Pyrimido[5,4-d]pyrimidine compounds, compositions comprising the same and uses thereof

Abstract

Compounds, compositions, and their use in treating proliferative diseases or conditions, e.g., the proliferative disease or condition is associated with a RAF gene mutation and / or a RAS gene mutation. The disclosed compounds are of Formula I, or a pharmaceutically acceptable salt or solvate thereof, wherein R 1 , R 2 , R 3 , X 1 , X 2 , X 3 , X 4 , and Y are as defined herein:

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 201,222, filed April 19, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present invention generally relates to pyrimido[5,4-d]pyrimidine compounds, pharmaceutical compositions comprising the same, and their use in the treatment and prevention of diseases characterized by dysregulation of the RAS-ERK pathway (e.g., cancer, RAS pathway diseases). Background Technology

[0004] The RAS-RAF-MEK-ERK (RAS: rat sarcoma; RAF: rapid-accelerating fibrosarcoma; MEK: mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase) signaling pathway (hereinafter referred to as the RAS-ERK pathway) plays a crucial role in transmitting proliferation signals generated by growth factor receptors from the plasma membrane to the nucleus. In most cancers, this pathway is dysregulated by activation of receptor tyrosine kinases (RTKs) (e.g., ERBB1, ERBB2, FLT3, RET, KIT), activation or inactivation of RAS regulators (SOS1 and NF1), and constitutive activating mutations in RAS genes (H-, K-, and NRAS; total in 30% of cancers) or BRAF genes (8% of cancers). KRAS mutations are particularly prevalent in pancreatic cancer (>90%), colorectal cancer (50%), and lung cancer (30%). In itself, BRAