alkyl group; and

[0148] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0149] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0150] In some embodiments, the compounds of the present invention have the structure of formula V, or a pharmaceutically acceptable salt thereof:

[0151]

[0152] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0153] R 2 It is an optional substituted C1-C6 alkyl group; and

[0154] R 3It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0155] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0156] In some embodiments, the compounds of the present invention have the structure of formula VI, or a pharmaceutically acceptable salt thereof:

[0157]

[0158] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0159] R 2 It is an optional substituted C1-C6 alkyl group; and

[0160] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0161] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0162] In some embodiments, the compounds of the present invention have the structure of formula VII, or a pharmaceutically acceptable salt thereof:

[0163]

[0164] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0165] R 2 It is an optional substituted C1-C6 alkyl group; and

[0166] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0167] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0168] In some embodiments, the compounds of the present invention have the structure of formula Va, formula Vb, formula Vc, or a pharmaceutically acceptable salt thereof:

[0169]

[0170] Each D indicates that the isotopic enrichment factor of deuterium is at least 5 hydrogens.

[0171] In some embodiments, the compounds of the present invention have the structure of formula Vd, formula Ve, formula Vf, or a pharmaceutically acceptable salt thereof:

[0172]

[0173] Each D indicates that the isotopic enrichment factor of deuterium is at least 5 hydrogens.

[0174] In some embodiments, A is an optionally substituted thiazole-diyl, optionally substituted oxazole-diyl, optionally substituted morpholine-diyl, optionally substituted pyrrolidine-diyl, optionally substituted piperidine-diyl, or optionally substituted phenylene. In some embodiments, A is an optionally substituted thiazole-diyl or optionally substituted morpholine-diyl. In some embodiments of the compounds of the present invention, A is an optionally substituted 5- to 10-membered heteroaryl group. In some embodiments, A is: In some implementation schemes, A is

[0175] In some embodiments of the compounds of the present invention, A is an optionally substituted phenylene. In some embodiments, A is: In some implementation schemes, A is

[0176] In some embodiments of the compounds of the present invention, A is an optionally substituted 3- to 6-membered heterocyclic alkylene group. In some embodiments, A is an optionally substituted 6-membered heterocyclic alkylene group. In some embodiments, A is selected from the following or their stereoisomers: In some embodiments, A is selected from the following or their stereoisomers:

[0177] In some embodiments of the compounds of the present invention, R 1 It is hydrogen or optionally substituted 3- to 10-membered heterocyclic alkyl. In some embodiments of the compounds of the present invention, R 1 It is an optionally substituted 3- to 10-membered heterocyclic alkyl group. In some embodiments of the compounds of the present invention, R 1 yes:

[0178] In some embodiments of the compounds of the present invention, R 1 yes: Each D indicates that the isotopic enrichment factor of deuterium is at least 5 hydrogens.

[0179] In some embodiments of the compounds of the present invention, R 2 yes:

[0180] In some embodiments of the compounds of the present invention, R 2 yes: Furthermore, each D indicates that the isotopic enrichment factor of deuterium is at least 5 for hydrogen.

[0181] In some embodiments of the compounds of the present invention, R 3 It is an optionally substituted C1-C6 alkyl or an optionally substituted 3- to 6-membered cycloalkyl. In some embodiments of the compounds of the present invention, R 3 It is an optional substituted C1-C6 alkyl group. In some embodiments, R 3 yes: In some implementation schemes, R 3 yes: In some implementation schemes, R 3 yes: Furthermore, each D indicates that the isotopic enrichment factor of deuterium is at least 5 for hydrogen.

[0182] In some embodiments of the compounds of the present invention, R 3 It is, or optionally, a substituted 3- to 6-membered cycloalkyl group. In some embodiments, R 3 yes: In some implementation schemes, R 3 yes:

[0183] In some embodiments of the compounds of the present invention, R 2 yes R 3 yes And A is

[0184] In some implementation schemes, R 2 yes R 3 yes And A is

[0185] In some embodiments of the compounds of the present invention, m is 1. In some embodiments, n is 1. In some embodiments, X 1 It is CH2. In some implementations, X 2 It is CH2. In some implementations, X 3 It is CH2. In some implementations, m is 1, n is 1, and X 1 X 2 and X 3 Each is CH2.

[0186] In some embodiments, the compounds of the present invention are selected from Table 1 or their pharmaceutically acceptable salts or stereoisomers.

[0187] Table 1: Some compounds of the present invention

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] It should be noted that bonds in some compounds are shown as straight lines or wedges. In some cases, the relative stereochemistry of the stereoisomers has been determined; in others, 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 table above are covered in this invention. In certain embodiments, the transisomers of the compounds in the table above are covered.

[0203] In some embodiments, compounds from Table 2 or pharmaceutically acceptable salts thereof are provided. In some embodiments, the compounds of the present invention are selected from Table 2 or pharmaceutically acceptable salts thereof or transisomers thereof.

[0204] Table 2: Some compounds of the present invention

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] It should be noted that bonds in some compounds are shown as straight lines or wedges. In some cases, a single embodiment number corresponds to a mixture of stereoisomers. All stereoisomers of the compounds in the table above are covered in this invention. In certain embodiments, the transisomers of the compounds in the table above are covered.

[0213] In some embodiments, the compounds of the present invention are selected from the compounds in Table 3 or their pharmaceutically acceptable salts or stereoisomers. In some embodiments, the compounds of the present invention are selected from the compounds in Table 3 or their pharmaceutically acceptable salts or transisomers.

[0214] In some embodiments, the compounds of the present invention are not selected from the compounds in Table 3. The compounds of the present invention are not selected from the compounds in Table 3 or their pharmaceutically acceptable salts or stereoisomers. In some embodiments, the compounds of the present invention are not selected from the compounds in Table 3 or their pharmaceutically acceptable salts or transisomers.

[0215] Table 3. Some compounds

[0216]

[0217]

[0218]

[0219] In some embodiments, the compounds of the present invention include a crosslinking group (e.g., an optionally substituted aziridine moiety) bound to an organic portion, which is a Ras-binding moiety, wherein, upon contact with a Ras protein, the compound binds to the Ras protein to form a conjugate. For example, the crosslinking group (e.g., an optionally substituted aziridine moiety) of the compound may bind to, for example, 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 residues of the K-Ras Switch-II binding pouch of the K-Ras protein. In some embodiments, the Ras-binding moiety is an H-Ras-binding moiety that binds to residues of the H-Ras Switch-II binding pouch of the H-Ras protein. In some embodiments, the Ras-binding moiety is an N-Ras-binding moiety that binds to residues of the N-Ras Switch-II binding pouch of the N-Ras protein. The molecular weight of the Ras-binding moiety is typically less than 1200 Da. For a description of the domains of the Ras protein, see, for example, Johnson et al., 292:12981-12993 (2017), which is incorporated herein by reference.

[0220] In some embodiments, the compounds of the present invention are or are used as prodrugs, such as for administration to cells or to a subject in need.

[0221] Pharmaceutical compositions are also provided, comprising the compounds of the present invention or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients.

[0222] A conjugate or salt thereof is also provided, the conjugate or salt thereof comprising the structure of formula III:

[0223] MP 1

[0224] Formula III

[0225] Where P 1 It is the unit price organic portion; and

[0226] M has the structure of formula IV:

[0227]

[0228] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0229] X 1 X 2 and X 3Each is independently selected from CH2, CF2, C=O, or O;

[0230] m is 1 or 2;

[0231] n is 0 or 1;

[0232] R 1 It is hydrogen, optionally substituted C1-C6 heteroalkyl or optionally substituted 3 to 10-membered heterocyclic alkyl;

[0233] R 2 It is an optional substituted C1-C6 alkyl group; and

[0234] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0235] Furthermore, each hydrogen atom in Formula IV is independently and optionally enriched with the isotope deuterium.

[0236] 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, M binds to an amino acid residue of the monovalent organic moiety.

[0237] In some embodiments, the compounds of the present invention have improved oral bioavailability (%F) compared to compounds known in the art. Methods for measuring oral bioavailability are known in the art, and one such method is provided below:

[0238] Oral bioavailability was determined in BALB / c mice. Following intravenous (IV) bolus and oral (PO) administration of the test compounds, approximately 30 μL of whole blood samples were collected at specified time points into tubes containing K₂EDTA. For some compounds, blood samples were centrifuged at 4600 rpm for approximately 5 minutes at 4°C, and plasma samples were stored at -80°C before bioanalysis. Blood or plasma samples were extracted by protein precipitation and analyzed by tandem mass spectrometry (UPLC MS / MS) using positive ion electrospray ionization on an AB Sciex Triple Quad 6500+ mass spectrometer in conjunction with an Acquity UPLC system.

[0239] All PK parameters can be obtained using non-compartmental analysis with WinNonlin, derived from data on blood (or plasma) concentration changes over time. Bioavailability (F%, also written as %F) is estimated using the following formula:

[0240]

[0241] AUC inf,PO It is the area under the curve of blood (or plasma) concentration over a time period from zero to infinity after PO administration.

[0242] AUC inf,IV It is the area under the curve of blood (or plasma) concentration over a period of time from zero to infinity after IV administration.

[0243] dose IV This is the total dose administered via IV.

[0244] dose PO It is the total dose of PO administered.

[0245] Generally, an F% (or %F) value exceeding 10% is preferred.

[0246] Of the 19 compounds in Table 1 described herein for testing oral bioavailability, all but three compounds had a %F greater than 10%. Furthermore, in the biochemical crosslinking assay described herein, all but three compounds crosslinked more than 60% of the K-Ras G12D residues over a 6-hour period. Thirteen of the tested compounds had a %F greater than 10% and crosslinked more than 60% of the K-Ras G12D residues. Without being bound by theory, the inventors propose that the N-methylazacyclopropane moiety of the compounds described herein is primarily responsible for this unexpected activity.

[0247] In some embodiments, the compounds of the present invention are selective for one or more specific Ras mutants relative to other Ras mutants or wild-types, compared to compounds known in the art. Methods for measuring this selectivity are known in the art, such as Ras-Raf binding assays, i.e., the methods provided herein:

[0248] The purpose of this biochemical assay was to measure the ability of the test compound to promote the formation of a ternary complex between a nucleotide-loaded Ras isoform and cyclic protein A; and to obtain the ternary complex disruption and BRAF. RBD The binding of the constructs suppresses Ras signal transduction through the RAF effector.

[0249] In an assay buffer containing 25 mM HEPES pH 7.3, 0.002% Tween 20, 0.1% BSA, 100 mM NaCl, and 5 mM MgCl2, label-free cyclophilic protein A, His6-K-Ras-GMPPNP (or other Ras variants), and GST-BRAF were added. RBDCompounds were combined in 384-well plates at final concentrations of 25 μM, 12.5 nM, and 50 nM, respectively. The compounds in each well were serially diluted 10 times (3-fold) starting from a final concentration of 30 μM. After incubation at 25°C for 3 hours, a mixture of anti-His Eu-W1024 and anti-GST allophycocyanin was added to the sample wells to achieve final concentrations of 10 nM and 50 nM, respectively, and the reaction was incubated for another 1.5 hours. The TR-FRET signal was read using a microplate reader (Ex 320 nm, Em 665 / 615 nm). Compounds that promoted the destruction of the K-Ras:RAF complex were identified as those that reduced the TR-FRET ratio relative to the DMSO control wells.

[0250] Therefore, in some embodiments, the compounds of the present invention are effective against KRAS relative to the Ras mutant or relative to the wild type. G12D It exhibits selectivity. The compounds of this invention also exhibit significant selectivity for other RAS mutants or combinations thereof disclosed herein.

[0251] In some embodiments, the compounds of the present invention are more potent against one or more specific Ras mutants than against other Ras mutants or wild-types compared to compounds known in the art. Methods for measuring this potency are known in the art, such as the pERK assay, the protocol of which is provided in the following examples. Therefore, in some embodiments, the compounds of the present invention are more potent against KRAS than compounds known in the art. G12D The compounds of this invention exhibit high potency. They also exhibit high potency against other RAS mutants or combinations thereof disclosed herein.

[0252] In some embodiments, compared to compounds known in the art, and relative to other Ras mutants or wild-types, the compounds of the present invention exhibit a stronger detrimental effect on cell viability against one or more specific Ras mutants. Methods for measuring cell viability are known in the art, such as those described herein. Cell viability assay:

[0253] Note – The following protocol describes a procedure for monitoring cell viability in K-Ras mutant cancer cell lines that respond to the compounds of this invention. Other RAS isoforms may also be used, but the number of cells seeded will vary depending on the cell line used.

[0254] The purpose of this cell assay is to use... 2.0 Reagent (Promega) The amount of ATP present at the quantitative endpoint was used to determine the effect of the tested compound on the proliferation of human Ras cancer cell lines (e.g., NCI-H358 (K-RasG12C), AsPC-1 (K-Ras G12D), and Capan-1 (K-Ras G12V)) during the 5-day treatment period.

[0255] Cells were seeded at 250 cells / well in 40 μL of growth medium in 384-well assay plates and incubated overnight at 37°C in a humid atmosphere containing 5% CO2. On the day of assay, firstly, the 10 mM stock solution of the test compound was diluted to 3 mM with 100% DMSO. 15 μL of the well-mixed compound solution was transferred to the next batch of wells containing 30 μL of 100% DMSO, and this process was repeated until nine serial dilutions of 3-fold concentration were prepared (the initial assay concentration was 10 μM). The test compound (132.5 nL) was directly applied to the assay plates containing cells. Each plate was shaken at 300 rpm for 15 seconds, centrifuged, and incubated at 37°C in a humid atmosphere containing 5% CO2 for 5 days. On day 5, the assay plates and their contents were equilibrated to room temperature for approximately 30 minutes. Add... 2.0 μL of reagent was added, and the contents of the plate were mixed on a cyclotron for 2 minutes, followed by incubation at room temperature for 10 minutes. Cold light was measured using a PerkinElmer Enspire. Data were normalized using the following formula: (sample signal / average DMSO) * 100. Four-parameter logistic fitting was used to fit the data.

[0256] Therefore, in some embodiments, the compounds of the present invention are targeted at KRAS compared to compounds known in the art. G12D The compounds exhibit a significant reduction in cell viability. Furthermore, the compounds of this invention also exhibit a significant reduction in cell viability against other RAS mutants or combinations thereof disclosed herein.

[0257] In some embodiments, the compounds of the present invention may exhibit higher metabolic stability, permeability, or solubility, or combinations thereof, compared to compounds known in the art. The compounds of the present invention may, for example, exhibit improved acid stability in simulated gastric juice stability assays. Methods for measuring these properties are known in the art. The compounds of the present invention may exhibit better Ras crosslinking compared to compounds known in the art. Methods for measuring Ras crosslinking are provided herein. In some embodiments, the compounds of the present invention may exhibit improvements in any or a combination of the following properties compared to compounds known in the art: selectivity, potency, cell viability, metabolic stability, acid stability, crosslinking, permeability, or solubility.

[0258] A method for treating a subject with cancer is also provided, comprising administering to the subject a therapeutically effective amount of the compound of the present invention, or a pharmaceutically acceptable salt thereof. The cancer may be, for example, pancreatic cancer, colorectal cancer, non-small cell lung cancer, acute myeloid leukemia, multiple myeloma, thyroid adenocarcinoma, myelodysplastic syndrome, or squamous cell lung cancer. In some embodiments, the cancer comprises a Ras mutation, such as K-Ras G12D or K-Ras G13D. Other Ras mutations are also described herein.

[0259] A method for treating Ras protein-related conditions in a subject of need is also provided, comprising administering to the subject a therapeutically effective amount of the compound of the present invention, or a pharmaceutically acceptable salt thereof.

[0260] A method for inhibiting Ras protein in cells is also provided, comprising contacting the cells with an effective amount of the 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 also described herein. The cells may be cancer cells, such as pancreatic cancer cells, colorectal cancer cells, non-small cell lung cancer cells, acute myeloid leukemia cells, multiple myeloma cells, thyroid adenocarcinoma cells, myelodysplastic syndrome cells, or squamous cell lung cancer cells. Other cancer types are also described herein. The cells may be in vivo or in vitro.

[0261] For the compounds of this invention, one stereoisomer may exhibit a stronger inhibitory effect than another stereoisomer. For example, one transisomer may exhibit an inhibitory effect, while another transisomer may exhibit very little or no inhibitory effect.

[0262] In some embodiments, the methods or uses described herein further include administering additional anticancer therapies. In some embodiments, the additional anticancer 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 anticancer therapy is an SHP2 inhibitor. Other additional anticancer therapies are also described herein.

[0263] Synthesis method

[0264] The compounds described herein can be prepared from commercially available starting materials or synthesized using known organic, inorganic, or enzymatic methods.

[0265] The compounds of this invention can be prepared by a variety of methods well known to those skilled in the art of organic synthesis. For example, the compounds of this invention can be synthesized using the methods described in the following embodiments, as well as synthetic methods known in synthetic organic chemistry techniques or modifications thereof as understood by those skilled in the art. These methods include, but are not limited to, those described in the following embodiments.

[0266] The compounds in Table 1 of this document were prepared using the methods disclosed herein, or using the methods disclosed herein in combination with the knowledge of those skilled in the art. The compounds in Table 2 were prepared using the methods disclosed herein, or using the methods disclosed herein in combination with the knowledge of those skilled in the art.

[0267] Option 1. General synthetic method for macrocyclic nitrogen-containing heterocyclic propanes

[0268]

[0269] As shown in Scheme 1, this class of compounds can be prepared by reacting a suitable amine (1) with a carboxylic acid (2) containing a protected amine in the presence of a standard amide coupling agent to give 3, followed by deprotection of the amine to produce 4. Azacyclopropane carboxylate (5) is coupled in the presence of a standard amide coupling agent to give the final compound (6).

[0270] Pharmaceutical Compositions and Methods of Use

[0271] Pharmaceutical Compositions and Administration

[0272] The compounds involved in this invention are Ras inhibitors and can be used to treat cancer. Therefore, one embodiment of this invention provides pharmaceutical compositions comprising the compounds of this invention or pharmaceutically acceptable salts thereof and pharmaceutically acceptable excipients, and methods for preparing such compositions using the compounds of this invention.

[0273] As used herein, the term "pharmaceutical composition" refers to a compound formulated with a pharmaceutically acceptable excipient, such as the compounds of this invention, or a pharmaceutically acceptable salt thereof.

[0274] In some embodiments, the compound is present in the pharmaceutical composition in a unit dose suitable for administration in a treatment regimen, and the compound shows a statistically significant likelihood of achieving the intended therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those suitable for administration by: oral administration, such as liquids (aqueous or non-aqueous solutions or suspensions), tablets (e.g., intended for buccal, sublingual, and systemic absorption), pills, powders, granules, or pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, such as sterile solutions or suspensions, or sustained-release formulations; surface administration, such as creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or mouth; intravaginal or rectal administration, such as pessaries, creams, or foams; sublingual administration; ocular administration; transdermal administration; or nasal, pulmonary, and other mucosal surfaces.

[0275] As used herein, “pharmaceuticalally acceptable excipient” means any inactive ingredient (e.g., a medium that suspends or dissolves an active compound) that is non-toxic and non-inflammatory in the body of a subject. Typical excipients include, for example: anti-adhesion agents, antioxidants, adhesives, coating agents, compression aids, disintegrants, dyes (pigments), softeners, emulsifiers, fillers (diluents), film-forming agents or coating agents, flavoring agents, fragrances, flow enhancers, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or water for hydration. Excipients include, but are not limited to: optionally substituted butylated hydroxytoluene (BHT), calcium carbonate, calcium hydrogen phosphate, calcium stearate, croscarmellose, croscarmellose, citric acid, croscarmellose, cysteine, ethyl cellulose, gelatin, optionally substituted hydroxypropyl cellulose, optionally substituted hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium glycolate starch, sorbitol, starch (corn starch), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. Those skilled in the art understand a variety of reagents and materials that can be used as excipients. See, for example, 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.

[0276] Unless explicitly stated otherwise, the compounds described herein, whether explicitly stated or not, may be provided or used in salt form, for example, in a pharmaceutically acceptable salt form. As used herein, the term "pharmaceutically acceptable salt" means a salt of a compound described herein that, to the extent of reasonable medical judgment, is suitable for use in contact with human tissues without excessive toxicity, irritation, anaphylactic reactions, etc., and is 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 Pharmaceutical Salts: Properties, Selection, and Use, (edited by P.H. Stahl and C. G. Germuth), Wiley-VCH, 2008. The salt may be prepared in situ during the final isolation and purification of the compounds described herein, or isolated by reacting a free basic group with a suitable organic acid.

[0277] The compounds of the present invention may have ionizable groups, thereby enabling their preparation into pharmaceutically acceptable salt forms. These salts may be acid addition salts involving inorganic or organic acids, or, if the compounds of the present invention are in acid form, the salts may be prepared from inorganic or organic bases. In some embodiments, the compounds are prepared or used in pharmaceutically acceptable salt forms, which are prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art, such as hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid, or tartaric acid, for forming acid addition salts; and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, etc., for forming base salts. Methods for preparing suitable salts are recognized in the art.

[0278] Representative acid addition salts include acetates, adipates, alginates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, hydrogen sulfates, borates, butates, camphorates, camphor sulfonates, citrates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, transbutenedioates, glucoheponicates, glycerol phosphates, hemisulfates, heptanates, hexanoates, hydrobromates, hydrochlorides, hydroiodates, and 2-optionally substituted hydroxy-ethyl groups. Alkane sulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methane sulfonates, 2-naphthalene sulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, dihydroxynaphthalates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, neopentanoates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, toluene sulfonates, undecanoates, valerates, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts, 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.

[0279] As used herein, the term "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to a human at any developmental stage. In some embodiments, "subject" refers to a human patient. In some embodiments, "subject" refers to a non-human animal. In some embodiments, a non-human animal is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate, or pig). In some embodiments, a subject includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, or insects. In some embodiments, a subject may be a transgenic animal, a genetically engineered animal, or a clone.

[0280] As used herein, the term "dosage form" refers to a physically discrete unit of a compound (e.g., the compound of the present invention) intended for administration to a subject. Each unit contains a predetermined amount of the compound. In some embodiments, the amount is an amount (or a portion thereof) of a unit dose suitable for administration according to a dosing regimen that is determined to be associated with a desired or beneficial outcome when administered to the relevant population (i.e., according to a therapeutic dosing regimen). Those skilled in the art will understand that the total amount of a therapeutic composition or compound administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0281] As used herein, the term "dosing regimen" refers to a set of unit doses (typically more than one unit dose) individually administered to a subject, said unit doses typically spaced at intervals of time. In some embodiments, a given therapeutic compound (e.g., the compound of the present invention) has a recommended dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each spaced at equal intervals; in some embodiments, the dosing regimen comprises multiple doses with at least two distinct time intervals separating the individual doses. In some embodiments, all doses within the dosing regimen are amounts of the same unit dose. In some embodiments, the different doses within the dosing regimen are different amounts. In some embodiments, the dosing regimen comprises a first dose in the amount of a first dose, followed by one or more additional doses in the amount of a second dose different from that first dose. In some embodiments, the dosing regimen comprises a first dose in the amount of a first dose, followed by one or more additional doses in the amount of a second dose identical to that first dose. In some embodiments, the dosing regimen, when administered to a relevant population (i.e., a therapeutic dosing regimen), is associated with desired or beneficial outcomes.

[0282] "Treatment regimen" refers to a dosage regimen in which a patient is administered a treatment to a population in relation to the desired or beneficial therapeutic outcome.

[0283] The term "treatment" ("treatment / treat / treating") in its broadest sense refers to any application of a substance (e.g., the compounds of this invention) that partially or completely relieves, improves, reduces, or inhibits one or more symptoms, features, or causes of a particular disease, condition, or disorder; delays its onset; reduces its severity; or decreases its occurrence. In some embodiments, such treatment may be administered to a subject who does not exhibit signs of the relevant disease, condition, or disorder, or to a subject who exhibits only early signs of the disease, condition, or disorder. Alternatively or additionally, in some embodiments, treatment may be administered to a subject exhibiting one or more defined signs of the relevant disease, condition, or disorder. In some embodiments, treatment may be used on a subject diagnosed with the relevant disease, condition, or disorder. In some embodiments, treatment may be used on a subject known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, condition, or disorder.

[0284] The term "therapeuticly effective amount" means an amount sufficient to treat a disease, condition, or disorder when administered according to a therapeutic dosing regimen to a population suffering from or susceptible to that disease, condition, or disorder. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence or severity of one or more symptoms of the disease, condition, or disorder, or delays its onset. Those skilled in the art will understand that the term "therapeuticly effective amount" does not actually require achieving the desired successful treatment in a particular individual. In fact, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a substantial number of subjects when administered to a patient requiring the treatment. It is particularly important to understand that a particular subject may actually be "therapeuticly effective" and "treatment-resistant." In some embodiments, the reference to a therapeutically effective amount may refer to an amount measured in one or more specific tissues (e.g., tissues affected by the disease, condition, or disorder) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). Those skilled in the art will understand that in some embodiments, a therapeutically effective amount may be formulated as a single dose or administered in a single dose. In some implementations, the therapeutically effective dose may be formulated as multiple doses, for example, as part of a dosing regimen, or administered in multiple doses.

[0285] For use as treatment of subjects, the compounds of the present invention or pharmaceutically acceptable salts thereof may be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the desired type of treatment, such as prevention, control, or treatment, the compounds or pharmaceutically acceptable salts thereof are formulated in accordance with the parameters described herein. An overview of such techniques can be found in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, edited by J. Swarbrick and J.C. Boylan, 1988–1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0286] The compositions can be prepared according to conventional mixing, granulation, or coating methods, and the pharmaceutical compositions of the present invention may contain, by weight or volume, about 0.1% to about 99%, about 5% to about 90%, or about 1% to about 20% of the compounds of the present invention or their pharmaceutically acceptable salts. In some embodiments, the amount of the compounds described herein or their pharmaceutically acceptable salts present may be, by weight, 1-95% of the total amount of the pharmaceutical composition.

[0287] The composition may be provided in dosage forms suitable for administration as follows: intra-articular, oral, parenteral (e.g., intravenous, intramuscular), rectal, skin, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intracystic, intraurethral, ​​intrathecal, epidural, ocular, or by injection, inhalation, or direct contact with the nasal, genitourinary, genital, or oral mucosa. Therefore, the pharmaceutical composition may be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels (including hydrogels), pastes, ointments, creams, plasters, solutions, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, formulations suitable for iontophoresis delivery, or aerosols. The composition may be formulated according to conventional pharmaceutical practice.

[0288] As used herein, the term "administration" means administering a composition (e.g., a compound or a formulation comprising a compound as described herein) to a subject or system. Administration to animal subjects (e.g., to humans) can be performed via any suitable route. For example, in some embodiments, administration can be via bronchial (including bronchial infusion), buccal, intestinal, intradermal, intraarterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intrasacral, mucosal, nasal, oral, rectal, subcutaneous, sublingual, surface, tracheal (including intratracheal infusion), percutaneous, vaginal, or vitreous administration.

[0289] Formulations can be prepared in a manner suitable for systemic or topical administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous, or subcutaneous) or those prepared for transdermal, transmucosal, or oral administration. Formulations will generally include diluents and, in some cases, adjuvants, buffers, preservatives, etc. Compounds or pharmaceutically acceptable salts thereof may also be administered as liposomal compositions or as microemulsions.

[0290] For injection, formulations can be prepared in conventional forms, such as liquid solutions or suspensions, or in solid forms, or emulsions, suitable for preparation as solutions or suspensions in liquids prior to injection. Suitable excipients include, for example, water, physiological saline, dextrose, glycerin, etc. These compositions may also contain a certain amount of non-toxic excipients, such as wetting agents or emulsifiers, pH buffers, etc., such as sodium acetate, dehydrated sorbitol monolaurate, etc.

[0291] Various sustained-release drug delivery systems have also been designed. See, for example, U.S. Patent No. 5,624,677.

[0292] Systemic administration may also include relatively non-invasive methods such as 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. It will be understood in the art that suitable forms include syrups, capsules, and tablets.

[0293] Each compound described herein, or its pharmaceutically acceptable salt, can be formulated in a variety of ways known in the art. For example, the first and second agents in a combination therapy can be formulated together or separately. Other modalities of combination therapy are also described herein.

[0294] Individual or separately formulated pharmaceutical preparations may be packaged together as a kit. Non-limiting examples include, but are not limited to, kits containing, for example, two pills, one pill and powder, suppositories, or liquids in vials, two types of topical creams, etc. The kit may include optional components to facilitate the administration of a unit dose to a subject, such as vials for reconstitution of the powder form, syringes, custom IV delivery systems, inhalers, etc. Additionally, the unit dose kit may contain instructions for the preparation and administration of the composition. The kit may be manufactured as a single-use unit dose for one subject, for multiple uses for a specific subject (at a constant dose, or where the potency of individual compounds or their pharmaceutically acceptable salts may vary with treatment progression); or the kit may contain multiple doses suitable for administration to multiple subjects (“integrated package”). The kit components may be assembled in cartons, blister packs, bottles, tubes, etc.

[0295] Formulations for oral use include tablets containing a mixture of an active ingredient and a non-toxic, pharmaceutically acceptable excipient. The excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating agents and disintegrants (e.g., cellulose derivatives, including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, alginate, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginate, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, optionally substituted hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, flow aids, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable excipients may include colorants, flavoring agents, plasticizers, humectants, buffers, etc.

[0296] Two or more compounds may be mixed together in tablets, capsules or other media, or they may be separated. In one embodiment, the first compound is contained on the inside of the tablet and the second compound is on the outside, thereby releasing the majority of the second compound before the first compound is released.

[0297] Formulations for oral use may also be provided as chewable tablets or as hard gelatin capsules, wherein 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, wherein the active ingredient is mixed with an aqueous or oil medium, such as peanut oil, liquid paraffin, or olive oil. Powders, granules, and fine pellets may be prepared using the ingredients mentioned above for tablets and capsules, in a conventional manner, using equipment such as mixers, fluidized bed apparatus, or spray drying equipment.

[0298] Controlled release through dissolution or diffusion can be achieved by appropriately coating the compound with tablets, capsules, fine granules, or granules, or by incorporating the compound or a pharmaceutically acceptable salt thereof into a suitable matrix. Controlled release coatings may include one or more of the coating substances mentioned above, such as shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glyceryl palmitate, ethyl cellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-optionally substituted hydroxymethacrylate, methacrylate hydrogel, 1,3-butanediol, ethylene glycol methacrylate, or polyethylene glycol. In controlled-release matrix formulations, matrix materials may also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene or halogenated fluorocarbons.

[0299] Liquid forms of compounds or pharmaceutically acceptable salts and compositions thereof that can be incorporated into the present invention for oral administration include aqueous solutions, appropriately flavored syrups, aqueous or oily suspensions, emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical mediators.

[0300] Generally, when administered to humans, the oral dose of any compound of the present invention or a pharmaceutically acceptable salt thereof will depend on the nature of the compound and can be readily determined by those skilled in the art. Dosages can be, for example, from about 0.001 mg to about 2000 mg daily, from about 1 mg to about 1000 mg daily, from about 5 mg to about 500 mg daily, from about 100 mg to about 1500 mg daily, from about 500 mg to about 1500 mg daily, from about 500 mg to about 2000 mg daily, or any range derived therefrom. In some embodiments, the daily dose range for oral administration may, for example, be from about 0.001 mg to about 2000 mg per kilogram of human body weight, administered in a single dose or divided doses. On the other hand, in some cases, doses outside these limits may be necessary.

[0301] In some embodiments, the pharmaceutical composition may further comprise additional compounds having antiproliferative activity. Depending on the administration mode, the compound or a pharmaceutically acceptable salt thereof will be formulated to suit the composition for delivery. Each compound or pharmaceutically acceptable salt thereof in the combination therapy may be formulated in a variety of ways known in the art. For example, the first and second agents in the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together so that the agents are administered simultaneously or nearly simultaneously.

[0302] It should be understood that the compounds and pharmaceutical compositions of the present invention can be formulated and used in combination therapies, i.e., the compounds and pharmaceutical compositions can be formulated together with one or more other desired therapeutic agents or medical procedures, or administered concurrently with, before, or after the administration of such one or more other desired therapeutic agents or medical procedures. The specific combination of each therapy (therapeutic agent or procedure) used in the combination regimen should take into account the compatibility of the desired therapeutic agent or procedure with the desired therapeutic effect to be achieved. It should also be understood that the therapies employed may achieve the desired effect for the same condition, or these therapies may achieve different effects (e.g., control any adverse effects).

[0303] As described in this article, each drug in the combination therapy can be administered independently, one to four times daily, for one day to one year, or even for the subject's lifetime. Chronic / long-term administration is also applicable.

[0304] How to use

[0305] In some embodiments, the present invention discloses a method for treating a disease or condition characterized by abnormal Ras activity caused by a Ras mutant. In some embodiments, the disease or condition is cancer.

[0306] Therefore, a method for treating cancer in a subject of need is also provided, the method comprising administering to the subject a therapeutically effective amount of the 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, appendix cancer, melanoma, acute myeloid leukemia, small bowel cancer, ampullary cancer, germ cell cancer, cervical cancer, cancer of unknown primary site, endometrial cancer, esophageal and gastric cancer, GI neuroendocrine cancer, ovarian cancer, sex cord stromal tumor cancer, hepatobiliary cancer, or bladder cancer. In some embodiments, the cancer is appendix cancer, endometrial cancer, or melanoma. A method for treating Ras protein-related conditions in a subject of need is also provided, the method comprising administering to the subject a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising such a compound or salt.

[0307] 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 variety of cancers, including tumors such as lung cancer, prostate cancer, breast cancer, brain cancer, skin cancer, cervical cancer, testicular cancer, etc. More specifically, cancers treatable by the compounds of the present invention or salts thereof, pharmaceutical compositions comprising such compounds or salts, and methods include, but are not limited to, tumor types such as: astrocytic, breast, cervical, colorectal, endometrial, esophageal, gastric, head and neck, hepatocellular, laryngeal, lung, oral cavity, ovarian, prostate, and thyroid carcinomas and sarcomas. Other cancers include, for example:

[0308] Heart-related sarcomas, such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyosarcoma, fibroma, lipoma, and teratoma.

[0309] Lung cancer, for example: bronchogenic carcinoma (squamous cell lung cancer, undifferentiated small cell lung cancer, undifferentiated large cell lung cancer, lung adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;

[0310] Gastrointestinal tract, such as: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, islet tumor, glucagonoma, gastrinoma, carcinoid tumor, vasoactive intestinal peptide tumor), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma);

[0311] Urogenital tract, such as: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminomatous seminoma, teratoma, embryonal carcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenoma-like tumor, lipoma);

[0312] The liver, for example: hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;

[0313] Bile duct cancer, such as gallbladder cancer, ampullary cancer, and bile duct cancer;

[0314] Skeletal tumors, such as: osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondrogenic osteoid), benign chondroma, chondroblastoma, chondromycinoid fibroma, osteoid osteoma, and giant cell tumor;

[0315] Nervous system, such as: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningeal sarcoma, glioma), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor), spinal neurofibroma, neurofibromatosis type 1, meningioma, glioma, sarcoma;

[0316] Gynecological conditions, such as: uterus (endometrial cancer, uterine cancer, endometrial cancer), cervix (cervical cancer, cervical precancerous dysplasia), ovary (ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-theca cell tumor, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), fallopian tube (cancer);

[0317] Hematopoietic system, such as: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders (e.g., myelofibrosis and myeloproliferative neoplasms), multiple myeloma, myelodyplasia syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma);

[0318] Skin conditions, such as: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, nevus dysplasia, lipoma, hemangioma, dermatofibroma, keloid, psoriasis; and

[0319] Adrenal glands, for example: neuroblastoma.

[0320] In some implementations, the Ras protein is wild-type (Ras WT Therefore, in some embodiments, the compounds of the present invention are used to treat patients with Ras WT (e.g. K-Ras) WT H-Ras WT or N-RasWT In methods for treating cancer patients, Ras protein is amplified (e.g., K-Ras) in some implementations. amp Therefore, in some embodiments, the compounds of the present invention are used to treat patients with Ras amp (K-Ras amp H-Ras amp or N-Ras amp In methods for treating patients with cancer. In some embodiments, the cancer contains Ras mutations, such as the Ras mutations described herein. In some embodiments, the mutation is selected from:

[0321] (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;

[0322] (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;

[0323] (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;

[0324] Or a combination of any of the foregoing. In some embodiments, the compound inhibits K-Ras G12C and K-Ras G12D. In some embodiments, the compound inhibits both K-Ras G12D and K-Ras G13D. In some embodiments, the compound inhibits both K-Ras G12V and K-Ras G12S. In some embodiments, the compound of the present invention inhibits Ras WT and one or more additional Ras mutations (e.g., K, H, or N-Ras). WT And K-Ras G12D). In some embodiments, the compounds of the present invention inhibit Ras ampand one or more additional Ras mutations (e.g., K-, H-, or N-Ras). amp And K-RasG12D).

[0325] Methods for detecting Ras mutations are known in the art. These methods include, but are not limited to, direct sequencing and the use of highly sensitive diagnostic assays (using CE-IVD markers), such as those described in Domagala et al., Pol J Pathol 3:145-164 (2012), which are incorporated herein by full reference, including TheraScreenPCR; AmoyDx; PNAClamp; RealQuality; EntroGen; LightMix; StripAssay; HybcellplexA; Devyser; Surveyor; Cobas; and TheraScreenPyro. See also, for example, WO2020 / 106640.

[0326] In some embodiments, the cancer is non-small cell lung cancer, and the Ras mutation includes a K-Ras mutation, such as K-RasG12D. In some embodiments, the cancer is colorectal cancer, and the Ras mutation includes a K-Ras mutation, such as K-RasG12D. In some embodiments, the cancer is pancreatic cancer, and the Ras mutation includes a K-Ras mutation, such as K-Ras G12D. In some embodiments, the cancer is pancreatic cancer, and the Ras mutation includes an N-Ras mutation, such as N-Ras G12D. In any of the foregoing, unless otherwise specified, the compound may also inhibit Ras... WT (e.g., K-, H-, or N-Ras) WT ) or Ras amp (e.g., K-, H-, or N-Ras) amp ).

[0327] A method for inhibiting Ras protein in cells is also provided, the method comprising contacting the cells with an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof. The compound or a pharmaceutically acceptable salt thereof can inhibit more than one type of Ras protein in the cells. A method for inhibiting RAF-Ras binding is also provided, the method comprising contacting the cells with an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof. The cells may be cancer cells. The cancer cells may belong to any type of cancer described herein. The cells may be in vivo or in vitro.

[0328] Combination therapy

[0329] The methods of the present invention may include the compounds of the present invention used alone or in combination with one or more additional therapies (e.g., non-pharmacological treatments or therapeutic agents). When administered alone, the dose of one or more of the additional therapies (e.g., non-pharmacological treatments or therapeutic agents) may be reduced relative to a standard dose. For example, the dose may be determined empirically based on the combination and arrangement of drugs, or inferred by isoradiometric measurements (e.g., Black et al., Neurology 65:S3-S6 (2005)).

[0330] The compounds of the present invention may be administered before, after, or simultaneously with one or more of the additional therapies. When combined, the dose of the compounds of the present invention and the dose of the one or more additional therapies (e.g., non-pharmacological treatments or therapeutic agents) provide a therapeutic effect (e.g., synergistic or additive therapeutic effect). The compounds of the present invention and additional therapies, such as anticancer agents, may be administered together, such as in the form of a single pharmaceutical composition, or separately, and when administered separately, the administration may occur simultaneously or sequentially. Such sequential administration may be proximate or staggered in time.

[0331] In some embodiments, the additional therapy is the administration of a side effect limiter (e.g., an agent intended to reduce the occurrence or severity of treatment side effects). For example, in some embodiments, the compounds of the present invention may also be used in combination with a therapeutic agent for treating nausea. Examples of agents that can be used to treat nausea include granisetron, metoclopramide, ondansetron, and prochlorperazine, or pharmaceutically acceptable salts thereof.

[0332] In some embodiments, the one or more additional therapies include non-pharmacological treatments (e.g., surgery or radiation therapy). In some embodiments, the one or more additional therapies include therapeutic agents (e.g., compounds or biologics as anti-angiogenic agents, signal transduction inhibitors, anti-proliferative agents, glycolysis inhibitors, or autophagy inhibitors). In some embodiments, the one or more additional therapies include non-pharmacological treatments (e.g., surgery or radiation therapy) and therapeutic agents (e.g., compounds or biologics as anti-angiogenic agents, signal transduction inhibitors, anti-proliferative agents, glycolysis inhibitors, or autophagy inhibitors). In other embodiments, the one or more additional therapies include two therapeutic agents. In still 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.

[0333] In this section on combination therapies, all references are incorporated by way of citation for the pharmaceutical agents described or their pharmaceutically acceptable salts, solvates, isomers (e.g., stereoisomers), prodrugs or tautomers, whether or not so explicitly stated.

[0334] Non-drug therapy

[0335] Examples of non-pharmacological treatments include, but are not limited to, radiotherapy, cryotherapy, hyperthermia, surgery (e.g., surgical removal of tumor tissue), and T-cell adoptive transfer (ACT) therapy.

[0336] In some embodiments, the compounds of the present invention can be used as postoperative adjuvant therapy. In some embodiments, the compounds of the present invention can be used as preoperative neoadjuvant therapy.

[0337] Radiation therapy can be used to inhibit abnormal cell growth or treat hyperproliferative conditions such as cancer in subjects (e.g., mammals, such as humans). Techniques for administering radiation therapy are known in the art. Radiation therapy can be administered by one or a combination of several methods, including but not limited to external beam therapy, internal radiation therapy, implantation radiation, stereotactic radiosurgery, whole-body radiation therapy, radiotherapy, and sustained or transient proximal therapy. As used herein, the term "proximal therapy" refers to radiation therapy delivered by inserting a radioactive material into or near a space defined by a tumor or other proliferative tissue disease site within the body. This term is intended, 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 Lu). Suitable radiation sources used as cell conditioning agents of the present invention include solids and liquids. As a non-limiting example, the radioactive source can be a radionuclide, such as I-125, I-131, Yb-169, Ir-192 as a solid source, I-125 as a solid source, or other radionuclides that emit photons, beta particles, gamma radiation, or other therapeutic rays. The radioactive material can also be a fluid made from a solution of any radionuclide, such as a solution of I-125 or I-131, or the radioactive fluid can be prepared using a slurry containing small solid radionuclide particles such as Au-198 or Y-90. Furthermore, the radionuclide can be embedded in a gel or radioactive microspheres.

[0338] In some embodiments, the compounds of the present invention can sensitize abnormal cells to radiotherapy, thereby killing or inhibiting the growth of such cells. Therefore, the present invention further relates to a method for sensitizing abnormal cells in a mammal to radiotherapy, the method comprising administering to the mammal a quantity of the compound of the present invention, the quantity of which effectively sensitizes the abnormal cells to radiotherapy. The amount of the compound in the method may be determined according to the manner used to determine the effective amount of the compound described herein. In some embodiments, the compounds of the present invention can be used as adjuvant therapy after radiotherapy or as neoadjuvant therapy before radiotherapy.

[0339] In some embodiments, the non-pharmacological treatment is adoptive T-cell transfer (ACT) therapy. In some embodiments, the T cells are activated T cells. The T cells may 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. The source of the T cells is obtained from the subject before the T cells are expanded and genetically modified. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, a variety of T cell lines available in the art may be used. In some embodiments, the T cells are autologous T cells. Before or after T cell genes are modified to express the desired protein (e.g., CAR), these T cells can generally be activated and expanded using methods described, for example, in the following U.S. patents: 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 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.

[0340] Therapeutic agents

[0341] The therapeutic agent can be a compound used to treat cancer or its related symptoms. The compounds of the present invention can be combined with a second, third, or fourth therapeutic agent, or more therapeutic agents. The compounds of the present invention can be combined with one or more therapeutic agents and one or more non-pharmacological therapies.

[0342] For example, the therapeutic agent may be a steroid. Steroids are known in the art. Therefore, in some embodiments, the one or more additional therapies include steroids. Suitable steroids may include, but are not limited to, acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, and cortisol. sone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort, fiucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortinbutyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednideneAcetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, hydrocortisone, loteprednoletabonate, mazipredone, medrysone, methylprednisone, methylprednisolone Mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 2,5-diethylaminoacetic acid, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, and their salts or derivatives.

[0343] Other examples of therapeutic agents that can be used in combination therapies with the compounds of the present invention include compounds described in the following patents: U.S. Patents 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 Patents. 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.

[0344] The therapeutic agent can be a biological agent (e.g., cytokines such as interferon or interleukins, such as IL-2) used to treat cancer or related symptoms. Biological agents are known in the art. In some embodiments, the biological agent is an immunoglobulin-based biological agent, such as a monoclonal antibody (e.g., a humanized antibody, a fully human antibody, an Fc fusion protein, or a functional fragment thereof) that activates a target to stimulate an anticancer response or antagonizes an antigen important for cancer. Antibody-drug conjugates are also included.

[0345] The therapeutic agent can be a T-cell checkpoint inhibitor. These checkpoint inhibitors are known in the art. 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, such as an Fc-receptor fusion protein. In some embodiments, the checkpoint inhibitor is an agent that interacts with a checkpoint protein, such as an antibody. In some embodiments, the checkpoint inhibitor is an agent that interacts with a ligand of a checkpoint protein, such as an antibody. In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor (e.g., an inhibitory antibody or a small molecule inhibitor) (e.g., an anti-CTLA-4 antibody or fusion protein). In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor or antagonist (e.g., an inhibitory antibody or a small molecule inhibitor). In some embodiments, the checkpoint inhibitor is a PD-L1 inhibitor or antagonist (e.g., an inhibitory antibody or a small molecule inhibitor). In some embodiments, the checkpoint inhibitor is a PD-L2 inhibitor or antagonist (e.g., an inhibitory antibody or an Fc fusion or a small molecule inhibitor) (e.g., a PD-L2 / Ig fusion protein). In some implementations, 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, B-7 family ligands, or combinations thereof. In some implementations, the checkpoint inhibitor is pembrolizumab, nivolumab, PDR001 (NVS), REGN2810 (Sanofi / Regeneron), PD-L1 antibodies such as avelumab, durvalumab, atezolizumab, pidilizumab, JNJ-63723283 (JNJ), BGB-A317 (BeiGene & Celgene), or P... Checkpoint inhibitors disclosed in reusser, M. et al. (2015) Nat. Rev. Neurol. include, but are not limited to, ipilimumab, tremelimumab, nivolumab, pembrolizumab, AMP224, AMP514 / MEDI0680, BMS936559, MED14736, MPDL3280A, MSB0010718C, BMS986016, IMP321, lirilumab, IPH2101, 1-7F9, and KW-6002.

[0346] Therapeutic agents may be anti-TIGIT antibodies, such as MBSA43, BMS-986207, MK-7684, COM902, AB154, MTIG7192A, or OMP-313M32 (etigilimab). Other anti-TIGIT antibodies are known in the art.

[0347] Therapeutic agents can be agents used to treat cancer or related symptoms (e.g., cytotoxic agents, non-peptide small molecules, or other compounds that can be used to treat cancer or related symptoms, collectively referred to as "anticancer agents"). Anticancer agents can be, for example, chemotherapeutic agents or targeted therapeutic agents. These agents are known in the art.

[0348] Anticancer agents include mitosis inhibitors, insertional 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, vincaalkaloids, epipodophyllotoxin, antibiotics, L-asparaginase, topoisomerase inhibitors, interferon, platinum coordination complexes, anthrone-substituted urea, methylhydrazine derivatives, adrenocortical inhibitors, adrenocortical steroids, progesterone, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. Other anticancer agents include leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. In some embodiments, the one or more additional therapies comprise two or more anticancer agents. The two or more anticancer agents may be used in a mixture for combined or separate administration. Suitable dosing regimens for the combined anticancer agents are known in the art and 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).

[0349] Other non-limiting examples of anticancer agents include (Imatinib Mesylate); (carfilzomib); (bortezomib); Casodex (bicalutamide); Gefitinib; alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; azacyclopropanes such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimine and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide, and tris(hydroxymethyl)melamine; polyacetyl (especially bullatasin). Cin) and bullatacinone; camptothecin (including its synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogues) (e.g., KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin A; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethami neoxidehydrochloride), melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracilmustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Antibiotics, such as acetylenic antibiotics (e.g., calicheamicin, such as calicheamicin γll and calicheamicin ωll (see, for example, Agnew, Chem. Intl. Ed Engl. 33:183-186 (1994)); dynemicin, such as dynemicin A; bisphosphonates, such as clodronate; esperamicin; neocarzinostatin chromophore and related chromogens; acetylenic antibiotic chromophores; aclacinomysin; actinomyces... Actinomycin, autramycin, azaserine, bleomycin, cactinomycin C, calicheamicin, carabicin, caminomycin, carminomycin, carzinophilin, chromomycins, dactinomycin D, daunorubicin, and ditocin. Detorubicin, 6-diazo-5-oxo-L-leucine, adriamycin (doxorubicin), N-morpholinodoxorubicin, cyanomorpholinodoxorubicin, (2-pyrrololino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (e.g., mitomycin C), mycophenolic acid, nogalamycin, ol Ivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogs, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-thioazolidin, carmofur, cytarabine, doxifluridine, enocitabine, and fluxuridine; and androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergic drugs, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolic acid. acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; ep... othilones, such as epsilone B; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;Podophyllinic acid; (2-ethylhydrazine; procarbazine); Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes, such as T-2 toxin, verracurin A, roridin A, and anguidine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; piperobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, such as... (paclitaxel) (A nanoparticle formulation of Pacific paclitaxel without polyoxyethylene hydrogenated castor oil or albumin-engineered paclitaxel) and (Docetaxel); Chloranbucil; Tamoxifen (Nolvadex) TM ); raloxifene; aromatase inhibitory 4(5)-imidazole; 4-hydroxytamoxifen; trioxifene; keoxifene; LY117018; onapristone; toremifene Flutamide, nilutamide, bicalutamide, leuprolide, goserelin; chlorambucil; Gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; esperamicins; capecitabine (e.g.) ), and pharmaceutically acceptable salts of any of the above.

[0350] Additional, non-restricted examples of anticancer agents include trastuzumab. Bevacizumab Cetuximab (cetuximabv) Rituximab ABVD, avicine, abagovomab, acridinecarboxamide, adecatumumab, demethoxygeldanamycin, alpharadin, alvocidib, thiosemicarbazone, amonafide, anthracene dione edione), antiCD22 immunotoxins, antitumor drugs (e.g., cell cycle nonspecific antitumor agents and other antitumor agents described herein), antitumor herbs, apaziquone, atipremod, azathioprine, belotecone, bendamustine, BIBW2992, biricodar, brostallicin, bryostatin, butionine sulfoxidesulfoximine), CBV (chemotherapy), calyculin, dichloroacetic acid, discormolide, elsamitrucin, enocitabine, eribulin, exatecan, exisulind, ferruginol, forodesine, fosfestrol, ICE chemotherapy regimen, IT-101, imexon, imiquimod, indolocarbazole, irofulven, lanicoquerone (l) aniquidar), larotaxel, lenalidomide, lucanthone, lurtotecan, mafosfamide, mitozolomide, naproxen, nedaplatin, olaparib, ortataxel, PAC-1, papaya, pixantrone, proteasome inhibitors, rebeccamycin, resiquimod, rubitecan, SN-38, salinosporamide A) Sapacitabine, Stanford V, swainsonine, talaporfin, tariquidar, tegafur-uracil, temodar, tesetaxel, triplatintetranitrate, tri(2-chloroethyl)amine, troxacitabine, uramustine, vadimezan, vinflunine, ZD6126, and zosuquidar.

[0351] Other non-limiting examples of anticancer agents include natural products such as vinca alkaloids (e.g., vincristine, vinorelbine, and vinorelbine), epipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., dactinomycin / actinomycin D), donomycin, and idarubicin), anthracycline, mitoxantrone, bleomycins, plicamycin (mithramycin), mitomycin, enzymes (e.g., L-asparaginase, which systemically metabolizes L-asparagine and removes cells that cannot synthesize asparagine), antiplatelet agents, antiproliferative / antimitotic alkylating agents such as nitrogen mustard (e.g., methylmuscarin, cyclophosphamide, and analogues melphalan and chlorambucil), ethyleneimine, and methylmelamine (e.g., hexamethyl... Melamine and thiotepa), CDK inhibitors (e.g., CDK4 / 6 inhibitors such as abemaciclib, ribociclib, palbociclib, seliciclib, UCN-01, P1446A-05, PD-0332991, dinaciclib, P27-00, AT-7519, RGB286638, and SCH727965), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine (BCNU) and analogues),And streptozocin), trazenes-dacarbazinine (DTIC), antiproliferative / antimitotic antimetabolites (such as folic acid analogs), pyrimidine analogs (such as fluorouracil, azuridine, and cytarabine), purine analogs and related inhibitors (such as mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine), aromatase inhibitors (such as anastrozole, exemestane, and letrozole), and platinum coordination complexes (such as cisplatin and carboplatin), procarbazine, hydroxyurea, mitotane, amylglutathione, histone deacetylase (HDAC) inhibitors (such as trichostatin, sodium butyrate, apicidan, and suberoylanilide hydroamic acid). acid), vorinostat, belinostat, LBH 589, romidepsin, ACY-1215 and panobinostat, mTOR inhibitors (e.g. vistusertib, temsirolimus, everolimus, ridaforolimus and sirolimus), KSP (Eg5) inhibitors (e.g. Array520), DNA binding agents (e.g., PI3K inhibitors such as PI3Kδ inhibitors (e.g., GS-1101 and TGR-1202), PI3Kδ and γ inhibitors (e.g., CAL-130), copanlisib, alpelisib, and idelalisib; multi-kinase inhibitors (e.g., TG02 and sorafenib); hormones (e.g., estrogens and hormone agonists, such as luteinizing 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 KOS 953), PI3K / Akt inhibitors (e.g., perifosine), Akt inhibitors (e.g., GSK-2141795), PKC inhibitors (e.g., enzastaurin), FTIs (e.g., Zarnestra) TM Anti-CD138 (e.g., BT062), Torcl / 2 specific kinase inhibitors (e.g., INK128), ER / UPR targets (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.

[0352] In some implementation schemes, the anticancer agent is selected from methicillin, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, etc. Sorafenib or any of the aforementioned analogues or derivative variants.

[0353] In some implementations, the anticancer agent is a HER2 inhibitor. HER2 inhibitors are known in the art. Non-limiting examples of HER2 inhibitors include monoclonal antibodies, such as trastuzumab. and pertuzumab Small molecule tyrosine kinase inhibitors, such as gefitinib Erlotinib Pilitinib, CP-654577, CP-724714, Canertinib (CI1033), HKI-272, Lapatinib (GW-572016); ), PKI-166, AEE788, BMS-599626, HKI-357, BIBW2992, ARRY-334543 and JNJ-26483327.

[0354] In some embodiments, the anticancer agent is an ALK inhibitor. ALK inhibitors are known in the art. 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 WO05016894.

[0355] In some implementations, the anticancer agent is an inhibitor of a downstream member of a receptor tyrosine kinase (RTK) / growth factor receptor (e.g., an SHP2 inhibitor, such as SHP099, TNO155, RMC-4550, RMC-4630, JAB-3068, JAB-3312, RLY-1971, ERAS-601, SH3809, PF-07284892, or BBP-398, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof). SOS1 inhibitors (e.g., BI-1701963, BI-3406, SDR5, BAY-293, or RMC-5845, or their pharmaceutically acceptable salts, solvates, isomers (e.g., stereoisomers), prodrugs, or tautomers), Raf inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, PTEN inhibitors, AKT inhibitors, or mTOR inhibitors (e.g., mTORC1 inhibitors or mTORC2 inhibitors). In some embodiments, the anticancer agent is JAB-3312.

[0356] In some implementations, the anticancer agent is an SOS1 inhibitor. SOS1 inhibitors are known in the art. In some implementations, the SOS1 inhibitor is selected from WO2022146698, WO2022081912, WO2022058344, WO2022026465, WO2022017519, WO2021173524, WO2021130731, WO2021127429, WO2021092115, WO2021105960, WO2021074227, WO2020180768, WO2020180770, WO2020173935, WO2020146470, WO2019201848, WO2019122129, WO 2018172250 and WO The compounds disclosed in 2018115380, or their pharmaceutically acceptable salts, solvates, isomers (e.g., stereoisomers), prodrugs, or tautomers. In some embodiments, the disclosed compounds are used in combination with SOS1 inhibitors to treat K-Ras G13D cancer.

[0357] In some embodiments, the anticancer agent is an additional Ras inhibitor or a Ras vaccine, or another therapeutic agent designed to directly or indirectly reduce the carcinogenic activity of Ras. These agents are known in the art. In some embodiments, the anticancer agent is an additional Ras inhibitor. In some embodiments, the Ras inhibitor targets Ras in an active or GTP-bound state. In some embodiments, the Ras inhibitor targets Ras in an inactive or GDP-bound state. In some implementations, the Ras inhibitor is, for example, a K-RasG12C inhibitor, such as AMG 510 (sottorazib), MRTX1257, MRTX849 (adagrasib), JNJ-74699157, LY3499446, ARS-1620, ARS-853, BPI-421286, LY3537982, JDQ443, AZ4625, JAB-21822, JAB-21000, IBI351, ERAS-3490, RMC-6291, or GDC-6036, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the Ras inhibitor is a K-Ras G12D inhibitor, such as MRTX1133 or JAB-22000, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the Ras inhibitor is a K-Ras G12V inhibitor, such as JAB-23000, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the Ras inhibitor is RMC-6236, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the Ras inhibitor is selected from the Ras(ON) inhibitors disclosed in the patents incorporated herein by reference in their entirety, or pharmaceutically acceptable salts, solvates, isomers (e.g., stereoisomers), prodrugs, or tautomers thereof: WO2021091982, WO2021091967, WO2021091956, and WO2020132597. Other examples of Ras inhibitors are known in the art, such as those disclosed in the patents incorporated herein by reference in their entirety: WO20220133038, WO2022133345, WO2022132200, WO2022119748, WO2022109485, WO2022109487, WO2022066805, WO2021190467, WO 2021185233, WO 2021180181, WO2021175199, 2021173923.WO2021169990, WO2021169963, WO2021168193, WO2021158071, WO2021155716, WO2021152149, WO2021150613, WO2021147967, WO2021147965, WO2021 143693, WO2021142252, WO2021141628, WO2021139748, WO2021139678, WO2021129824, WO2021129820, WO2021127404, WO2021126816, WO2021126799, WO2021124222, WO2021121371, WO2021121367, WO2021121330, WO2020050890, WO2020047192, WO2020035031, WO2020028706, WO2019241157, WO20192 W O2019051291, WO2018218070, WO2018217651, WO2018218071, WO2018218069, WO2018206539, WO2018143315, WO2018140600, WO2018140599, WO201814 0598, WO2018140514, WO2018140513, WO2018140512, WO2018119183, WO2018112420, WO2018068017, WO2018064510, WO2017201161, WO2017172979, WO WO2017100546, WO2017087528, WO2017058807, WO2017058805, WO2017058728, WO2017058902, WO2017058792, WO2017058768, WO2017058915, WO2017015562, WO2016168540, WO2016164675, WO2016049568, WO2016049524, WO2015054572, WO2014152588, WO2014143659 and WO2013155223, or pharmaceutically acceptable salts, solvates,Isomers (e.g., stereoisomers), prodrugs, or tautomers.

[0358] In some embodiments, therapeutic agents that can be combined with the compounds of the present invention are MAP kinase (MAPK) pathway inhibitors (or "MAPK inhibitors"). These agents are known in the art. MAPK inhibitors include, but are not limited to, one or more MAPK inhibitors described in Cancer (Basel) September 2015; 7(3):1758-1784. For example, MAPK inhibitors may be selected from one or more of the following: trametinib, binimetinib, selumetinib, cobimetinib, LERAFAON (NeoPharm), ISIS 5132; vemurafenib, pimaertib, TAK733, RO4987655 (CH4987655); CI-1040; PD-0325901; CH5126766; MAP855; AZD6244; refametinib (RDEA 119 / BAY) 86-9766); GDC-0973 / XL581; AZD8330 (ARRY-424704 / ARRY-704); RO5126766 (Roche, described in PLoSOne. 25 November 2014; 9(11)); and GSK1120212 (or JTP-74057, described in Clin CancerRes. 1 March 2011; 17(5):989-1000). MAPK inhibitors may be PLX8394, LXH254, GDC-5573 or LY3009120.

[0359] In some embodiments, the anticancer agent is a disruptor or an inhibitor of the RAS-RAF-ERK, PI3K-AKT-TOR, or PI3K-AKT signaling pathway. These agents are known in the art. PI3K / AKT inhibitors may include, but are not limited to, one or more PI3K / AKT inhibitors described in Cancer (Basel) 2015 Sep; 7(3):1758-1784. For example, the PI3K / AKT inhibitor may be selected from one or more of the following: NVP-BEZ235; BGT226; XL765 / SAR245409; SF1126; GDC-0980; PI-103; PF-04691502; PKI-587; GSK2126458.

[0360] In some implementations, the anticancer agent is a PD-1 or PD-L1 antagonist. These agents are known in the art.

[0361] In some implementations, additional therapeutic agents include ALK inhibitors, HER2 inhibitors, EGFR inhibitors, IGF-1R inhibitors, MEK inhibitors, PI3K inhibitors, AKT inhibitors, TOR inhibitors, MCL-1 inhibitors, BCL-2 inhibitors, SHP2 inhibitors, proteasome inhibitors, and immunotherapy. In some implementations, additional therapeutic agents include FGFR inhibitors, PARP inhibitors, BET inhibitors, PRMT5i inhibitors, MAT2A inhibitors, VEGF inhibitors, and HDAC inhibitors. In some implementations, the therapeutic agent may be a pan-RTK inhibitor, such as afatinib.

[0362] IGF-1R inhibitors are known in the art and include linsitinib or a pharmaceutically acceptable salt thereof.

[0363] EGFR inhibitors are known in the art and include, but are not limited to, small molecule antagonists, antibody inhibitors, or specific antisense nucleotides or siRNAs. Useful antibody inhibitors for EGFR include cetuximab. Panitumumab Zalutumumab, nimotuzumab, and matuzumab. Other antibody-based EGFR inhibitors include any anti-EGFR antibody or antibody fragment that can partially or completely block the activation of EGFR by natural ligands. 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. EGFR inhibitors can be monoclonal antibodies such as Mab E7.6.3 (Yang, 1999, ibid.) or Mab C225 (ATCC accession number HB-8508) or antibodies or antibody fragments that have binding specificity to EGFR.

[0364] Small molecule antagonists of EGFR include gefitinib Erlotinib and lapatinib See, for example, 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 implementations, the EGFR inhibitor is osimertinib. Other non-limiting examples of small molecule EGFR inhibitors include any EGFR inhibitor described in the following patent publications, and all pharmaceutically acceptable salts of such EGFR inhibitors: EP05722; EP0566226; WO96 / 33980; U.S. Patent No. 5,747,498; WO96 / 30347; EP0787772; WO97 / 30034; WO97 / 30044; WO97 / 38994; WO97 / 49688; EP 837063; WO98 / 02434; WO97 / 38983; WO95 / 19774; WO95 / 19970; WO97 / 13771; WO98 / 02437; WO98 / 02438; WO97 / 32881; DE 19629652; WO98 / 33798; WO97 / 32880; WO97 / 32880; EP682027; WO97 / 02266; WO97 / 27199; WO98 / 07726; WO97 / 34895; WO96 / 31510; WO98 / 14449; WO98 / 14450; WO98 / 14451; WO95 / 09847; WO97 / 19065; WO98 / 17662; U.S. Patent No. 5,789,427; U.S. Patent No. 5,650,415; U.S. Patent No. 5,656,643; WO99 / 35146; WO99 / 35132; WO99 / 07701; and WO92 / 20642. Other non-limiting examples of small molecule EGFR inhibitors include any EGFR inhibitor 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 includes HER1 (EGFR, ERBB1), HER2 (NEU, ERBB2), HER3 (ERBB3), and HER (ERBB4).

[0365] MEK inhibitors are known in the art, and include, but are not limited to, pimasetinib, selumetinib, and cobimetinib. Trametinib and Bimetinib In some embodiments, the MEK inhibitor targets a MEK mutation selected from the following type I MEK1 mutations: D67N, P124L, P124S, and L177V. In some embodiments, the MEK mutation is selected from the following type II MEK1 mutations: ΔE51-Q58, ΔF53-Q58, E203K, L177M, C121S, F53L, K57E, Q56P, and K57N.

[0366] PI3K inhibitors are known in the art and include, but are not limited to, wortmannin; 17-hydroxywortmannin analogs as described in WO06 / 044453; 4-[2-(1H-indazol-4-yl)-6-[[4-(methanesulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as pictilisib or GDC-0941, and described in WO09 / 036082 and WO09 / 055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (also known as BEZ) 235 or NVP-BEZ235, and described in WO06 / 122806); (S)-l-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxyprop-1-one (described in WO08 / 070740); LY294002(2-(4-morpholino)-8-phenyl-4H-l-benzopyran-4-one (purchased from Axon Medchem); PI 103 hydrochloride (3-[4-(4-morpholinylpyridino[3',2':4,5]furano[3,2-d]pyrimidin-2-yl]phenol hydrochloride (purchased from Axon Medchem); PIK75 (2-methyl-5-nitro-2-[(6-bromoimidazolo[1,2-a]pyridin-3-yl)methylene]-1-methylhydrazine-benzenesulfonic acid monohydrochloride) (purchased from Axon Medchem); PIK 90 (N-(7,8-dimethoxy-2,3-dihydro-imidazo[l,2-c]quinazolin-5-yl)-nicotinamide (purchased from Axon Medchem); AS-252424 (5-[l-[5-(4-fluoro-2-hydroxy-phenyl)-furan-2-yl]-methyl-(Z)-ylidene]-thiazodin-2,4-dione (purchased from Axon Medchem); TGX-221 (7-methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one (purchased from Axon Medchem); XL-765; and XL-147.Other PI3K inhibitors include demethoxyviridin, perifosine, CAL101, PX-866, BEZ235, SF1126, INK1117, IPI-145, BKM120, XL147, XL765, Palomid529, GSK1059615, ZSTK474, PWT33597, IC87114, TGI00-115, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136.

[0367] AKT inhibitors are known in the art and include, but are not limited to, Akt-1-1 (inhibiting Aktl) (Barnett et al., Biochem. J. 2005, 385(Pt.2):399-408); Akt-1-1,2 (inhibiting Akl 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]pyridyl compounds (e.g., WO 05 / 011700); indole-3-methanol and its derivatives (e.g., U.S. Patent No. 6,656,963; Sarkar and Li J). Nutr. 2004, 134(12 Supplement): 3493S-3498S); perifoxine (e.g., interfering 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).

[0368] mTOR inhibitors are known in the art and include, but are not limited to, ATP-competitive mTORC1 / mTORC2 inhibitors, such as PI-103, PP242, PP30; Torin 1; FKBP12 enhancers; 4H-1-benzopyran-4-one derivatives; and rapamycin (also known as sirolimus) and its derivatives, including temsirolimus. Everolimus WO94 / 09010); deforolimus (also known as deforolimus or AP23573); rapalogs, such as those disclosed in WO98 / 02441 and WO01 / 14387, such as AP23464 and AP23841; 40-(2-hydroxyethyl)rapalog; 40-[3-hydroxy(hydroxymethyl)methylpropionate]-rapalog (also known as CC1779); 40-epio-(tetrazole)-rapalog (also known as ABT578); 32-deoxyrapalog; 16-pentyneoxy-32(S)-dihydrorapalog; derivatives disclosed in WO05 / 005434; United States Derivatives disclosed in patents No. 5,258,389, 5,118,677, 5,118,678, 5,100,883, 5,151,413, 5,120,842 and 5,256,790, as well as in WO94 / 090101, WO92 / 05179, WO93 / 111130, WO94 / 02136, WO94 / 02485, WO95 / 14023, WO94 / 02136, WO95 / 16691, WO96 / 41807, WO96 / 41807 and WO2018204416; and phosphorus-containing rapamycin derivatives (e.g. WO05 / 016252). In some implementations, the mTOR inhibitor is a bisteric inhibitor (see, for example, WO2018204416, WO2019212990 and WO2019212991), such as RMC-5552, having the following structure

[0369]

[0370] BRAF inhibitors that can be used in combination with the compounds of the present invention are known in the art and include, for example, vemurafenib, dabrafenib, and encorafenib. BRAF may contain type 3 BRAF mutations. In some embodiments, the type 3 BRAF mutation is 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.

[0371] MCL-1 inhibitors are known in the art and include, but are not limited to, AMG-176, MIK665, and S63845. Myeloid leukemia-1 (MCL-1) protein is a key anti-apoptotic member of the B-cell lymphoma-2 (BCL-2) protein family. Overexpression of MCL-1 is closely associated with tumor progression and resistance to targeted therapies, not only conventional chemotherapy but also BCL-2 inhibitors such as ABT-263.

[0372] In some embodiments, the additional therapeutic agent is an SHP2 inhibitor. SHP2 inhibitors are known in the art. SHP2 is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene, which facilitates a variety of cellular functions, including 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 subcellular localization and functional regulation of SHP2. The molecule exists in a stable, inactive, self-inhibiting conformation through a binding network involving residues from the N-SH2 and PTP domains. Stimulation with cytokines or growth factors, such as those acting via receptor tyrosine kinases (RTKs), exposes the catalytic site, leading to enzymatic activation of SHP2.

[0373] SHP2 is involved in signal transduction via the RAS-mitogen-activated protein kinase (MAPK) pathway, namely the JAK-STAT or phosphoinositol 3-kinase-AKT pathway. Mutations in the PTPN11 gene and subsequently in SHP2 have been identified in several human developmental disorders, such as Noonan Syndrome and Leopard Syndrome, and human cancers, such as juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, and breast, lung, and colon cancers. Some of these mutations destabilize the autoinhibitory conformation of SHP2 and promote autoactivation or enhanced growth factor-driven activation of SHP2. Therefore, SHP2 represents a target of particular interest for developing novel therapies for treating a variety of diseases, including cancer. It has been shown that combinations of SHP2 inhibitors (e.g., RMC-4550 or SHP099) with RAS pathway inhibitors (e.g., MEK inhibitors) can inhibit the proliferation of various cancer cell lines (e.g., pancreatic, lung, ovarian, and breast cancer) in vitro. Therefore, combination therapy involving SHP2 inhibitors and RAS pathway inhibitors can be a general strategy for preventing tumor resistance in a variety of malignancies.

[0374] Non-limiting examples of such SHP2 inhibitors known in the art include: 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 PCT applications: WO2022135568, WO 2021176072, WO 2021171261, WO 2021149817, WO2021148010, WO2021147879, WO 2021143823, WO 2021143701, WO2021143680, WO 2021121397, WO2021119525, WO 2021115286, WO2021110796, WO 2021088945, WO 2021073439, WO2021061706, WO2021061515, WO 2021043077, WO 2021033153、WO 2021028362、WO2021033153、WO 2021028362、WO 2021018287、WO 2020259679、WO2020249079、WO2020210384、WO 2020201991、WO 2020181283, WO2020177653, WO 2020165734, WO2020165733, WO 2020165732, WO2020156243, WO 2020156242, WO 2020108590, WO2020104635, WO2020094104, WO 2020094018, WO 2020081848, WO 2020073949, WO2020073945, WO 2020072656, WO 2020065453, WO 2020065452, WO2020063760, WO2020061103, WO 2020061101, WO 2020033828, WO2020033286, WO 2020022323, WO2019233810, WO 2019213318, WO2019183367, WO 2019183364, WO 2019182960, WO2019167000, WO2019165073, WO 2019158019, WO 2019152454, WO 2019051469, WO2019051084, WO2018218133, WO 2018172984, WO 2018160731, WO2018136265, WO2018136264, WO 2018130928, WO 2018129402, WO2018081091, WO 2018057884, WO2018013597, WO 2017216706, WO2017211303, WO 2017210134, WO 2017156397, WO2017100279, WO2017079723, WO 2017078499, WO 2016203406, WO WO2016203405, WO2016203404, WO 2016196591, WO 2016191328, WO 2015107495, WO2015107494, WO2015107493, WO 2014176488, WO 2014113584, US20210085677, US10858359, US10934302, US10954243, US10988466, US11001561, US11033547, US11034705 or US11044675, or pharmaceutically acceptable salts, solvates, isomers (e.g., stereoisomers), prodrugs or tautomers thereof, are incorporated herein by reference.

[0375] 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, such as a non-covalent allosteric inhibitor. In some embodiments, the SHP2 inhibitor is a covalent SHP2 inhibitor, such as an inhibitor targeting a cysteine ​​residue (C333) located 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 having the following structure:

[0376] Or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is RMC-4550 having the following structure:

[0377]

[0378] Or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is RMC-4630 having the following structure:

[0379]

[0380] Or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is JAB-3068 having the following structure:

[0381]

[0382] Or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is JAB-3312. In some embodiments, the SHP2 inhibitor is the following compound:

[0383]

[0384] Or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is RLY-1971 having the following structure:

[0385]

[0386] Or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is ERAS-601, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is BBP-398, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof. In some embodiments, the SHP2 inhibitor is SH3809. In some embodiments, the SHP2 inhibitor is PF-07284892, or a pharmaceutically acceptable salt, solvate, isomer (e.g., stereoisomer), prodrug, or tautomer thereof.

[0387] In some embodiments, the additional therapeutic agent is selected from the group consisting of: MEK inhibitors, HER2 inhibitors, SHP2 inhibitors, CDK4 / 6 inhibitors, mTOR inhibitors, SOS1 inhibitors, and PD-L1 inhibitors. In some embodiments, the additional therapeutic agent is selected from the group consisting of: MEK inhibitors, SHP2 inhibitors, and PD-L1 inhibitors. See, for example, Hallin et al., Cancer Discovery, DOI:10.1158 / 2159-8290 (October 28, 2019), and Canon et al., Nature, 575:217 (2019). In some embodiments, the Ras inhibitor of the present invention is used in combination with MEK inhibitors and SOS1 inhibitors. In some embodiments, the Ras inhibitor of the present invention is used in combination with PD-L1 inhibitors and SOS1 inhibitors. In some embodiments, the Ras inhibitor of the present invention is used in combination with PD-L1 inhibitors and SHP2 inhibitors. In some embodiments, the Ras inhibitor of the present invention is used in combination with MEK inhibitors and SHP2 inhibitors. In some embodiments, the Ras inhibitor of the present invention is used in combination with an SHP2 inhibitor and a Ras inhibitor that inhibits multiple Ras isoforms and / or mutants (e.g., RMC-6236). In some embodiments, the cancer is colorectal cancer, and treatment comprises administering a combination of the Ras inhibitor of the present invention with a second or third therapeutic agent, such as an SHP2 inhibitor and a Ras inhibitor that inhibits multiple Ras isoforms and / or mutants. In some embodiments, the cancer is cholangiocarcinoma, and treatment comprises administering the Ras inhibitor of the present invention, sorafenib, and a chemotherapy agent. In some embodiments, the cancer is gastric cancer, and treatment comprises administering the Ras inhibitor of the present invention and an FGFR inhibitor (e.g., FGFR2i or FGFR4i). In some embodiments, the Ras inhibitor of the present invention is used in combination with immunotherapy, optionally in combination with a chemotherapy agent.

[0388] Proteasome inhibitors are known in the art, and include, but are not limited to, carfilzomib. Bortezomib And opozomib.

[0389] Immunotherapy includes, but is not limited to, monoclonal antibodies, immunomodulatory imides (IMiDs), GITR agonists, genetically engineered T cells (e.g., CAR-T cells), bispecific antibodies (e.g., BiTE), and anti-PD-1 agents, anti-PD-L1 agents, anti-CTLA4 agents, anti-LAGI1 agents, and anti-OX40 agents. Other immunotherapies are known in the art.

[0390] Immunomodulatory agents (IMiDs) are a class of immunomodulatory drugs containing imide groups (drugs that regulate immune responses). IMiD drugs include thalidomide and its analogues (lenalidomide, pomalidomide, and apremilast).

[0391] Exemplary anti-PD-1 antibodies and their methods of use are described in Goldberg et al., Blood 2007, 110(1):186-192; Thompson et al., Clin. Cancer Res. 2007, 13(6):1757-1761; and WO06 / 121168A1), and are also described elsewhere in this article.

[0392] FGFR inhibitors are known in the art, such as pemigatinib and erdafitinib, which include FGFR2 inhibitors and FGFR4 inhibitors. See, for example, Cancers (Basel), June 2021; 13(12)2968.

[0393] BET inhibitors are known in the art, such as romidasine, pabistal, and belistat. See, for example, British J. Cancer 124:1478 (2021).

[0394] PRMT5 inhibitors are known in the art, such as PF-0693999, PJ-68, and MRTX1719. See, for example, Biomed. Pharmacotherapy 144:112252 (2021).

[0395] MAT2A inhibitors are known in the art, such as AG-270 and IDE397. See, for example, Exp Opin TherPatents (2022) DOI:10.1080 / 13543776.2022.2119127.

[0396] GITR agonists are known in the art and include, but are not limited to, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as the GITR fusion proteins described in U.S. Patent Nos. 6,111,090, 8,586,023, WO2010 / 003118, and WO2011 / 090754; or, for example, U.S. Patent Nos. 7,025,962, EP1947183, 7,812,135; 8,388,967; 8,591,886; 7,618,632, EP1947183, WO2010 / 003118, and WO2011 / 090754. The anti-GITR antibodies described in 1866339 and WO2011 / 028683, WO2013 / 039954, WO05 / 007190, WO07 / 133822, WO05 / 055808, WO99 / 40196, WO01 / 03720, WO99 / 20758, WO06 / 083289, WO05 / 115451 and WO2011 / 051726.

[0397] 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 are known in the art and include, but are not limited to, chemical compositions, antibodies, antigen-binding regions, radionuclides, and combinations and conjugates thereof synthesized in vitro. Anti-angiogenic agents can be agonists, antagonists, allosteric modulators, toxins, or more generally can be used to inhibit or stimulate their targets (e.g., receptor or enzyme activation or inhibition), thereby promoting cell death or arresting cell growth. In some embodiments, one or more additional therapies include an anti-angiogenic agent.

[0398] Anti-angiogenic 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 anti-angiogenic agents include rapamycin, tesiromolimus (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 / 52889, WO99 / 29667, WO99007675, EP0606046, EP0780386, EP1786785, EP1181017, EP0818442, EP1004578 and US20090012085, as well as U.S. Patent Nos. 5,863,949 and 5,861,510. Preferred MMP-2 and MMP-9 inhibitors are inhibitors with very low or no MMP-1 inhibitory activity. More preferably, they are inhibitors that selectively inhibit MMP-2 or AMP-9 relative to other matrix metalloproteinases (i.e., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specific examples of MMP inhibitors include AG-3340, RO 32-3555, and RS13-0830.

[0399] Other exemplary anti-angiogenic agents include kinase domain KDR (kinase domain receptor) inhibitors (e.g., antibodies and antigen-binding domains that specifically bind to kinase domain receptors), and anti-VEGF agents (e.g., antibodies or antigen-binding domains that specifically bind to VEGF (e.g., bevacizumab) or soluble VEGF receptors or their ligand-binding domains), such as VEGF-TRAP. TM And anti-VEGF receptor agents (e.g., antibodies or antigen-binding domains that specifically bind to the VEGF receptor), VEGF inhibitors, EGFR inhibitors (e.g., antibodies or antigen-binding domains that specifically bind to EGFR), such as (panitumumab), erlotinib Anti-Ang1 and anti-Ang2 agents (e.g., antibodies or antigen-binding domains that specifically bind to Angle and Ang2 or their receptors, such as Tie2 / Tek), and anti-Tie2 kinase inhibitors (e.g., antibodies or antigen-binding domains that specifically bind to Tie2 kinase). Other anti-angiogenic agents include Camppath, IL-8, β-FGF, Tek antagonists (US2003 / 0162712; US6,413,932), anti-TWEAK agents (e.g., antibodies or antigen-binding domains that specifically bind, or soluble TWEAK receptor antagonists; see US6,727,225), and ADAM disintegrin domains that antagonize the binding of integrins to their ligands (US...). 2002 / 0042368), specifically binding anti-eph receptor or anti-pterygium antibody or antigen-binding region (US Patent Nos. 5,981,245, 5,728,813, 5,969,110, 6,596,852, 6,232,447, 6,057,124 and members of their patent families), and anti-PDGF-BB antagonists (e.g., specifically binding antibody or antigen-binding region), as well as antibodies or antigen-binding regions specifically binding to PDGF-BB ligands, and PDGFR kinase inhibitors (e.g., antibodies or antigen-binding regions specifically binding to PDGFR kinases). Additional anti-angiogenic agents include: SD-7784 (Pfizer, USA); cilengitide (Merck KGaA, Germany, EPO 0770622); pegaptanib octasodium (Gilead Sciences, USA); alphastatin (BioActa, UK); M-PGA (Celgene, USA, US 5712291); ilomastat (Arriva, USA, US5892112); emaxanib (Pfizer, USA, US 5792783); vatalanib (Novartis, Switzerland); 2-methoxyestradiol (EntreMed, USA); TLC ELL-12 (Elan, Ireland); and anecrolave ​​acetate. acetate) (Alcon, USA); α-D148 Mab (Amgen, USA); CEP-7055 (Cephalon, USA); anti-Vn Mab (Crucell, Netherlands), DAC anti-angiogenic agent (ConjuChem, Canada);Angiocidin (InKine Pharmaceutical, USA); KM-2550 (Kyowa Hakko, Japan); SU-0879 (Pfizer, USA); CGP-79787 (Novartis, Switzerland, EP 0970070); ARGENT technology (Ariad, USA); YIGSR-Stealth (Johnson & Johnson, USA); Fibrinogen-E fragment (BioActa, UK); Angiogenesis inhibitor (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 (IVAX, USA); BeneFin (LaneLabs, USA); Tz-93 (Tsumura, Japan); TAN-1120 (Takeda, Japan); FR-111142 (Fujisawa, Japan, JP02233610); Platelet-4 (RepliGen, USA, EP407122); Vascular endothelial growth factor antagonist (Borean, Denmark); Bevacizumab (pINN) (Genentech, USA); Angiogenesis inhibitor (SUGEN, USA); XL784 (Exelixis, USA); XL 647 (Exelixis, USA); Second-generation α5β3 integrin MAb (Applied Molecular Evolution, USA and Medlmmune, USA); Enzastaurin hydrochloride hydrochloride (Lilly, USA); CEP 7055 (Cephalon, USA and Sanofi-Synthelabo, France); BC 1 (Genoa Institute of Cancer Research, Italy); rBPI21 and BPI-derived anti-angiogenic agents (XOMA, USA); PI 88 (Progen, Australia); silengitide (Merck KGaA, Germany);Munich Technical University, Germany, Scripps Clinic and Research Foundation, USA); AVE 8062 (Ajinomoto, Japan); AS1404 (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); ZD6474 (AstraZeneca, UK); ZD 6126 (Angiogene Pharmaceuticals, UK); PPI2458 (Praecis, USA); AZD 9935 (AstraZeneca, UK); AZD 2171 (AstraZeneca, UK); pINN (Novartis, Switzerland and Schering) AG (Germany); Tissue factor pathway inhibitor (EntreMed, USA); Pinn (Gilead Sciences, USA); Xanthorrhizol (Yonsei University, South Korea); Gene-based VEGF-2 vaccine (Scripps Clinic and Research Foundation, USA); SPV5.2 (Supratek, Canada); SDX 103 (University of California, 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 (British Columbia University, Canada); CDP 791 (Celltech Group, UK); Atiprimod (pINN) (GlaxoSmithKline, UK); E 7820 (Eisai, Japan);CYC 381 (Harvard University, USA); AE941 (Aeterna, Canada); Angiogenesis vaccine (EntreMed, USA); Urokinase plasminogen activator inhibitor (Dendreon, USA); Oglufanide (pINN) (Melmotte, USA); HIF-1α inhibitor (Xenova, UK); CEP 5214 (Cephalon, USA); BAYRES2622 (Bayer, Germany); InKine (InKine, USA); A6 (Angstrom, USA); KR 31372 (Korea Research Institute of Chemical Technology, South Korea); GW2286 (GlaxoSmithKline, UK); EHT0101 (ExonHit, France); CP 868596 (Pfizer, USA); CP 564959 (OSI, USA); CP 547632 (Pfizer, USA); 786034 (GlaxoSmithKline, UK); KRN 633 (KirinBrewery, Japan); Drug delivery system, intraocular 2-methoxyestradiol; Angnex (Maastricht University, Netherlands, and Minnesota University, USA); ABT510 (Abbott, USA); AAL993 (Novartis, Switzerland); VEGI (ProteomTech, USA); Tumor necrosis factor-alpha inhibitor; SU 11248 (Pfizer, USA and SUGENUSA); ABT 518 (Abbott, USA); YH16 (Yantai Rongchang, China); S-3APG (Boston Children's) Hospital, USA and EntreMed, USA); MAb, KDR (ImClone Systems, USA); MAb, α5β (Protein Design, USA); KDR kinase inhibitor (Celltech Group, UK and Johnson & Johnson, USA); GFB 116 (South Florida University, USA and Yale University, USA); CS 706 (Sankyo, Japan);Cobretastatin A4 prodrug (Arizona State University, USA); Chondroitinase AC (IBEX, Canada); BAYRES2690 (Bayer, Germany); AGM 1470 (Harvard University, USA, Takeda, Japan, and TAP, USA); AG 13925 (Agouron, USA); Tetrathiomolybdate (University of Michigan, USA); GCS100 (Wayne State University, USA); CV247 (Ivy Medical, UK); CKD 732 (Chong KunDang, South Korea); Irsogladine (Nippon Shinyaku, Japan); RG 13577 (Aventis, France); WX360 (Wilex, Germany); Squalamine (Genaera, USA); RPI4610 (Sirna, USA); Heparinase inhibitor (InSight, Israel); KL3106 (Kolon, South Korea); and Honokiol (Emory University, USA); ZKCDK (Schering AG, Germany); ZKAngio (Schering AG, Germany); ZK229561 (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 FLT1 (vascular endothelial growth factor receptor 1) (Merck & Co, USA); Tie-2 ligand (Regeneron, USA); and thromboprotein 1 inhibitor (Allegheny Health, Education and Research Foundation, USA).

[0400] Other examples of therapeutic agents that can be used in combination with the compounds of the present invention include agents that specifically bind to and inhibit the activity of growth factors (e.g., antibodies, antigen-binding domains, or soluble receptors), such as antagonists of hepatocyte growth factor (HGF, also known as scatter factor), and antibodies or antigen-binding domains that specifically bind to the receptor c-Met. These agents are known in the art.

[0401] 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 are known in the art and include, but are not limited to, chloroquine, 3-methyladenine, and hydroxychloroquine (Plaquenil). TM This includes bafilomycin A1, 5-amino-4-imidazolamide riboside (AICAR), leucocyanidin, 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 vincristine. Additionally, antisense RNA or siRNA that inhibits protein expression, including but not limited to ATG5 (involved in autophagy), may be used. In some embodiments, one or more of these additional therapies include autophagy inhibitors.

[0402] Another example of a therapeutic agent that can be used in combination with the compounds of the present invention is an antitumor agent, which is known in the art. In some embodiments, the one or more additional therapies include an antitumor agent. Non-limiting examples of antitumor agents include acemannan, aclarubicin, aldesleukin, alemtuzumab, alitretinoin, hexamethylmelamine, aifostine, amrubicin, amsacrine, anagrelide, anastrozole, ancer, ancestim, arglabin, arsenic trioxide, BAM-002 (Novelos), bexarotene, bicalutamide, broxuridine, capecitabine, celmoleukin, cetrorelix, cladribine, clotrimazole, and cytarabine phosphate. Cytarabineocfosfate, DA3030 (Dong-A), daclizumab, denileukindiftitox, deslorelin, dexrazoxane, dilazep, docetaxel, docosanol, doxercalciferol, doxifluridine Doxorubicin, bromocriptine, carmustine, cytarabine, fluorouracil, diclofenac (HIT), interferon-alpha, doxorubicin, tretinoin, edelfosine, edrecolomab, eflornithine, emitefur, epirubicin, beta-epoetin beta, etoposide phosphatePhosphate), exemestane, exisulind, fadrozole, filgrastim, finasteride, fludarabine phosphate, formestane, fotemustine, gallium nitrate, gemcitabine, gemtuzumab zogamicin, gimeracil / oteracil / tegafur combination, glycopine, goserelin, heptaplatin, human chorionic gonadotropin, human fetal alpha-fetoprotein, ibandronic acidInterferon-1β, idarubicin, imiquimod, interferon α, natural interferon α, interferon α-2, interferon α-2a, interferon α-2b, interferon α-Nl, interferon α-n3, compound interferon-1, natural interferon α, interferon β, interferon β-la, interferon β-lb, interferon γ, natural interferon γ-la, interferon γ-lb), interleukin-1β, iobenguanine, irinotecan, isolax Irsogladine, lanreotide, LC9018 (Yakult), leflunomide, lenograstim, lentinan sulfate, letrozole, leukocyte alpha interferon, leuprorelin, levamisole + fluorouracil, liarozole, lobaplatin, lonidamine damine), lovastatin, masoprocol, melarsoprol, metoclopramide, mifepristone, miltefosine, mirimostim, mismatched double-stranded RNA, mitoxantrone, dibromoceroxyl, mitoxantrone, molgramostim, nafarelin, naloxone + pentazocine, nartograstim, nedaplatin, niluamide, noscapine, novel erythropoietin, NSC631570 octreotide, oprelvekin, osaterone, oxaliplatin, paclitaxel, pamidronate acid), pegaspargase, pegylated interferon α-2b, pentosan polysulfates sodium, pentostatin, picibanil, pirubicin, rabbit anti-thymocyte multiclonal antibody, pegylated interferon α-2a, porfimer sodium, raloxifene, raltitrexed, rasburiembodiment, rhenium ethodoleate Re186. RII (Retinamide), Rituximab, Romurtide, Samarium lexidronam (153Sm), Sargramostim, Sizofiran, Sobuzoxane, Sonermin, Strontium-89 Chloride, Suramin, Tasonermin, Tazarotene, Tegafur, Temoporfin, Temozolomide, Teniposide, Tetrachlorodecaoxide, Thalidomide, Thymalfasin, Thyrotropin A, Topotecan, Toremifene, Tositumomab-iodine 131) Trastuzumab, treosulfan, retinoic acid, trilostane, trimetrexate, triptorelin, natural tumor necrosis factor α, ubenimex, bladder cancer vaccine, Maruyama vaccine, melanoma lysis product vaccine, valrubicin, verteporfin, vinorelbine, virulizin, zinostatinstimalamer, or zoledronic acid; abarelix; AE 941 (Aeterna), ambamustine, antisense oligonucleotides, bcl-2 (Genta), APC 8015 (Dendreon), decitabine, dexaminoglutethimide, diaziquone, EL 532 (Elan), EM 800 (Endorecherche), eniluracil, etanidazole, fenretinide, filgrastim, SD01 (Amgen), fulvestrant, galocitabine, gastrin-17 immunogen, HLA-B7 gene therapy (Vical), granulocyte-macrophage community-stimulating factorColonystimulating factor), histamine dihydrochloride, ibritumomab tiuxetan, ilomastat, IM862 (Cytran), interleukin-2, iproxifene, LDI200 (Milkhaus), leridistim, lintuzumab, CA 125MAb (Biomira), cancer MAb (Japan Pharmaceutical Development), HER-2 and Fc MAb (Medarex), Idiotype 105AD7MAb (CRC Technology), Idiotype CEAMAb (Trilex), LYM-1-Iodine-131MAb (Techniclone), Polymorphic Epithelial Mucin-Yttrium 90MAb (Antisoma), Marimastat, Menogaril, Mitumomab, Motexafingadolinium, MX6 (Galderma), Nelarabine, Nolatrexed, P30 protein, Pegvisomant, Pemetrexed, Porfiromycin, Prinomastat, RL 0903 (Shire), Rubitecan, Satraplatin, Sodium Phenylacetate, Sparfosic acid, SRL 172 (SR Pharma), SU5416 (SUGEN), TA077 (Tanabe), tetrathiomolybdate, thaliblastine, thrombopoietin, tinethyletiopurpurin, tirapazamine, cancer vaccine (Biomira), melanoma vaccine (New York University), melanoma vaccine (Sloan Kettering Institute), melanoma tumor lysis product vaccine (New York Medical College), viral melanoma cell lysis product vaccine (Royal Newcastle Hospital), or valspodar.

[0403] Additional examples of therapeutic agents that can be used in combination with the compounds of the present invention include ipilimumab. Trimelimumab; Galiximab; Nivolumab, also known as BMS-936558 Pembrolizumab Avelumab 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; Atecicept; CP-870893; Lucarumumab; Dacetuzumab; Muromonab-CD3; Ipilumumab; MEDI4736 MSB0010718C; AMP224; Adalimumab ADO-trastuzumab emtansine Aflibercept alemtuzumab Baliximab belimumab Baliximab belimumab brentuximab vedotin Canakinumab Polyethylene glycol combined with certolizumab (bpegol) Daktarizumab daratumumab denosumab eculizumab efalizumab Gemtuzumab / Ozogamicin golimumab ibritumomab tiuxetan infliximab Motavizumab Natalizumab obinutuzumab ofatumumab Omalizumab palivizumab pertuzumab pertuzumab ranibizumab raxibacumab Tocilizumab Tositumomab; Tositumomab-i-131; Tositumomab and Tositumomab-i-131 ustekinumab AMG 102; AMG 386; AMG 479; AMG 655; AMG 706; AMG 745; and AMG 951.

[0404] Depending on the condition being treated, the compounds described herein may be used in combination with the pharmaceutical agents disclosed herein or other suitable agents. Therefore, in some embodiments, one or more compounds of the present invention will be administered co-administered with other therapies described herein. When used in combination therapy, the compounds described herein may be administered simultaneously or separately with a second pharmaceutical agent. This combination administration may include simultaneous administration of two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, the compounds described herein may be formulated together with any pharmaceutical agent described herein into the same dosage form and administered simultaneously. Alternatively, the compounds of the present invention may be administered simultaneously with any therapy described herein, wherein the two agents are present in separate formulations. In another alternative, the compounds of the present invention may be administered first, followed by any therapy described herein, or vice versa. In some embodiments of separate administration, the compounds of the present invention and any therapy described herein are administered at intervals of minutes, hours, or days.

[0405] In some embodiments of any of the methods described herein, a first therapy (e.g., the compound of the present invention) and one or more additional therapies are administered simultaneously or sequentially in any order. The first therapeutic agent may be administered immediately before or after the administration of the one or more additional therapies, for 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, 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, or up to 1-7 days, 1-14 days, 1-21 days, or 1-30 days.

[0406] The invention is further characterized by a kit comprising (a) a pharmaceutical composition comprising the agents described herein (e.g., compounds of the present invention) and (b) a packaging insert with instructions on performing any of the methods described herein. In some embodiments, the kit comprises (a) a pharmaceutical composition comprising the agents described herein (e.g., compounds of the present invention), (b) one or more additional therapies (e.g., non-pharmacological treatments or therapeutic agents), and (c) a packaging insert with instructions on performing any of the methods described herein.

[0407] Since one aspect of the invention covers the treatment of diseases or their related symptoms with combinations of separately administerable pharmaceutically active compounds, the invention also relates to the combination of independent pharmaceutical compositions in the form of a kit. The kit may contain two independent pharmaceutical compositions: the compound of the invention and one or more additional therapies. The kit may include a container for containing the independent compositions, such as a dispensing vial or a dispenser foil package. Additional examples of containers include syringes, boxes, and bags. In some embodiments, the kit may include instructions on the use of the independent components. The kit format is particularly advantageous when the independent components are preferably administered in different dosage forms (e.g., oral or parenteral), at different dose intervals, or when the prescribing healthcare professional wishes to adjust the individual components in the combination.

[0408] Implementation plan with numbering

[0409] 1. A compound or a pharmaceutically acceptable salt thereof having the structure of Formula I:

[0410]

[0411] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0412] X1 X 2 and X 3 Each is independently selected from CH2, CHF, CF2, C=O, or O;

[0413] m is 1 or 2;

[0414] n is 0 or 1;

[0415] R 1 It is hydrogen, optionally substituted C1-C6 heteroalkyl or optionally substituted 3 to 10-membered heterocyclic alkyl;

[0416] R 2 It is an optional substituted C1-C6 alkyl group; and

[0417] R 3 It is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted 3- to 6-membered cycloalkyl, or an optionally substituted heterocycloalkyl.

[0418] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0419] 2. A compound or a pharmaceutically acceptable salt thereof having the structure of Formula I:

[0420]

[0421] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0422] X 1 X 2 and X 3 Each is independently selected from CH2, CF2, C=O, or O;

[0423] m is 1 or 2;

[0424] n is 0 or 1;

[0425] R 1 It is hydrogen, optionally substituted C1-C6 heteroalkyl or optionally substituted 3 to 10-membered heterocyclic alkyl;

[0426] R 2 It is an optional substituted C1-C6 alkyl group; and

[0427] R 3 It can be a C1-C6 alkyl group that has been optionally substituted, a 3- to 6-membered cycloalkyl group that has been optionally substituted, or a heterocyclic alkyl group that has been optionally substituted.

[0428] Furthermore, each hydrogen atom is independently and optionally enriched with the isotope deuterium.

[0429] 3. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 1 or 2, having the structure of any one of formula Ia, formula Ib or formula Ic:

[0430]

[0431] Each D indicates that the isotopic enrichment factor of deuterium is at least 5 hydrogens.

[0432] 4. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 3, wherein R 1 It is hydrogen or optionally substituted 3 to 10-membered heterocyclic alkyl groups.

[0433] 5. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 4, wherein R 1 It is an optional substituted 3 to 10-membered heterocyclic alkyl group.

[0434] 6. The compound as described in embodiment 5 or a pharmaceutically acceptable salt thereof, wherein R 1 yes:

[0435] 7. The compound as described in embodiment 5 or a pharmaceutically acceptable salt thereof, wherein R 1 yes: Furthermore, each D indicates that the isotopic enrichment factor of deuterium is at least 5 for hydrogen.

[0436] 8. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 7, wherein m is 1.

[0437] 9. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 8, wherein n is 1.

[0438] 10. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 9, wherein X 1 X 2 and X 3 Each is CH2.

[0439] 11. The compound or a pharmaceutically acceptable salt thereof as described in Embodiment 1, having the structure of Formula II:

[0440]

[0441] 12. The compound or a pharmaceutically acceptable salt thereof as described in Embodiment 1, having the structure of Formula V:

[0442]

[0443] 13. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 12, having the structure of any one of formula Va, formula Vb or formula Vc:

[0444]

[0445]

[0446] Each D indicates that the isotopic enrichment factor of deuterium is at least 5 hydrogens.

[0447] 14. The compound or a pharmaceutically acceptable salt thereof as described in Embodiment 12, having the structure of any one of the formulas Vd, Ve, or Vf:

[0448]

[0449]

[0450] Each D indicates that the isotopic enrichment factor of deuterium is at least 5 hydrogens.

[0451] 15. The compound or a pharmaceutically acceptable salt thereof as described in Embodiment 1, having the structure of Formula VI:

[0452]

[0453]

[0454] 16. The compound or a pharmaceutically acceptable salt thereof as described in Embodiment 1, having the structure of Formula VII:

[0455]

[0456] 17. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 16, wherein A is optionally substituted thiazole-diyl, optionally substituted oxazole-diyl, optionally substituted morpholine-diyl, optionally substituted pyrrolidine-diyl, optionally substituted piperidine-diyl, or optionally substituted phenylene.

[0457] 18. The compound as described in Embodiment 17 or a pharmaceutically acceptable salt thereof, wherein A is optionally substituted thiazol-diyl or optionally substituted morpholine-diyl.

[0458] 19. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 16, wherein A is an optionally substituted 5 to 10-membered heteroaryl group.

[0459] 20. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 19, wherein A is:

[0460] 21. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 20, wherein A is:

[0461] 22. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 16, wherein A is an optionally substituted phenylene.

[0462] 23. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 22, wherein A is:

[0463] 24. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 23, wherein A is:

[0464] 25. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 16, wherein A is an optionally substituted 3- to 6-membered heterocyclic alkyl group.

[0465] 26. The compound as described in embodiment 25 or a pharmaceutically acceptable salt thereof, wherein A is an optionally substituted 6-membered heterocyclic alkyl group.

[0466] 27. A compound or a pharmaceutically acceptable salt thereof as described in embodiment 25, wherein A is selected from the following or their stereoisomers:

[0467] 28. A compound or a pharmaceutically acceptable salt thereof as described in embodiment 26, wherein A is selected from the following or their stereoisomers:

[0468] 29. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 28, wherein R 2 yes:

[0469] 30. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 28, wherein R 2 yes: Furthermore, each D indicates that the isotopic enrichment factor of deuterium is at least 5 for hydrogen.

[0470] 31. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 30, wherein R 3 It is an optional substituted C1-C6 alkyl or an optional substituted 3- to 6-membered cycloalkyl.

[0471] 32. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 31, wherein R 3 It is an optional substituted C1-C6 alkyl group.

[0472] 33. The compound as described in embodiment 32 or a pharmaceutically acceptable salt thereof, wherein R 3 yes:

[0473] 34. The compound as described in embodiment 33 or a pharmaceutically acceptable salt thereof, wherein R 3 yes:

[0474] 35. The compound as described in embodiment 32 or a pharmaceutically acceptable salt thereof, wherein R 3 yes: Furthermore, each D indicates that the isotopic enrichment factor of deuterium is at least 5 for hydrogen.

[0475] 36. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 31, wherein R 3 It is or optionally substituted with a 3- to 6-membered cycloalkyl group.

[0476] 37. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 36, wherein R 3 yes:

[0477] 38. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 36, wherein R 3 It is or can be optionally substituted with a 5-membered cycloalkyl group.

[0478] 39. The compound or a pharmaceutically acceptable salt thereof as described in embodiment 38, wherein R 3 yes:

[0479] 40. The compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 11 to 16, wherein:

[0480] R 2 yes

[0481] R 3 yes and

[0482] A is

[0483] 41. The compound as described in any one of embodiments 11 to 16, wherein:

[0484] R 2 yes

[0485] R 3 yes and

[0486] A is

[0487] 42. A compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1 to 41, wherein said compound is not one of the compounds in Table 3.

[0488] 43. A compound or a pharmaceutically acceptable salt thereof having the structure of the compounds listed in Table 1 or Table 2.

[0489] 44. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof, as described in any one of embodiments 1 to 43, and a pharmaceutically acceptable excipient.

[0490] 45. A method of treating cancer in a subject in need, the method comprising administering to the subject a therapeutically effective amount of any one of embodiments 1 to 43, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of embodiment 44.

[0491] 46. ​​The method as described in embodiment 45, wherein the cancer is pancreatic cancer, non-small cell lung cancer, colorectal cancer, or endometrial cancer.

[0492] 47. The method as described in embodiment 45 or 46, wherein the cancer comprises a Ras mutation.

[0493] 48. The method of embodiment 47, wherein the Ras mutation is K-Ras G12D or K-Ras G13D.

[0494] 49. A method for treating a subject with Ras protein-related disease, the method comprising administering to the subject a therapeutically effective amount of any one of embodiments 1 to 43, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of embodiment 44.

[0495] 50. A method for inhibiting Ras protein in cells, the method comprising contacting the cells with an effective amount of the compound of any one of embodiments 1 to 43 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of embodiment 44.

[0496] 51. The method as described in embodiment 49 or 50, wherein the Ras protein is K-Ras G12D or K-Ras G13D.

[0497] 52. The method as described in embodiment 50 or 51, wherein the cell is a cancer cell.

[0498] 53. The method of embodiment 52, wherein the cancer cells are pancreatic cancer cells, non-small cell lung cancer cells, colorectal cancer cells, or endometrial cells.

[0499] 54. The method or use as described in any one of embodiments 45 to 53, wherein the method further comprises administering additional anticancer therapy.

[0500] 55. The method of embodiment 54, wherein the additional anticancer 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.

[0501] 56. The method as described in embodiment 54 or 55, wherein the additional anticancer therapy is an SHP2 inhibitor.

[0502] 57. A conjugate or a salt thereof, comprising the structure of formula III:

[0503] MP 1

[0504] Formula III

[0505] Where P 1 It is the unit price organic portion; and

[0506] M has the structure of formula IV:

[0507]

[0508] Wherein A is an optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 3- to 6-membered heterocyclic alkylene, optionally substituted 6-membered arylene, or optionally substituted 5- to 10-membered heterocyclic arylene;

[0509] X 1 X 2 and X 3 Each is independently selected from CH2, CHF, CF2, C=O, or O;

[0510] m is 1 or 2;

[0511] n is 0 or 1;

[0512] R 1 It is hydrogen, optionally substituted C1-C6 heteroalkyl or optionally substituted 3 to 10-membered heterocyclic alkyl;

[0513] R 2 It is an optional substituted C1-C6 alkyl group; and

[0514] R 3It can be an optionally substituted C1-C6 alkyl, an optionally substituted 3- to 6-membered cycloalkyl, an optionally substituted C1-C6 heteroalkyl, or an optionally substituted heterocycloalkyl.

[0515] Furthermore, each hydrogen atom in Formula IV is independently and optionally enriched with the isotope deuterium.

[0516] 58. The conjugate or salt thereof as described in embodiment 57, wherein A is optionally substituted thiazole-diyl, optionally substituted oxazole-diyl, optionally substituted morpholine-diyl, optionally substituted pyrrolidine-diyl, optionally substituted piperidine-diyl, or optionally substituted phenylene.

[0517] 59. The conjugate or salt thereof as described in embodiment 57 or 58, wherein R 1 yes:

[0518]

[0519] 60. The conjugate or salt thereof as described in any one of embodiments 57 to 59, wherein m is 1, n is 1, and X 1 X 2 and X 3 Each is CH2.

[0520] 61. The conjugate or salt thereof as described in any one of embodiments 57 to 60, wherein the monovalent organic portion is a protein.

[0521] 62. The conjugate or its salt as described in embodiment 61, wherein the protein is a Ras protein.

[0522] 63. The conjugate or salt thereof as described in embodiment 62, wherein the Ras protein is K-Ras G12D or K-Ras G13D.

[0523] 64. The conjugate as described in any one of embodiments 57 to 63, wherein M is bound to an amino acid residue of the monovalent organic moiety.

[0524] Example

[0525] The present invention will be further illustrated by the following embodiments and synthetic embodiments, which should not be construed as limiting the scope or spirit of the invention to the specific procedures described herein. It should be understood that the provided embodiments are intended to illustrate certain implementations and are not intended to limit the scope of the invention thereon. It should also be understood that various other embodiments, modifications, and equivalents conceived by those skilled in the art may also be invoked without departing from the spirit of the invention or the scope of the appended claims.

[0526] Chemical synthesis

[0527] The following examples and definitions used elsewhere in this document are as follows:

[0528] CH2Cl2, DCM, methylene chloride, dichloromethane

[0529] CH3CN, MeCN acetonitrile

[0530] CuI (copper iodide I)

[0531] DIPEA (diisopropylethylamine)

[0532] DMF N,N-dimethylformamide

[0533] EtOAc (ethyl acetate)

[0534] h hours

[0535] H2O water

[0536] HCl hydrochloric acid

[0537] K3PO4 Potassium phosphate (tribasic form)

[0538] MeOH (methanol)

[0539] Na2SO4 Sodium sulfate

[0540] NMP N-methylpyrrolidone

[0541] Pd(dppf)Cl2 [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloro

[0542] Synthetic intermediates

[0543] Intermediate 1: Synthesis of 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylprop-1-ol

[0544]

[0545] Step 1: Synthesis of 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylprop-1-one

[0546] Under a nitrogen atmosphere, at 0°C, a 1M SnCl4 solution in DCM (137 mL, 137 mmol) was slowly added to a mixture of 3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropionyl chloride (65 g, 137 mmol, crude) in DCM (120 mL). The mixture was stirred at 0°C for 30 minutes, followed by dropwise addition of a solution of 5-bromo-1H-indole (26.8 g, 137 mmol) in DCM (40 mL). The mixture was stirred at 0°C for 45 minutes, then 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)C 29 H 32 The calculated m / z[M+Na] value for BrNO2SiNa is 556.1; the experimental value is 556.3.

[0547] Step 2: Synthesis of 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylprop-1-one

[0548] Under a nitrogen atmosphere, LiBH4 (6.1 g, 281 mmol) was added to a mixture of 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylprop-1-one (50 g, 93.6 mmol) and THF (100 mL) at 0 °C. The mixture was heated to 60 °C and stirred for 20 hours, followed by the addition of MeOH (10 mL) and EtOAc (100 mL). The mixture was washed with brine (50 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with DCM (50 mL), cooled to 10 °C, and dihydropyridine (9.5 g, 37.4 mmol) and TsOH·H2O (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)C 29 H 34 The calculated m / z[M+H] value of BrNOSi is 519.2; the experimental value is 520.1.

[0549] Step 3: Synthesis of 5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-2-iodo-1H-indole

[0550] AgOTf (888 mg, 3.5 mmol) was added to a mixture of 1-(5-bromo-1H-indol-3-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylprop-1-one (1.5 g, 2.9 mmol) and I2 (731 mg, 2.9 mmol) in THF (15 mL) at room temperature. The mixture was stirred at room temperature for 2 hours, then diluted with EtOAc (200 mL) and washed with saturated Na2S2O3 aqueous solution (100 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 as a solid (900 mg, 72% yield).

[0551] Step 4: Synthesis of (1S)-1-(3-bromopyridin-2-yl)ethanol

[0552] Under an Ar atmosphere, at 0 °C, (4S,5S)-2-chloro-2-methyl-1-(4-methylbenzenesulfonyl)-4,5-diphenyl-1,3-diaza-2-rutheniumcyclopentaneisopropyltoluene (3.9 g, 6.0 mmol) was added fractionally to a stirred mixture of HCO2H (66.3 g, 1.44 mol) in Et3N (728 mL, 7.2 mol). The mixture was heated to 40 °C and stirred for 15 min, then cooled to room temperature and 1-(3-bromopyridin-2-yl)acetone (120 g, 600 mmol) was added fractionally. The mixture was heated to 40 °C and stirred for another 2 h, then 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 Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain an oily product (100 g, 74% yield). Calculated m / z [M+H] for LCMS (ESI)C7H8BrNO: 201.98; Experimental: 201.9.

[0553] Step 5: Synthesis of 3-bromo-2-[(1S)-1-methoxyethyl]pyridine

[0554] At 0 °C, fractionally add a 60% dispersion (14.25 g, 594 mmol) of NaH in oil to a stirred mixture of (1S)-1-(3-bromopyridin-2-yl)ethanol (100 g, 495 mmol) in DMF (1 L). Stir the mixture at 0 °C for 1 hour. Add MeI (140.5 g, 990 mmol) dropwise at 0 °C, and heat the mixture to room temperature while stirring for 2 hours. Cool the mixture to 0 °C and add a saturated aqueous solution of NH4Cl (5 L). Extract the mixture with EtOAc (3 × 1.5 L), dry to anhydrous Na2SO4, and filter. Concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography to give an oily product (90 g, 75% yield). LCMS(ESI)C8H 10 Calculated m / z[M+H] value of BrNO: 215.99; Experimental value: 215.9.

[0555] Step 6: Synthesis of 2-[(1S)-1-methoxyethyl]-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)pyridine

[0556] Under an Ar atmosphere, at room temperature, bis(pinacol)diboron (127 g, 500 mmol), KOAc (81.8 g, 833 mmol), and Pd(dppf)Cl2 (30.5 g, 41.7 mmol) were added to a stirred mixture of 3-bromo-2-[(1S)-1-methoxyethyl]pyridine (90 g, 417 mmol) in toluene (900 mL). The mixture was heated to 100 °C and stirred for 3 hours. The filtrate was concentrated under reduced pressure, and the residue was purified by Al2O3 column chromatography to give a semi-solid product (100 g, 63% yield). LCMS(ESI)C 14 H 22 The calculated value of m / z[M+H] for BNO3 is 264.17; the experimental value is 264.1.

[0557] Step 7: Synthesis of 5-bromo-3-[3-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylpropyl]-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-1H-indole

[0558] Under an Ar atmosphere, at room temperature, fractionally add K₂CO₃ (74.8 g, 541 mmol), Pd(dppf)Cl₂ (15.9 g, 21.7 mmol), and H₂O (280 mL) 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-dioxaborhecyclopentan-2-yl)pyridine (100 g, 380 mmol) in dioxane (1.4 L). The mixture is heated to 85 °C and stirred for 4 hours, followed by the addition of cold H₂O (5 L) and extraction with EtOAc (3 × 2 L). The combined organic layers were washed with brine (2 × 1 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 as a solid (71 g, 45% yield). LCMS(ESI)C 37 H 43 The calculated m / z[M+H] value of BrN2O2Si is 655.23; the experimental value is 655.1.

[0559] 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

[0560] Under a nitrogen atmosphere and at 0 °C, fractionally added Cs₂CO₃ (70.6 g, 217 mmol) and EtI (33.8 g, 217 mmol) 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). The mixture was heated to room temperature and stirred for 16 hours, followed by the addition of H₂O (4 L) and extraction with EtOAc (3 × 1.5 L). The combined organic layers were washed with brine (2 × 1 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 an oily product (66 g, 80% yield). LCMS(ESI)C 39 H 47 The calculated m / z[M+H] value of BrN2O2Si is 683.26; the experimental value is 683.3.

[0561] Step 9: Synthesis of 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylprop-1-ol

[0562] Under a nitrogen atmosphere, fractionally add 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) to a stirred mixture of TBAF (172.6 g, 660 mmol) in THF (660 mL) at room temperature. The mixture is heated to 50 °C and stirred for 16 hours, cooled, diluted with H₂O (5 L), and extracted with EtOAc (3 × 1.5 L). The combined organic layers are washed with brine (2 × 1 L), dried over anhydrous Na₂SO₄, and filtered. After filtration, the filtrate is concentrated under reduced pressure, and the residue is purified by silica gel column chromatography to give a solid product (30 g, 62% yield). LCMS(ESI)C 23 H 29 The calculated m / z[M+H] value of BrN2O2 is 445.14; the experimental value is 445.1.

[0563] Intermediate 2: Substitute synthesis via the Fisher Indole Route.

[0564]

[0565] Step 1: Synthesis of 5-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-2,2-dimethyl-5-oxovalerate

[0566] Under a nitrogen atmosphere, a 2.5 M hexane solution (333 mL, 833 mmol) of n-BuLi was added dropwise over 15 minutes at -10 °C to a mixture of i-PrMgCl (2 M THF solution, 0.5 L). The mixture was stirred at -10 °C for 30 minutes, followed by the dropwise addition of 3-bromo-2-[(1S)-1-methoxyethyl]pyridine (180 g, 833 mmol) to THF (0.5 L) over 30 minutes at -10 °C. The resulting mixture was heated to -5 °C and stirred for 1 hour, followed by the dropwise addition of 3,3-dimethyloxane-2,6-dione (118 g, 833 mmol) to THF (1.2 L) over 30 minutes at -5 °C. The mixture was heated to 0°C and stirred for 1.5 hours, then quenched at 0°C by adding a pre-cooled dioxane solution of 4M HCl (0.6 L) to adjust the pH to approximately 5. The mixture was diluted with 3 L of H₂O at 0°C and extracted with EtOAc (3 × 2.5 L). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give a solid product (87 g, 34% yield). LCMS(ESI)C15 H 21 The calculated value of m / z[M+H] for NO4 is 280.15; the experimental value is 280.1.

[0567] Step 2: Synthesis of 3-(5-bromo-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-1H-indol-3-yl)-2,2-dimethylpropionic acid and (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropionic acid ethyl ester

[0568] Under a nitrogen atmosphere, fractionally add (4-bromophenyl)hydrazine hydrochloride (68.7 g, 307 mmol) to a mixture of 5-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-2,2-dimethyl-5-oxopentanoic acid (78 g, 279 mmol) in EtOH (0.78 L) at room temperature. Heat the mixture to 85 °C and stir for 2 hours, then cool to room temperature, followed by dropwise addition of a 4 M HCl solution of dioxane (69.8 mL, 279 mmol). Heat the mixture to 85 °C and stir for another 3 hours, then concentrate under reduced pressure and dissolve the residue in TFA (0.78 L). Heat the mixture to 60 °C and stir for 1.5 hours, concentrate under reduced pressure, and adjust the residue to approximately pH 5 with a saturated NaHCO3 aqueous solution, then extract with EtOAc (3 × 1.5 L). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. The residue was then purified by silica gel column chromatography to obtain the product (78 g, crude). LCMS(ESI)C 21 H 23 The calculated m / z[M+H] value of BrN2O3 is 430.1 and C 23 H 27 The calculated m / z [M+H] value of BrN2O3 is 459.12; the experimental values ​​are 431.1 (carboxylic acid) and 459.1.

[0569] Step 3: Synthesis of ethyl 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylpropionate

[0570] Under a nitrogen atmosphere, Cs₂CO₃ (449 g, 1.38 mol) was added fractionally to a mixture of 3-(5-bromo-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]-1H-indol-3-yl)-2,2-dimethylpropionic acid and (S)-3-(5-bromo-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropionic acid ethyl ester (198 g, 459 mmol) in DMF (1.8 L) at 0 °C. Then, EtI (215 g, 1.38 mmol) was added dropwise to DMF (200 mL) at 0 °C. The mixture was heated to room temperature and stirred for 4 hours, then diluted with brine (5 L) and extracted with EtOAc (3 × 2.5 L). The combined organic layers were washed with brine (2 × 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 as a solid (160 g, 57% yield). LCMS(ESI)C 25 H 31 The calculated m / z[M+H] value of BrN2O3 is 487.17; the experimental value is 487.2.

[0571] Step 4: Synthesis of 3-(5-bromo-1-ethyl-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol

[0572] Under a nitrogen atmosphere, LiBH4 (28.6 g, 1.3 mol) was added to a mixture of ethyl 3-(5-bromo-1-ethyl-2-[2-[(1S)-1-methoxyethyl]pyridin-3-yl]indol-3-yl)-2,2-dimethylpropionate (160 g, 328 mmol) in THF (1.6 L) at 0 °C. The mixture was heated to 60 °C and held for 16 hours, then cooled and quenched with a pre-cooled (0 °C) saturated aqueous solution of NH4Cl (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 Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give two trans-restricted isomers of 3-(5-bromo-1-ethyl-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (in single trans-restricted isomer form) (60 g, 38% yield) and (40 g, 26% yield), both in solid form. LCMS (ESI) m / z: C 23 H 29 The calculated value of [M+H] for BrN2O2 is 445.14; the experimental value is 445.2.

[0573] Intermediate 3: Synthesis (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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazineza-2(1,3)-benzocyclodecaban-5,7-dione

[0574]

[0575] Step 1: Synthesis of methyl (S)-3-(3-bromophenyl)-2-((tert-butoxycarbonyl)amino)propionate

[0576] At room temperature, NaHCO3 (48.8 g, 581.1 mmol) and MeI (61.9 g, 435.8 mmol) were added to a solution of (2S)-3-(3-bromophenyl)-2-[(tert-butoxycarbonyl)amino]propionic acid (100 g, 290 mmol) in DMF (1 L). The reaction mixture was stirred for 16 hours, then quenched with H2O (1 L) and extracted with EtOAc (3 × 1 L). The combined organic layers were washed with brine (3 × 500 mL), dried over Na2SO4, 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)C 15 H 20 The calculated m / z[M+Na] value of BrNO4 is 380.05; the experimental value is 380.0.

[0577] Step 2: Synthesis of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)propionate

[0578] KOAc (73.97 g, 753.70 mmol) and Pd(dppf)Cl2 (22.06 g, 30.15 mmol) were added to a stirred solution of (2S)-3-(3-bromophenyl)-2-[(tert-butoxycarbonyl)amino]propionate (108 g, 301.5 mmol) and bis(pinacol)diboron (99.53 g, 391.93 mmol) in dioxane (3.2 L). The reaction mixture was heated to 90 °C and maintained for 3 h, then cooled to room temperature and extracted with EtOAc (2 × 3 L). The combined organic layers were washed with brine (3 × 800 mL), dried over Na2SO4, 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)C 21 H 32 The calculated value of m / z[M+Na] for BNO6 is 428.22; the experimental value is 428.1.

[0579] Step 3: Synthesis of methyl (S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propionate

[0580] Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl]propionate (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 H₂O (300 mL) were added to a mixture of K₂CO₃ (64.11 g, 463.85 mmol) and Pd(DtBPF)Cl₂ (15.12 g, 23.19 mmol). The reaction mixture was heated to 70 °C and stirred for 4 hours. The reaction mixture was extracted with EtOAc (2 × 2 L) and the combined organic layers were washed with brine (3 × 600 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20% EtOAc / petroleum ether) to obtain the product (130 g, crude). LCMS(ESI)C 30 H 38 The calculated m / z[M+H] value of N2O6 is 523.28; the experimental value is 523.1.

[0581] 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)propionate

[0582] At -10 °C, AgOTf (70.0 g, 272.7 mmol) and NaHCO3 (22.9 g, 272.65 mmol) were added to a solution of (2S)-3-(3-[3-[3-(acetyloxy)-2,2-dimethylpropyl]-1H-indol-5-yl]phenyl)-2-[(tert-butoxycarbonyl)amino]propionate (95.0 g, 181.8 mmol) and iodine (36.91 g, 145.41 mmol) in THF (1 L). The reaction mixture was stirred for 30 min, followed by quenching with 100 mL of saturated aqueous solution of Na2S2O3 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 Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / petroleum ether) to give the desired product (49.3 g, 41.8% yield). LCMS(ESI)C 30 H 37 The calculated m / z[M+H] value of IN2O6 is 649.18; the experimental value is 649.1.

[0583] Step 5: Synthesis of (S)-2-((tert-butoxycarbonyl)amino)-3-(3-(3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)propionic acid

[0584] A solution of methyl (2S)-3-(3-[3-[3-(acetyloxy)-2,2-dimethylpropyl]-2-iodo-1H-indol-5-yl]phenyl)-2-[(tert-butoxycarbonyl)amino]propionate (60 g, 92.5 mmol) in THF (600 mL) was added to a solution of LiOH·H₂O (19.41 g, 462.5 mmol) in H₂O (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 Na₂SO₄, filtered, and concentrated under reduced pressure to give the product (45 g, 82.1% yield). LCMS(ESI)C 27 H 33 The calculated m / z[M+Na] value of IN2O6 is 615.13; the experimental value is 615.1.

[0585] Step 6: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0586] NMM (40.97 g, 405.08 mmol), HOBt (2.05 g, 15.19 mmol), and EDCI (19.41 g, 101.27 mmol) were added to a solution of (2S)-2-[(tert-butoxycarbonyl)amino]-3-[3-[3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl]phenyl]propionic acid (30 g, 50.6 mmol) and (3S)-1,2-diazacyclohexane-3-carboxylate (10.9 g, 75.9 mmol) in DCM (400 mL). The reaction mixture was stirred overnight, then washed with saturated aqueous solution of NH4Cl (2 × 200 mL) and brine (2 × 200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the product (14 g, 38.5% yield). LCMS(ESI)C 33 H 43 The calculated m / z[M+H] value of IN4O6 is 718.23; the experimental value is 719.4.

[0587] 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)propionyl)hexahydropyridazine-3-carboxylic acid

[0588] At 0 °C, a solution of methyl (92 g, 128.0 mmol) of LiOH·H₂O in H₂O (640.10 mmol) was added to a solution of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(3-hydroxy-2,2-dimethylpropyl)-2-iodo-1H-indol-5-yl)phenyl)propionyl)hexahydropyridazine-3-carboxylate in THF (920 mL). The reaction mixture was stirred for 2 hours, followed by concentration under reduced pressure to give the product (90 g, crude product). LCMS(ESI)C 32 H 41 The calculated m / z[M+H] value of IN4O6 is 705.22; the experimental value is 705.1.

[0589] Step 8: Synthesis ((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)-indolaza-6(1,3)-pyridazinaza-2(1,3)-benzocyclodecaeno-4-yl) tert-butyl carbamate

[0590] At 0 °C, HOBt (34.52 g, 255.46 mmol), DIPEA (330.17 g, 2554.62 mmol), and EDCI (367.29 g, 1915.96 mmol) were added 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]propionyl]-1,2-diazacyclohexane-3-carboxylic acid (90 g, 127.73 mmol) in DCM (10 L). The reaction mixture was stirred for 16 hours, followed by concentration 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 Na2SO4, 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)C 32 H 39 The calculated m / z[M+H] value of IN4O5 is 687.21; the experimental value is 687.1.

[0591] Step 9: Synthesis ((6) 3 S,4S)-10,10-dimethyl-5,7-dioxo-12-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)-6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(1,3)-benzocyclodecaeno-4-yl) tert-butyl carbamate

[0592] At room temperature, add ((6) to a 1L round-bottom flask 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)-indolia-6(1,3)-pyridazinia-2(1,3)-benzocyclodecan-4-yl) tert-butyl 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 added dropwise to the mixture at room temperature with stirring. The resulting solution was stirred at 60 °C for 3 hours. 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 as a solid (22 g, 90% yield). LCMS(ESI)C 38 H 51 The calculated m / z[M+H] value of BN4O7 is 687.3; the experimental value is 687.4.

[0593] Step 10: Synthesis ((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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(1,3)-benzocyclodecaeno-4-yl) tert-butyl carbamate

[0594] Under N2 atmosphere, ((6) 3 S,4S)-10,10-dimethyl-5,7-dioxo-1 2 -(4,4,5,5-Tetramethyl-1,3,2-dioxaborane-2-yl)-6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1A mixture of tert-butyl carbamate (2.0 g, 2.8 mmol), 3-bromo-2-[(1S)-1-methoxyethyl]pyridine (0.60 g, 2.8 mmol), Pd(dppf)Cl2 (0.39 g, 0.5 mmol), and K3PO4 (1.2 g, 6.0 mmol) in dioxane (50 mL) and H2O (10 mL) was heated to 70 °C and stirred for 2 hours. The mixture was diluted with H2O (50 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (3 × 50 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 a solid product (1.5 g, 74% yield). LCMS(ESI)C 40 H 49 The calculated m / z[M+H] value of N5O6 is 695.4; the experimental value is 696.5.

[0595] Step 11: Synthesis ((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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(1,3)-benzocyclodecaeno-4-yl) tert-butyl carbamate

[0596] At 0℃, towards ((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 1A solution of tert-butyl carbamate (20 g, 28.7 mmol) in DMF (150 mL) and Cs₂CO₃ (18.7 g, 57.5 mmol) in DMF was added to 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 H₂O (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 Na₂SO₄, 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 transisomer (5.78 g, 25.7% yield) in solid form. LCMS(ESI)C 42 H 53 The calculated m / z[M+H] value of N5O6 is 724.4; the experimental value is 724.6.

[0597] Step 12: Synthesis (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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazineza-2(1,3)-benzocyclodecaban-5,7-dione

[0598] At 0℃, ((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)-indolaza-6(1,3)-pyridazinaza-2(1,3)-benzocyclodecana-4-yl)carbamate tert-butyl carbamate (1.3 g, 1.7 mmol) in TFA (10 mL) and DCM (20 mL) was stirred for 2 hours. The mixture was concentrated under reduced pressure to give a solid product (1.30 g, crude product). LCMS(ESI)C 37 H 45 The calculated m / z[M+H] value of N5O4 is 623.3; the experimental value is 624.4.

[0599] Intermediate 4: Synthesis ((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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-4-yl) tert-butyl carbamate

[0600]

[0601] Step 1: Synthesis of (S)-3-(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propionic acid

[0602] At room temperature, LiOH (21.64 g, 903.6 mmol) was added to a solution of methyl (2S)-3-(4-bromo-1,3-thiazolyl-2-yl)-2-[(tert-butoxycarbonyl)amino]propionate (110 g, 301.2 mmol) in THF (500 mL) and H₂O (200 mL). The resulting solution was stirred for 1 hour, followed by concentration under reduced pressure. The residue was adjusted to pH 6 with 1 M HCl, and then extracted with DCM (3 × 500 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the desired product (108 g, crude). LCMS(ESI)C 11 H 15 Calculated m / z[M+H] value of BrN2O4S: 351.00; Experimental value: 351.0.

[0603] Step 2: Synthesis of (S)-1-((S)-3-(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0604] At 0 °C, (3S)-1,2-diazacyclohexane-3-carboxylic acid methyl bis(trifluoroacetic acid) 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 to a solution of (S)-3-(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propionic acid (70 g, 199.3 mmol), DCM (500 mL). The resulting solution was heated to room temperature and stirred for 1 hour. The reaction was then quenched with H2O (500 mL) and extracted with EtOAc (3 × 500 mL). The combined organic layers were dried over Na2SO4, 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)C 17 H 25 The calculated m / z[M+H] value of BrN4O5S is 477.08; the experimental value is 477.1.

[0605] Step 3: Synthesis of (S)-3-(1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborphane-2-yl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol

[0606] At room temperature, bis(pinacol)diboron (51.31 g, 202.1 mmol), Pd(dppf)Cl2 (9.86 g, 13.48 mmol), and KOAc (26.44 g, 269.4 mmol) were added to a solution of 3-(5-bromo-1-ethyl-2-(2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (60 g, 134.7 mmol) in toluene (500 mL). The reaction mixture was then heated to 90 °C and stirred for 2 hours. 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) yielded the desired product (60.6 g, 94% yield). LCMS(ESI)C 29 H 41 The calculated m / z[M+H] value of BN2O4 is 493.32; the experimental value is 493.3.

[0607] 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)pyridin-3-yl)-1H-indol-5-yl)thiazolyl)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0608] At room temperature, a solution of (S)-3-(1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborpine-2-yl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (30 g, 60.9 mmol) in toluene (600 mL), dioxane (200 mL), and H2O (200 mL) was prepared by adding methyl (S)-1-((S)-3-(4-bromothiazol-2-yl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylate (43.62 g, 91.4 mmol), K3PO4 (32.23 g, 152.3 mmol), and Pd(dppf)Cl2 (8.91 g, 12.18 mmol) to the solution of (S)-3-(1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborpine-2-yl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (30 g, 60.9 mmol) in toluene (600 mL), dioxane (200 mL), and H2O (200 mL). The resulting solution was heated to 70°C and stirred overnight. The reaction mixture was then cooled to room temperature and quenched with H₂O (200 mL). The resulting mixture was extracted with EtOAc (3 × 1000 mL), and the combined organic layers were dried over Na₂SO₄, 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: C 40 H 54 The calculated [M+H] value of N6O7S is 763.39; the experimental value is 763.3.

[0609] 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)thiazolyl)propionyl)hexahydropyridazine-3-carboxylic acid

[0610] At room temperature, methyl hexahydropyridazine-3-carboxylate (39.7 g, 52.0 mmol) in THF (400 mL) and H₂O (100 mL) was added 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)thiazolyl)propionyl)hexahydropyridazine-3-carboxylate (39.7 g, 52.0 mmol) and added to LiOH·H₂O (3.74 g, 156.2 mmol). The resulting mixture was stirred for 1.5 hours 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)C 39 H 52 The calculated m / z[M+H] value of N6O7S is 749.37; the experimental value is 749.4.

[0611] Step 6: Synthesis ((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 H-8-oxa-2(4,2)-thiazolyl-1(5,3)-indole-6(1,3)-pyridazine-hexacycloundecaban-4-yl)tert-butyl carbamate

[0612] At 0 °C, EDCI (271.63 g, 1416.9 mmol) was added 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)thiazolyl)propionyl)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). The resulting mixture was heated to room temperature and stirred overnight. The reaction mixture was then quenched with H₂O 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)C 39 H 50 The calculated m / z[M+H] value of N6O6S is 731.36; the experimental value is 731.3.

[0613] Intermediate 5: Synthesis (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 -Decahydrogen-1 1 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(5,1)-pyridazinecyclodecaban-5,7-dione

[0614]

[0615] Step 1: Synthesis of methyl 2-((tert-butoxycarbonyl)amino)acrylate

[0616] DIPEA (17 g, 137 mmol) was added to a solution of (tert-butoxycarbonyl)-L-serine methyl ester (10 g, 45 mmol) in anhydrous MeCN (150 mL). The reaction mixture was stirred at 45 °C for 2 hours to give the product in solution form. LCMS(ESI)C9H 15The calculated value of m / z[M+Na] for NO4 is 201.1; the experimental value is 224.1.

[0617] Step 2: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)acrylate

[0618] At 0 °C, DMAP (13 g, 90 mmol) and (Boc)₂O (26 g, 120 mmol) were added to a solution of methyl 2-((tert-butoxycarbonyl)amino)acrylate (12 g, 60 mmol) in anhydrous MeCN (150 mL). The reaction was stirred for 6 h, followed by quenching with H₂O (100 mL) and extraction with DCM (3 × 200 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give a solid product (12.5 g, 65% yield). LCMS(ESI)C 14 H 23 The calculated value of m / z[M+Na] for NO6 is 301.2; the experimental value is 324.1.

[0619] Step 3: Synthesis of methyl 2-(bis(tert-butoxycarbonyl)amino)-3-(5-bromo-3,6-dihydropyridine-1(2H)-yl)propionate

[0620] Under an Ar atmosphere, methyl 2-{bis[(tert-butoxy)carbonyl]amino}prop-2-enoate (22 g, 74 mmol) was added to a mixture of 5-bromo-1,2,3,6-tetrahydropyridine (8.0 g, 49 mmol) and MeOH (120 mL). The mixture was stirred for 16 hours, then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give an oily product (12 g, 47% yield). LCMS(ESI)C 19 H 31 The calculated m / z[M+H] value of BrN2O6 is 462.1; the experimental value is 463.1.

[0621] Step 4: Synthesis of 3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propionic acid

[0622] LiOH (3.6 g, 151 mmol) was added to a mixture of methyl 2-(bis(tert-butoxycarbonyl)amino)-3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)propionate (14 g, 30 mmol) in dioxane (30 mL) and H₂O (12 mL). The mixture was heated to 35 °C and stirred for 12 hours, followed by the addition of 1 M HCl and adjustment of the pH to approximately 3–4. The mixture was extracted with DCM (2 × 300 mL), and the combined organic layers were dried over anhydrous Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure to give a solid product (10 g, 85% yield). LCMS(ESI)C 13 H 21 The calculated m / z[M+H] value of BrN2O4 is 348.1; the experimental value is 349.0.

[0623] Step 5: Synthesis of (3S)-1-(3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0624] HATU (13 g, 34 mmol) was added to a mixture of 3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propionic acid (10 g, 30 mmol), DIPEA (12 g, 93 mmol), and (3S)-1,2-diazacyclohexane-3-carboxylate (5.4 g, 37 mmol) in DMF (100 mL) at 0 °C under an Ar atmosphere. The mixture was stirred for 2 hours at 0 °C, followed by the addition of H2O and extraction with EtOAc (2 × 300 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by reversed-phase chromatography to give a solid product (9.0 g, 55% yield). LCMS(ESI)C 19 H 31 The calculated m / z[M+H] value of BrN4O5 is 474.1; the experimental value is 475.1.

[0625] Step 6: 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)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0626] Under an Ar atmosphere, a mixture of (3S)-1-(3-(5-bromo-3,6-dihydropyridin-1(2H)-yl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylate (9.0 g, 18 mmol), K2CO3 (4.5 g, 32 mmol), Pd(dppf)Cl2.DCM (1.4 g, 2 mmol), and 3-(1-ethyl-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)indol-3-yl)-2,2-dimethylprop-1-ol (9.8 g, 20 mmol) in dioxane (90 mL) and H2O (10 mL) was heated to 75 °C and stirred for 2 hours. H₂O was added and the mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were dried over Na₂SO₄, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product as a solid (4.0 g, 25% yield). LCMS(ESI)C 42 H 60 The calculated m / z[M+H] value of N6O7 is 760.5; the experimental value is 761.4.

[0627] 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)propionyl)hexahydropyridazine-3-carboxylic acid

[0628] At 0 °C, LiOH (0.60 g, 27 mmol) was added 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)propionyl)hexahydropyridazine-3-carboxylate (4.1 g, 5.0 mmol) in THF (35 mL). The mixture was stirred at 0 °C for 1.5 h, followed by the addition of 1 M HCl to adjust the pH to approximately 6-7. The mixture was then extracted with EtOAc (3 × 200 mL). The combined organic layers were dried over Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to give a solid product (3.6 g, 80% yield). LCMS(ESI)C 41 H 58 The calculated m / z[M+H] value of N6O7 is 746.4; the experimental value is 747.4.

[0629] Step 8: Synthesis ((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 -Decahydrogen-1 1 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(5,1)-pyridazine-1 / ...

[0630] Under an Ar atmosphere, EDCI-HCl (28 g, 140 mmol) and HOBt (6.5 g, 50 mmol) were added 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)propionyl)hexahydropyridazine-3-carboxylic acid (3.6 g, 5.0 mmol) and DIPEA (24 g, 190 mmol) in DCM (700 mL). The mixture was heated to 30 °C and stirred at 30 °C for 16 hours, followed by concentration under reduced pressure. The residue was diluted with EtOAc (200 mL) and washed with H2O (2 × 200 mL) and brine (200 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 a solid product (1.45 g, 40% yield). LCMS(ESI)C 41 H 56 The calculated m / z[M+H] value of N6O6 is 728.4; the experimental value is 729.4.

[0631] Step 9: Synthesis (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 64 6 5 6 6 -Decahydrogen-1 1 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(5,1)-pyridazinecyclodecaban-5,7-dione

[0632] At 0℃, towards ((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 -Decahydrogen-1 1 TFA (0.3 mL) was added to a mixture of H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(5,1)-pyridazine undecano-4-yl)carbamate (130 mg, 0.20 mmol) in DCM (1.0 mL). The mixture was heated to room temperature and stirred for 2 hours, followed by concentration under reduced pressure to obtain the product, which was used directly in the next step without further purification. LCMS(ESI)C 36 H 48 The calculated m / z[M+H] value of N6O4 is 628.4; the experimental value is 629.4.

[0633] Intermediate 6: Synthesis (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 H-8-oxa-2(4,2)-morpholina-1(5,3)-indola-6(1,3)-pyridazine-cycloundecaban-5,7-dione

[0634]

[0635] Step 1: Synthesis of (2R)-2-formylmorpholino-4-ylcarboxylate tert-butyl ester

[0636] At room temperature, TEMPO (715 mg, 4.6 mmol) and NaHCO3 (58 g, 690 mmol) were added to a solution of (2R)-2-(hydroxymethyl)morpholino-4-ylcarboxylate (50 g, 230 mmol) in EtOAc (1 L). The mixture was cooled to -50 °C, and then TCCA (56 g, 241 mmol) was added dropwise to EtOAc (100 mL) over 30 minutes. The reaction mixture was heated to 5 °C and maintained for 2 hours, then quenched with 10% Na2S2O3 (200 mL) and stirred for 20 minutes. 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 H2O (100 mL) and brine (100 mL), and then dried over anhydrous Na2SO4. The organic layers were concentrated under reduced pressure to give an oily product (50 g, crude).

[0637] Step 2: Synthesize (S,Z)-2-(2-(((benzoxy)carbonyl)amino)-3-methoxy-3-oxoprop-1-en-1-yl)morpholin-4-carboxylic acid tert-butyl ester

[0638] Tetramethylguanidine (35 g, 306 mmol) was added to a solution of (2R)-2-formylmorpholino-4-ylcarboxylate (49 g, 153 mmol) and methyl 2-{[(benzyloxy)carbonyl]amino}-2-(dimethoxyphosphoryl)acetate (60 g, 183 mmol) in MeCN (300 mL) at 0–10 °C. The reaction mixture was stirred at 10 °C for 30 min, then heated to room temperature and maintained for 2 h. The reaction mixture was diluted with DCM (200 mL) and washed with 10% citric acid (200 mL) and 10% NaHCO3 aqueous solution (200 mL). The organic phase was concentrated under reduced pressure and purified by silica gel column chromatography to give a solid product (36 g, 90% yield). LCMS(ESI)C 21 H 28 The calculated m / z [M+Na] value for N₂O₄ is 420.2; the experimental value is 443.1.

[0639] Step 3: Synthesis of tert-butyl (S)-2-((S)-2-(((benzoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)morpholine-4-carboxylic acid ester

[0640] Add (S,S)-Et-DUPHOS-Rh (500 mg, 0.7 mmol) to a solution of (S,Z)-2-(2-(((benzyloxy)carbonyl)amino)-3-methoxy-3-oxopropyl-1-en-1-yl)morpholin-4-carboxylate (49 g, 0.12 mol) in MeOH (500 mL). Stir the mixture at room temperature for 48 hours under a H2 (60 psi) atmosphere. The reaction mixture was concentrated and purified by silica gel column chromatography to give the product as a solid (44 g, 90% yield). LCMS(ESI)C 21 H 30 The calculated m / z[M+Na] value of N2O7 is 422.2; the experimental value is 445.2.

[0641] Step 4: Synthesis of methyl (S)-2-(((benzoxy)carbonyl)amino)-3-((S)-morpholin-2-yl)propionate

[0642] At 15°C, HCl / EtOAc (25 mL) was added to a stirred solution of (S)-2-((S)-2-(((benzoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)morpholine-4-carboxylic acid tert-butyl ester (2.2 g, 5.2 mmol) in EtOAc (2 mL). The reaction was stirred at 15°C for 2 hours, followed by concentration under reduced pressure to give an oily product (1.51 g, 90% yield). LCMS(ESI)C 16 H 22 The calculated value of m / z[M+H] for N2O5 is 322.1; the experimental value is 323.2.

[0643] 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

[0644] At 0 °C, TBSCl (50.7 g, 0.34 mol) in DCM (200 mL) was added to a solution of 3-(5-bromo-1-ethyl-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-3-yl)-2,2-dimethylprop-1-ol (100 g, 0.22 mol) and imidazole (30.6 g, 0.45 mol) in DCM (800 mL). The reaction was stirred at room temperature for 2 hours. The resulting solution was washed with H2O (3 × 300 mL) and brine (2 × 200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give a solid product (138 g, 90% yield). LCMS(ESI)C 29 H 43The calculated m / z[M+H] value of BrN2O2Si is 558.2; the experimental value is 559.2.

[0645] 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]propionate

[0646] Methyl (2S)-2-{[(benzyloxy)carbonyl]amino}-3-[(2S)-morpholin-2-yl]propionate was added 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). (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 the addition of RuPhos-Pd-G2 (3.5 g, 4.4 mmol) at 105 °C under N2 atmosphere. The reaction mixture was stirred at 105 °C for 6 hours under 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 a solid product (55 g, 73% yield). LCMS(ESI)C 45 H 64 The calculated m / z[M+H] value of N4O7Si is 800.5; the experimental value is 801.5.

[0647] 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]propionic acid

[0648] At room temperature, 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]propionate (10 g, 12 mmol) in THF (270 mL) was added to LiOH (1.3 g, 31 mmol) in H₂O (45 mL). The reaction was stirred at room temperature for 2 hours, followed by pH adjustment to 4–5 with 1 N HCl at 0–5 °C. The resulting mixture was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine and dried over anhydrous Na₂SO₄. The organic phase was then concentrated under reduced pressure to give a solid product (9.5 g, 97% yield). LCMS(ESI)C 44 H 62 The calculated m / z[M+H] value of N4O7Si is 786.4; the experimental value is 787.4.

[0649] Step 8: Synthesis of (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)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0650] Methyl (S)-hexahydropyridazine-3-carboxylate (2 g, 14 mmol) was added 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]propionic acid (10 g, 12.7 mmol) in DMF (150 mL). The mixture was then cooled to 0 °C, and DIPEA (32.8 g, 254 mmol) was added, followed by HATU (9.7 g, 25.4 mmol) at 0–5 °C. The reaction mixture was stirred for 1 hour at 0–5 °C. The resulting mixture was diluted with EtOAc (500 mL) and H2O (200 mL). The organic layer was separated and washed with H₂O (2 × 100 mL) and brine (100 mL), then 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 obtain the product. LCMS(ESI)C 50 H 72 The calculated m / z[M+H] value of N6O8Si is 912.5; the experimental value is 913.4.

[0651] Step 9: 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)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0652] At room temperature, a mixture of tetrabutylammonium fluoride (1M in THF, 180mL, 180mmol) and AcOH (11g, 200mmol) was added to a solution of (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)propionyl)hexahydropyridazine-3-carboxylate (8.5g, 9mmol) in THF (8mL). The reaction mixture was stirred at 75°C for 3 hours. The resulting mixture was diluted with EtOAc (150mL) and washed with H2O (6 x 20mL). The organic phase was concentrated under reduced pressure to give a solid product (7.4 g, 100% yield). LCMS(ESI)C 44 H 58 The calculated m / z[M+H] value of N6O8 is 799.4; the experimental value is 798.4.

[0653] 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)propionyl)hexahydropyridazine-3-carboxylic acid

[0654] LiOH (600 mg, 25 mmol) in H₂O (30 mL) was added to a solution 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)propionyl)hexahydropyridazine-3-carboxylate (8 g, 10 mmol) in THF (200 mL). The reaction mixture was stirred for 1 hour at room temperature, then treated with 1N HCl at 0–5 °C to adjust the pH to 4–5, and extracted with EtOAc (2 × 500 mL). The organic phase was washed with brine and concentrated under reduced pressure to give a solid product (8 g, crude). LCMS(ESI)C 43 H 56 The calculated value of m / z[M+H] for N6O8 is 784.4; the experimental value is 785.4.

[0655] Step 11: Synthesize to obtain ((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 H-8-oxa-2(4,2)-morpholina-1(5,3)-indola-6(1,3)-pyridazine-hexacycloundecaban-4-yl)carbamate

[0656] Under an argon atmosphere, at room temperature, EDCI (88 g, 458 mmol) and HOBt (27.6 g, 204 mmol) were added to a stirred solution 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)propionyl)hexahydropyridazine-3-carboxylic acid (8 g, 10.2 mmol) and DIPEA (59 g, 459 mmol) in DCM (800 mL). The reaction mixture was stirred for 16 hours at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give a solid product (5 g, 66% yield); LCMS(ESI)C 43 H54 The calculated m / z[M+H] value of N6O7 is 766.4; the experimental value is 767.4.

[0657] Step 12: Synthesis (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 H-8-oxa-2(4,2)-morpholina-1(5,3)-indola-6(1,3)-pyridazine-cycloundecaban-5,7-dione

[0658] In an H2 atmosphere, at room temperature, to ((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 A solution of methyl carbamate (400 mg, 0.5 mmol) in MeOH (20 mL) was mixed with Pd / C (200 mg) and ammonium acetate (834 mg, 16 mmol) and stirred for 2 hours. The resulting mixture was filtered and concentrated under reduced pressure. The residue was redissolved in DCM (20 mL) and washed with H₂O (5 mL × 2), followed by concentration under reduced pressure to give a solid product (320 mg, 97% yield). LCMS(ESI)C 35 H 48 The calculated m / z[M+H] value of N6O5 is 632.4; the experimental value is 633.3.

[0659] Intermediate 7: Synthesis ((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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-4-yl) tert-butyl carbamate

[0660]

[0661] Step 1: Synthesis of (S)-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)boronic acid

[0662] Under an argon atmosphere, dtbpy (14.91 g, 55.5 mmol) and chloro(1,5-cyclooctadiene)iridium(I) dimer (7.46 g, 11.1 mmol) were added to a stirred solution of (S)-3-bromo-2-(1-methoxyethyl)pyridine (80.0 g, 370.24 mmol) and bis(pinacol)diborone (141.03 g, 555.3 mmol) in THF (320 mL). The resulting mixture was stirred at 75 °C for 16 hours. The mixture was concentrated under reduced pressure, and the resulting residue was dissolved in EtOAc (200 mL) and adjusted to pH 10 with a solution of Na2CO3 (40 g) and NaOH (10 g) in H2O (600 mL). The aqueous layer was extracted with EtOAc (800 mL), followed by acidification of the aqueous phase to pH 6 with HCl (6N), precipitating the desired product as a solid (50 g, 52% yield). LCMS(ESI)C8H 11 Calculated m / z[M+H] value of BBrNO3: 260.01; Experimental value: 260.0.

[0663] Step 2: Synthesis of (S)-3-bromo-5-iodo-2-(1-methoxyethyl)pyridine

[0664] NIS (49.78 g, 221.2 mmol) was added to a stirred solution of (S)-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)boronic acid (23.0 g, 88.5 mmol) in MeCN (230 mL) at room temperature. The resulting mixture was stirred overnight at 80 °C under an argon atmosphere. The mixture was concentrated under reduced pressure, and the residue was dissolved in DCM (2.1 L) and washed with Na2S2O3 (3 × 500 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the desired product (20 g, 66% yield). Calculated m / z [M+H] for LCMS (ESI)C8H9BrINO: 341.90; Experimental: 341.7.

[0665] Step 3: Synthesis of (S)-4-(5-bromo-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylic acid methyl ester

[0666] To a 3L, three-necked round-bottom flask purged with and maintained under an inert argon atmosphere, add 147 g (429.8 mmol) of 3-bromo-5-iodo-2-[(1S)-1-methoxyethyl]pyridine, 94.69 g (429.8 mmol) of piperazine-1-carboxylic acid methyl ester, 4.83 g (21.4 mmol) of Pd(OAc)₂, 5.35 g (8.6 mmol) of BINAP, 350.14 g (1074.6 mmol) of Cs₂CO₃, and 1 L of toluene. The resulting solution was stirred overnight in an oil bath at 100 °C. The reaction mixture was then cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / hexane) to give a solid product (135 g, 65% yield). LCMS(ESI)C 20 H 24 The calculated m / z[M+H] value of BrN3O3 is 433.1; the experimental value is 434.1.

[0667] Step 4: Synthesis of (S)-4-(6-(1-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborphane-2-yl)pyridin-3-yl)piperazine-1-carboxylic acid methyl ester

[0668] To a 3-L round-bottom flask purged with and maintained under an inert argon atmosphere, add methyl 4-[5-bromo-6-[(1S)-1-methoxyethyl]pyridin-3-yl]piperazine-1-carboxylic acid (135 g, 310.8 mmol), bis(pinacol)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). Stir the resulting solution in an oil bath at 90 °C for 2 days. Cool the reaction mixture to room temperature. Concentrate the resulting mixture under reduced pressure. Purify the residue by neutral alumina column chromatography (30% EtOAC / / hexane) to give a solid product (167 g, crude). LCMS(ESI)C 26 H 36 The calculated value of m / z[M+H] for BN3O5 is 481.3; the experimental value is 482.1.

[0669] Step 5: Synthesis of (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-carboxylic acid methyl ester

[0670] To a 3-L, 3-necked round-bottom flask purged with and maintained under an inert argon atmosphere, add (S)-4-(6-(1-methoxyethyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborphane-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)Cl2 (25.38 g, 34.6 mmol), dioxane (600 mL), H2O (200 mL), K3PO4 (184.09 g, 867.2 mmol), and toluene (200 mL). Stir the resulting solution overnight in an oil bath at 70 °C. Next, the reaction mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% EtOAc / hexane) to give a solid product (146 g, 48% yield). LCMS(ESI)C 49 H 57 The calculated m / z[M+H] value of BrN4O4Si is 872.3; the experimental value is 873.3.

[0671] Step 6: Synthesis of (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-carboxylic acid methyl ester

[0672] Under a nitrogen atmosphere, methyl iodoethane (52.11 g, 334.0 mmol) was added fractionally to a stirred mixture of (S)-4-(5-(5-bromo-3-(3-((tert-butyldiphenylsilyl)oxy)-2,2-dimethylpropyl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazin-1-carboxylic acid (146 g, 167.0 mmol) and Cs₂CO₃ (163.28 g, 501.1 mmol) in DMF (1200 mL) at 0 °C. The final reaction mixture was stirred for 12 hours at room temperature. The resulting mixture was diluted with EtOAc (1 L) and washed with brine (3 × 1.5 L). The organic layer was dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to obtain a solid product (143g, crude product), which was used directly in the next step without further purification. LCMS(ESI)C 51 H 61 The calculated m / z[M+H] value of BrN4O4Si is 900.4; the experimental value is 901.4.

[0673] Step 7: Synthesis of (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-carboxylic acid methyl ester

[0674] CsF (72.24 g, 475.5 mmol) was added to a stirred mixture of (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)piperazin-1-carboxylic acid methyl ester (143 g, 158.5 mmol) in DMF (1250 mL). The reaction mixture was then stirred for 2 days at 60 °C under a N2 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 transisomers, A (38 g, 36% yield) and B (34 g, 34% yield), both as solids. LCMS(ESI)C 35 H 43The calculated m / z[M+H] value of BrN4O4 is 663.2; the experimental value is also 662.2.

[0675] Step 8: Synthesis of (S)-4-(5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylic acid methyl ester

[0676] To a 500-mL three-necked round-bottom flask purged with and maintained under an inert nitrogen atmosphere, add (S)-4-(5-(5-bromo-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazin-1-carboxylic acid methyl ester (14 g, 21.1 mmol), bis(pinacol)diboron (5.89 g, 23.21 mmol), Pd(dppf)Cl2 (1.54 g, 2.1 mmol), KOAc (5.18 g, 52.7 mmol), and toluene (150 mL). Stir the resulting solution in an oil bath at 90 °C for 5 hours. Cool the reaction mixture to room temperature and then concentrate under reduced pressure. Purify the residue by silica gel column chromatography (30% EtOAc / petroleum ether) to give the product as a solid (12 g, 76% yield). LCMS(ESI)C 41 H 55 The calculated value of m / z[M+H] for BN4O6 is 710.4; the experimental value is 711.3.

[0677] Step 9: Synthesis of (S)-1-((S)-3-(4-(2-(5-(4-((benzoxy)carbonyl)piperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-5-yl)thiazolyl-2-yl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0678] To a 250-mL round-bottom flask purged with and maintained under an inert argon atmosphere, place methyl benzoate (S)-4-(5-(1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)-1H-indol-2-yl)-6-(1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylic acid (10.8 g, 15.2 mmol), (3S)- Methyl 1-[(2S)-3-(4-bromo-1,3-thiazolyl-2-yl)-2-[(tert-butoxycarbonyl)amino]propionyl]-1,2-diazacyclohexane-3-carboxylate (7.98 g, 16.7 mmol), Pd(dtbpf)Cl2 (0.99 g, 1.52 mmol), K3PO4 (8.06 g, 37.9 mmol), toluene (60 mL), dioxane (20 mL), H2O (20 mL). The resulting solution was stirred in an oil bath at 70 °C 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 as a solid (8 g, 51% yield). LCMS(ESI)C 52 H 68 The calculated m / z[M+H] value of N8O9S is 980.5; the experimental value is 980.9.

[0679] Step 10: Synthesis of (S)-1-((S)-3-(4-(2-(5-(4-((benzoxy)carbonyl)piperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-5-yl)thiazolyl-2-yl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylic acid

[0680] Under a nitrogen atmosphere, LiOH (2.45 g, 61.1 mmol) was added to a stirred mixture 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)thiazolyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylate (12 g, 12.23 mmol) in THF (100 mL) / H2O (100 mL), and the resulting mixture was stirred for 2 hours at room temperature. THF was removed under reduced pressure. The pH of the aqueous phase was acidified to 5 with 1N HCl at 0 °C. The aqueous layer was extracted with DCM (3 × 100 mL). The organic phase was concentrated under reduced pressure to give a solid product (10 g, 85% yield). LCMS(ESI)C 51 H 66 The calculated m / z[M+H] value of N8O9S is 966.5; the experimental value is 967.0.

[0681] Step 11: Synthesize 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)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-1-cyclodecaban-1 2 methyl 6-((S)-1-methoxyethyl)pyridin-3-yl)piperazine-1-carboxylic acid methyl ester

[0682] To a 3-L round-bottom flask purged with and maintained under an inert nitrogen atmosphere, add (S)-1-((S)-3-(4-(2-(5-(4-((benzoxy)carbonyl)piperazin-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-1H-indol-5-yl)thiazolyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazin-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 overnight at room temperature, followed by concentration under reduced pressure. The resulting solution was diluted with DCM (1 L) and washed with 1 M HCl (3 × 1 L) and H₂O (3 × 1 L). The organic layer was then concentrated under reduced pressure and purified by silica gel column chromatography (50% EtOAc / hexane) to give a solid product (10.4 g, 55% yield). LCMS(ESI)C 51 H 64 The calculated value of m / z[M+H] for N8O8S is 948.5; the experimental value is 949.3.

[0683] Step 12: Synthesis ((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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-4-yl) tert-butyl carbamate

[0684] Add 4-(5-(6) to a 250-mL round-bottom flask purged with and maintained under an inert nitrogen atmosphere. 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 1H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-1-cyclodecaban-1 2 Benzyl 6-((S)-1-methoxyethyl)pyridin-3-yl)piperazin-1-carboxylic acid (10.40 g, 10.9 mmol), Pd(OH)₂ / C (5 g, 46.9 mmol), MeOH (100 mL). The resulting solution was stirred for 3 hours at room temperature under a 2 atm H₂ atmosphere. The solid was filtered off and the filter cake was washed with MeOH (3 × 100 mL). The combined organic phases were concentrated under reduced pressure to give the product as a solid (8.5 g, 90% yield). LCMS(ESI)C 43 H 58 The calculated m / z[M+H] value of N8O6S is 814.4; the experimental value is 815.3.

[0685] Step 13: Synthesis ((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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-4-yl) tert-butyl carbamate

[0686] Place (6) into a 1000-mL round-bottom flask purged with and maintained by an inert nitrogen atmosphere. 3 S,4S,Z)-1 1 -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 1H-8-oxa-2(4,2)-thiazo-1(5,3)-indolia-6(1,3)-pyridazine-cycloundecan-4-yl)carbamate tert-butyl ester (8.5 g, 10.4 mmol), MeOH (100 mL), and AcOH (1.88 g, 31.2 mmol) were stirred for 15 minutes, followed by the addition of HCHO (1.88 g, 23.15 mmol, 37% aqueous solution) and NaBH3CN (788 mg, 12.5 mmol) at room temperature. The resulting solution was stirred for 3 hours. The mixture was then quenched with H2O (100 mL) and concentrated under reduced pressure to remove MeOH. The resulting solution was diluted with DCM (300 mL) and washed with H2O (3 × 100 mL). The solution was concentrated under reduced pressure to give the product as a solid (8.2 g, 90% yield). LCMS(ESI)C 44 H 60 The calculated m / z[M+H] value of N8O6S is 828.4; the experimental value is 829.3.

[0687] Intermediate 8: Synthesis (6) 3 (S,4S,Z)-4-amino-1 1 -Ethyl-1 2 -(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-5,7-dione

[0688]

[0689] 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

[0690] Under an argon atmosphere and at 0 °C, fractionally add DMAP (2.74 g, 22.452 mmol) and Ac₂O (27.50 g, 269.420 mmol) to a stirred solution of (S)-3-(5-bromo-1-ethyl-2-(2-(1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylprop-1-ol (100 g, 224.517 mmol) and Et₃N (45.44 g, 449.034 mmol) in DCM (1 L). Stir the resulting mixture at room temperature for 3 hours. Concentrate the mixture under reduced pressure and then dilute with EtOAc (1000 mL). Wash the resulting mixture with 1 M HCl (500 mL), followed by washing with saturated NaHCO₃ (500 mL) and brine (500 mL), and dry over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by grinding with petroleum ether (500 mL) to give a product as a white solid (93.3 g, 85% yield). LCMS(ESI)C 25 H 31 The calculated m / z[M+H] value for BrN₂O₃ is 487.16; the experimental value is 489.2.

[0691] 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

[0692] Under an argon atmosphere, at room temperature, fractionally add dtbpy (7.71 g, 28.711 mmol) and chloro(1,5-cyclooctadiene)iridium(I) dimer (6.43 g, 9.570 mmol) 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). The resulting mixture was stirred overnight at 75 °C. The mixture was then concentrated under reduced pressure to give an oily product (190 g, crude product). LCMS(ESI)C 25 H 32 The calculated m / z [M+H] value of BBrN2O5 is 531.17; the experimental value is 533.3.

[0693] 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

[0694] A fractional amount of NaI (186.22 g, 1242.354 mmol) in H₂O (225 mL) was added 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 trihydrate-T (349.96 g, 1242.354 mmol) in THF (550 mL) at 0 °C. The resulting mixture was stirred overnight at 50 °C under argon atmosphere. The mixture was concentrated under reduced pressure and then washed with CHCl₃ (500 mL). The mixture was filtered, and the filter cake was washed with CHCl₃ (3 × 250 mL). The filtrate was extracted with CHCl₃ (3 × 500 mL). The combined organic layers were washed with Na₂S₂O₃ (500 mL), followed by washing with brine (2 × 200 mL) and drying over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (18% EtOAc / petroleum ether) to give a solid product (24 g, 18% yield). LCMS (ESI) m / z [M+H]; C 25 H 30 Calculated value of BrIN2O3: 613.06; Experimental value: 614.7

[0695] Step 4: Synthesis of 3-(5-bromo-1-ethyl-2-(5-(((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate

[0696] Under an argon atmosphere, at room temperature, Pd(OAc)₂ (329.44 mg, 1.467 mmol) was added to a stirred solution of acetic acid 3-(5-bromo-1-ethyl-2-{5-iodo-2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-3-yl) in toluene (63 mL). The resulting mixture was stirred at 100 °C for 6 hours. 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 a solid product (6.9 g, 75% yield). LCMS(ESI)C32 H 43 The calculated m / z[M+H] value of BrN4O4 is 627.25; the experimental value is 627.4.

[0697] Step 5: Synthesis of 3-(1-ethyl-2-(5-((S)-hexahydropyrazino[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-dioxaborhecyclopentan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate

[0698] Under an argon atmosphere, at room temperature, Pd(dppf)Cl2 (0.37 g, 0.512 mmol) was added in fractions to a stirred solution of acetic acid 3-(5-bromo-1-ethyl-2-(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl ester (3.2 g, 5.115 mmol), KOAc (1.51 g, 15.345 mmol) and 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1,3,2-dioxaborhexacyclopentanane (2.60 g, 10.230 mmol) in toluene (48 mL). The resulting mixture was stirred at 90°C for 1.5 hours. 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 a solid product (3.0 g, 88% yield). LCMS(ESI)C 38 H 55 The calculated m / z[M+H] value for BN4O6 is 675.43; the experimental value is 675.1.

[0699] Step 6: Synthesis of (S)-1-((S)-3-(4-(3-(3-acetoxy-2,2-dimethylpropyl)-1-ethyl-2-(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazolyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0700] Under an argon atmosphere, dioxane (17.82 mL, 210.307 mmol) and H₂O (17.82 mL) were added to a stirred mixture of 3-(1-ethyl-2-(5-((S)-hexahydropyrazino[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-dioxaborhecyclopentan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (5 g, 7.433 mmol) and K₃PO₄ (4.26 g, 20.067 mmol) in toluene (54 mL) at room temperature. 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. 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 a solid product (4.6 g, 66% yield). LCMS(ESI)C 49 H 68 The calculated m / z [M+H] value for N8O9S is 945.49; the experimental value is 945.7.

[0701] Step 7: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-2-(5-(((S)-hexahydropyrazino[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)thiazolyl-2-yl)propionyl)hexahydropyridazine-3-carboxylic acid

[0702] At 0 °C, methyl hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazolyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylate (6 g, 6.361 mmol) in a stirred solution of (S)-1-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)thiazolyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylate (6 g, 6.361 mmol) in THF (43 mL) was added to LiOH·H₂O (573.92 mg, 13.677 mmol). The resulting mixture was stirred for 16 hours at room temperature. The mixture was acidified to pH 6 with HCl (aqueous solution). 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 obtain a solid product (4 g, crude product). LCMS(ESI)C 45 H 60 Calculated m / z[M+H] value for N8O9S: 889.43; Experimental value: 889.7

[0703] Step 8: Synthesis ((6) 3 S,4S,Z)-1 1 -Ethyl-1 2 -(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-4-yl) tert-butyl carbamate

[0704] Under an argon atmosphere, at 0°C, EDCI (25.93 g, 135.27 mmol) was added dropwise to a stirred solution of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(1-ethyl-2-(5-(((S)-hexahydropyrazino[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)thiazolyl)propionyl)hexahydropyridazin-3-carboxylic acid (4 g, 4.51 mmol), HOBt (6.09 g, 45.09 mmol), and DIPEA (23.31 g, 180.36 mmol) in a DCM (200 mL) solution. The resulting mixture was stirred for 16 hours at room temperature, followed by concentration under reduced pressure. The reaction was quenched with H₂O at 0 °C and extracted with EtOAc (500 mL). The combined organic layers were washed with brine 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 a solid product (2.0 g, 52% yield). LCMS(ESI)C 46 H 62 Calculated m / z[M+H] value for N8O7S: 870.4; Experimental value: 871.8

[0705] Step 9: Synthesis (6) 3 (S,4S,Z)-4-amino-1 1 -Ethyl-1 2 -(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-5,7-dione

[0706] Under an argon atmosphere, at 0°C, towards ((6) 3 S,4S,Z)-1 1 -Ethyl-1 2 -(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 62 6 3 6 4 6 5 6 6 -hexahydro-1 1 TFA (1 mL) was added dropwise to a stirred solution of H-8-oxa-2(4,2)-thiazo-1(5,3)-indolia-6(1,3)-pyridazine-cycloundecan-4-yl)carbamate (316 mg, 0.345 mmol) in a DCM (3 mL). The resulting mixture was stirred for 2 hours at room temperature. The mixture was alkalized to pH 8 with saturated aqueous NaHCO3 solution. The resulting mixture was extracted with EtOAc (3 × 80 mL). The combined organic layers were washed with brine (3 × 40 mL) and dried over anhydrous Na2SO4. 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)C 41 H 54 Calculated m / z[M+H] value for N8O5S: 771.4; Experimental value: 771.6

[0707] Intermediate 9: Synthesis (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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-5,7-dione

[0708]

[0709] 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

[0710] Under an argon atmosphere, at room temperature, Pd(OAc)₂ (329.44 mg, 1.467 mmol) was added fractionally to a stirred solution of acetic acid 3-(5-bromo-1-ethyl-2-{5-iodo-2-[(1S)-1-methoxyethyl]pyridin-3-yl}indol-3-yl) in toluene (63 mL). The resulting mixture was stirred at 100 °C for 6 hours, then 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 a solid product (6 g, 65% yield). LCMS(ESI)C 33 H 45 The calculated m / z[M+H] value of BrN4O3 is 625.28; the experimental value is 627.4.

[0711] 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-dioxaborhecyclopentan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate

[0712] Under an argon atmosphere, at room temperature, Pd(dppf)Cl2 (0.37 g, 0.512 mmol) was added fractionally 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-dioxaborhexacyclopentan-2-yl)-1,3,2-dioxaborhexacyclopentane (2.60 g, 10.230 mmol) in toluene (48 mL). The resulting mixture was stirred at 90 °C for 1.5 hours. 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 a solid product (3.1 g, 81% yield). LCMS(ESI)C 39 H 57The calculated m / z[M+H] value for BN4O5 is 673.45; the experimental value is 673.4.

[0713] Step 3: Synthesis of (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)thiazolyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0714] Under an argon atmosphere, at room temperature, 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-dioxaborhecyclopentan-2-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (5 g, 7.433 mmol), (S)-1-(( Methyl hexahydropyridazine-3-carboxylate (3.89 g, 8.176 mmol) and K3PO4 (4.26 g, 20.067 mmol) were added to a stirred mixture of toluene (54 mL), dioxane (18 mL), and H2O (18 mL), followed by Pd(dtbpf)Cl2 (969 mg, 1.486 mmol). The 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 Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (8% MeOH / DCM) to give a solid product (6.8 g, 83% yield). LCMS(ESI)C 50 H 70 The calculated m / z[M+H] value for N8O8S is 943.51; the experimental value is 943.4.

[0715] 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-pyridino[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1H-indol-5-yl)thiazolyl)propionyl)hexahydropyridazine-3-carboxylic acid

[0716] Under an argon atmosphere and at 0 °C, methyl hexahydropyridazine-3-carboxylate (6 g, 6.361 mmol) was added dropwise to a stirred solution of (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)thiazolyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylate (6 g, 6.361 mmol) in THF (43 mL). The resulting mixture was stirred for 16 hours at room temperature. The mixture was acidified to pH 6 with HCl (aqueous solution). 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 obtain a solid product (4 g, crude product). LCMS(ESI)C 47 H 66 The calculated m / z[M+H] value for N8O7S is 887.49; the experimental value is 887.6.

[0717] Step 5: Synthesis ((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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-4-yl) tert-butyl carbamate

[0718] Under an argon atmosphere, at 0°C, EDCI (25.93 g, 135.270 mmol) was added dropwise to a stirred solution 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)thiazolyl)propionyl)hexahydropyridazin-3-carboxylic acid (4 g, 4.509 mmol), HOBt (6.09 g, 45.090 mmol) and DIPEA (23.31 g, 180.360 mmol) in a DCM (200 mL) solution containing (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)thiazolyl)propionyl)hexahydropyridazin-3-carboxylic acid (4 g, 4.509 mmol), HOBt (6.09 g, 45.090 mmol) and DIPEA (23.31 g, 180.360 mmol) in a DCM (200 mL) solution. The resulting mixture was stirred for 16 hours at room temperature. The mixture was concentrated under reduced pressure and quenched with H₂O at 0 °C, then extracted with EtOAc (500 mL). The combined organic layers were washed with brine 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 a solid product (2.0 g, 49% yield). LCMS(ESI)C 47 H 64 Calculated m / z[M+H] value for N8O6S: 869.47; Experimental value: 869.8

[0719] Step 6: Synthesis (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 H-8-oxa-2(4,2)-thiazo-1(5,3)-indolaz-6(1,3)-pyridazine-cycloundecaban-5,7-dione

[0720] At 0℃, towards ((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 26 3 6 4 6 5 6 6 -hexahydro-1 1 900 mg (1.035 mmol) of tert-butyl carbamate (H-8-oxa-2(4,2)-thiazo-1(5,3)-indolia-6(1,3)-pyridazine-cycloundecan-4-yl)carbamate (900 mg, 1.035 mmol) was added dropwise to a stirred solution in a DCM (9 mL) with 3 mL of TFA. The resulting mixture was stirred for 2 hours at room temperature. The mixture was alkalized to pH 8 with saturated aqueous NaHCO3 solution and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous Na2SO4. 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)C 42 H 56 Calculated m / z[M+H] value for N8O4S: 769.42; Experimental value: 769.5

[0721] Intermediate 10: Synthesis (6) 3 S,4S)-4-amino-1 2 -(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazineza-2(1,3)-benzocyclodecaban-5,7-dione

[0722]

[0723] 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

[0724] Under an argon atmosphere and at 0 °C, DMAP (1.46 g, 0.012 mol) and acetic anhydride (14.7 g, 144 mmol) were added dropwise 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-dimethylprop-1-ol (60 g, 0.12 mol) and Et3N (24.33 g, 0.24 mol) in DCM (600 mL). The resulting mixture was stirred for 2 hours at room temperature. The mixture was concentrated under reduced pressure and washed with HCl (500 mL). The resulting mixture was washed with a saturated aqueous solution of NaHCO3 (500 mL). The combined organic layers were washed with brine (500 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to obtain an oily product (59.6 g, 92% yield). LCMS(ESI)C 25 H 28 The calculated m / z[M+H] value of BrF3N2O3 is 541.13; the experimental value is 543.2.

[0725] 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

[0726] Under an argon atmosphere, at room temperature, dtbpy (4.10 g, 15.266 mmol) and chloro(1,5-cyclooctadiene)iridium(I) dimer (3.42 g, 5.089 mmol) were added fractionally 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-dioxaborhexacyclopentan-2-yl)-1,3,2-dioxaborhexacyclopentane (38.77 g, 152.656 mmol) in THF (40 mL). The resulting mixture was stirred at 75 °C for 5 hours. The resulting mixture was concentrated under reduced pressure to obtain an oily product (102.4 g, crude product). LCMS(ESI)C 25 H 29 The calculated m / z [M+H] value of BBrF3N2O5 is 585.14; the experimental value is 585.2.

[0727] Step 3: Synthesis 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

[0728] Under an argon atmosphere, at 0 °C, NaI (104.91 g, 699.896 mmol) was added dropwise 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]imino (197 g, 699.896 mmol) in THF (258 mL) in H₂O (129 mL). The resulting mixture was stirred at 55 °C for 16 hours. The resulting mixture was concentrated under reduced pressure and extracted with CH₃Cl (2 × 200 mL). The combined organic layers were washed with brine (2 × 20 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20% EtOAc / petroleum ether) to give a solid product (15.3 g, 26% yield). LCMS(ESI)C 32 H 40 The calculated m / z[M+H] value of BrF3N4O4 is 666.0; the experimental value is 667.3.

[0729] Step 4: Synthesis of (S)-1-((S)-3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-((S)-hexahydropyrazino[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)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0730] Under an argon atmosphere, at room temperature, fractionally add Cs₂CO₃ (5932.38 mg, 18.207 mmol) and BINAP (125.97 mg, 0.202 mmol) 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). Fractionally add Pd(OAc)₂ (90.84 mg, 0.405 mmol) to the mixture. Stir the resulting mixture at 90 °C for 16 hours. The mixture was cooled to room temperature and then filtered. The filter cake was washed with EtOAc (2 × 20 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (10% MeOH / DCM) to give a solid product (2.3 g, 83% yield). LCMS(ESI)C 32 H 40 The calculated m / z[M+H] value of BrF3N4O4 is 681.23; the experimental value is 681.4.

[0731] Step 5: Synthesis of (S)-1-((S)-3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-((S)-hexahydropyrazino[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)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0732] In an air atmosphere at room temperature, methyl hexahydropyrazinoyl (2.33 g, 4.512 mmol) and K3PO4 (1.59 g, 7.490 mmol) were added to a 250 mL three-necked round-bottom flask. At room temperature, fractionally added Pd(dtbpf)Cl2 (0.29 g, 0.451 mmol) to a stirred mixture of H2O (8.20 mL) and dioxane (8.20 mL) in toluene. The resulting mixture was stirred at 65 °C for 3 hours. The 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 Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (3→4% MeOH / DCM) to give a solid product (2.7 g, 90% yield). LCMS(ESI)C 52 H 68 Calculated m / z[M+H] value for F3N7O9: 991.5; Experimental value: 992.7

[0733] Step 6: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(2-(5-(((S)-hexahydropyrazino[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)propionyl)hexahydropyridazine-3-carboxylic acid

[0734] At room temperature, methyl (S)-1-((S)-3-(3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(5-((S)-hexahydropyrazino[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)propionyl)hexahydropyridazine-3-carboxylate (3 g, 3.024 mmol) and THF (30 mL) were added in fractions to H₂O (12.7 mL) at 0 °C. The resulting mixture was stirred for 16 hours at room temperature. The mixture was acidified to pH 5 with 1N HCl. The resulting mixture was extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with brine (2 × 100 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to give a solid product (2.7 g, crude product). LCMS(ESI)C 49 H 64 The calculated m / z[M+H] value for F3N7O8 is 936.48; the experimental value is 936.7.

[0735] Step 7: Synthesis ((6) 3 S,4S)-12-(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(1,3)-benzocyclodecaeno-4-yl) tert-butyl carbamate

[0736] At room temperature, (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(2-(5-(((S)-hexahydropyrazino[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)propionyl)hexahydropyridazin-3-carboxylic acid (3.12 g, 3.333 mmol) and DCM (624 mL) were added to the mixture in fractions at 0 °C. The resulting mixture was then stirred for 30 minutes. At room temperature, EDCI (19.17 g, 99.990 mmol) was added in fractions to the above mixture over 16 hours. 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 × 100 mL). The combined organic layers were washed with brine (3 × 100 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 → 4% MeOH / DCM) to give a solid product (3 g, 98% yield). LCMS(ESI)C 49 H 62 The calculated m / z[M+H] value for F3N7O7 is 918.47; the experimental value is 918.8.

[0737] Step 8: Synthesis (6) 3 S,4S)-4-amino-1 2 -(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazineza-2(1,3)-benzocyclodecaban-5,7-dione

[0738] Under an argon atmosphere, at 0°C, towards ((6) 3S,4S)-12-(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-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 tert-butyl carbamate (930 mg, 1.013 mmol) was added dropwise to a stirred solution in DCM (15 mL) containing TFA (5 mL, 67.315 mmol) dissolved in DCM (5 mL). The mixture was stirred at 0 °C for 2 hours. The residue was alkalized to pH 8 with saturated aqueous NaHCO3 solution. The resulting mixture was extracted with DCM, and the combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give a solid product (880 mg, crude). LCMS(ESI)C 44 H 54 The calculated m / z[M+H] value for F3N7O5 is 818.42; the experimental value is 818.6.

[0739] Intermediate 11: Synthesis (6 3 S,4S)-4-amino-12-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyridino[1,2-a]pyrazin-2-yl)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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazineza-2(1,3)-benzocyclodecaban-5,7-dione

[0740]

[0741] Step 1: 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

[0742] At room temperature, 3-(5-bromo-2-{2-[(1S)-1-methoxyethyl]pyridin-3-yl}-1-(2,2,2-trifluoroethyl)indol-3-yl)-2,2-dimethylpropyl acetate (10 g, 18.470 mmol), 4,4,5,5-tetramethyl-2-(tetramethyl-1,3,2-dioxaborhexacyclopentan-2-yl)-1,3,2-dioxaborhexacyclopentane (8.44 g, 33.25 mmol), and dtbpy (0.89 g, 3.325 mmol) were added to a 100 mL three-necked round-bottom flask. Then, 1,5-cyclooctadiene iridium(I) dimer (0.74 g, 1.108 mmol) and THF (40 mL) were added to the mixture. The resulting mixture was stirred at 80 °C for 16 hours. The mixture was then concentrated under reduced pressure. The crude product was used directly in the next step without further purification. LCMS(ESI)C 25 H 29 The calculated m / z [M+H] value of BBrF3N2O5 is 585.14; the experimental value is 585.0.

[0743] Step 2: Synthesis 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

[0744] Under a nitrogen atmosphere, at 0 °C, sodium chloro[(4-methylbenzene)sulfonyl]iminocyanate (68.93 g, 244.688 mmol) and NaI (36.68 g, 244.688 mmol) in H₂O (44.75 mL) were added dropwise 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 (17.9 g, 30.586 mmol) in THF (89.5 mL). The resulting mixture was stirred for another 20 minutes at room temperature, followed by heating to 50 °C and maintaining the temperature for 16 hours. The resulting mixture was concentrated under reduced pressure and washed with CHCl₃ (300 mL). After filtration, the filter cake was washed with CHCl₃ (3 × 100 mL). The filtrate was extracted with CHCl3 (3 × 200 mL). The combined organic layers were washed with Na2S2O3 (300 mL) and brine (2 × 150 mL), and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (16% EtOAc / petroleum ether) to give a solid product (6.6 g, 32% yield). LCMS(ESI)C 25 H27 The calculated m / z [M+H] value of BrF3IN2O3 is 667.03; the experimental value is 668.7.

[0745] Step 3: Synthesis of 3-(5-bromo-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate

[0746] Cs₂CO₃ (3076.05 mg, 9.441 mmol), BINAP (65.32 mg, 0.105 mmol), and Pd(OAc)₂ (47.10 mg, 0.210 mmol) were added 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 (1.4 g, 2.098 mmol) and (R)-octahydro-2H-pyrido[1,2-a]pyrazine (353.04 mg, 2.518 mmol) in toluene (10 mL). The resulting mixture was stirred overnight at 90 °C under an argon atmosphere. The reaction was quenched with H₂O (100 mL). The resulting mixture was extracted with DCM (3 × 100 mL). The combined organic layers were washed with H₂O (3 × 100 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% MeOH / DCM) to give an oily product (1 g, 49% yield). LCMS(ESI)C 33 H 42 The calculated m / z[M+H] value of BrF3N4O3 is 679.25; the experimental value is 679.5.

[0747] Step 4: Synthesis of (S)-1-((S)-3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylic acid methyl ester

[0748] 3-(5-bromo-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate (1 g, 1.471 mmol) and (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxane) Methyl hexaborane-2-yl)phenyl)propionyl)hexahydropyridazine-3-carboxylate (913.62 mg, 1.765 mmol) was added to a stirred mixture in toluene (9 mL), followed by dioxane (6 mL), H₂O (3 mL), K₃PO₄ (780.82 mg, 3.678 mmol), and Pd(dtbpf)Cl₂ (95.90 mg, 0.147 mmol). The mixture was stirred at 70 °C for 2 hours under a nitrogen atmosphere. The mixture was alkalized to pH 8 with saturated aqueous NaHCO₃. The resulting mixture was extracted with DCM (3 × 30 mL). The combined organic layers were washed with H₂O (3 × 30 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20% EtOAc / petroleum ether) to give a solid product (1.2 g, 74% yield). LCMS(ESI)C 53 H 70 The calculated m / z[M+H] value for F3N7O8 is 990.53; the experimental value is 990.8.

[0749] Step 5: Synthesis of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(3-(3-(3-hydroxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyridino[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)propionyl)hexahydropyridazine-3-carboxylic acid

[0750] At 0 °C, fractionally added H₂O (6 mL) was added to a stirred mixture of (S)-1-((S)-3-(3-(3-acetoxy-2,2-dimethylpropyl)-2-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylate (1.2 g, 1.212 mmol) and LiOH (252 mg, 10.523 mmol) in THF (6 mL). The resulting mixture was stirred overnight at 0 °C. The mixture was then acidified to pH 7 with 1N HCl (aqueous solution). The aqueous layer was extracted with DCM (3 × 30 mL). The combined organic layers were washed with H₂O (30 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to give the product as a solid (1.2 g, 84% yield). LCMS(ESI)C 50 H 66 Calculated m / z[M+H] value for F3N7O7: 934.51; Experimental value: 935.0

[0751] Step 6: Synthesis ((6) 3 S,4S)-12-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(1,3)-benzocyclodecaeno-4-yl) tert-butyl carbamate

[0752] At 0 °C, fractionally add HOBt (0.87 g, 6.425 mmol) and EDCI-HCl (5.58 g, 35.980 mmol) to a stirred mixture of (S)-1-((S)-2-((tert-butoxycarbonyl)amino)-3-(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)-1-(2,2,2-trifluoroethyl)-1H-indol-5-yl)phenyl)propionyl)hexahydropyridazin-3-carboxylic acid (1.2 g, 1.285 mmol) and DIPEA (7.83 mL, 44.975 mmol) in a DCM (100 mL). The resulting mixture is stirred overnight at 0 °C. The mixture was diluted with DCM (30 mL). The combined organic layers were washed with H2O (3 × 30 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% MeOH / DCM) to give the product as a solid (850 mg, 65% yield). LCMS(ESI)C 50 H 64 Calculated m / z[M+H] value for F3N7O6: 916.49; Experimental value: 917.0

[0753] Step 7: Synthesis (6) 3 S,4S)-4-amino-12-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyridino[1,2-a]pyrazin-2-yl)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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazineza-2(1,3)-benzocyclodecaban-5,7-dione

[0754] At 0℃, towards ((6) 3 S,4S)-12-(2-((S)-1-methoxyethyl)-5-((R)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-1 1 -(2,2,2-trifluoroethyl)-6 1 6 2 6 3 6 46 5 6 6 -hexahydro-1 1 TFA (4 mL) was added to a stirred mixture of H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazinaza-2(1,3)-benzocyclodecaneta-4-yl)carbamate (1000 mg, 1.092 mmol) in DCM (4 mL). The mixture was stirred at 0 °C for 1 hour under a nitrogen atmosphere. The mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with a saturated aqueous solution of NaHCO3. The aqueous layer was extracted with DCM (3 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give a solid product (800 mg, 80% yield). LCMS(ESI)C 45 H 56 Calculated m / z[M+H] value of F3N7O4: 816.44; Experimental value: 816.6

[0755] Intermediate 12: Synthesis (6) 3 S,4S)-4-amino-1 1 -Ethyl-1 2 -(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-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 H-8-oxa-1(5,3)-indolaza-6(1,3)-pyridazineza-2(1,3)-benzocyclodecaban-5,7-dione

[0756]

[0757] Step 1: Synthesis of methyl (S)-1-((S)-3-(3-(3-acetoxy-2,2-dimethylpropyl)-1-ethyl-2-(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-5-yl)phenyl)-2-((tert-butoxycarbonyl)amino)propionyl)hexahydropyridazine-3-carboxylate

[0758] Under an argon atmosphere, at room temperature, 14.2 g (22.625 mmol) of 3-(5-bromo-1-ethyl-2-(5-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-1H-indol-3-yl)-2,2-dimethylpropyl acetate was added to a 500 mL three-necked round-bottom flask. Methyl hexahydropyridazine-3-carboxylate (17.56 g, 33.938 mmol), dioxane (150 mL) containing H₂O (30 mL), and Pd(dtbpf)Cl₂ (1.47 g, 2.263 mmol) were used. The mixture was stirred at 65 °C for 3 hours and then cooled to room temperature. The mixture was filtered, and the filter cake was washed with EtOAc (2 × 200 mL). The filtrate was concentrated under reduced pressure and then extracted with EtOAc (2 × 200 mL). The combined organic layers were washed with brine (2 × 250 mL) and dried over anhydrous Na₂...

Claims

1. A compound having the structure of Formula I: or a pharmaceutically acceptable salt thereof, wherein A is morpholine-diyl; X 1 , X 2 and X 3 are each CH2; m is 1; n is 1; R 1 is hydrogen, optionally substituted C1-C6heteroalkyl, or optionally substituted 3- to 10-membered heterocycloalkyl; R 2 is optionally substituted C1-C6alkyl; and R 3 is cyclopentyl.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein A is 3. A compound having the structure of: or a pharmaceutically acceptable salt thereof.

4. A compound having the structure of:

5. A pharmaceutical composition comprising a compound having the structure of: or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

6. A pharmaceutical composition comprising a compound having the structure of: and a pharmaceutically acceptable excipient.

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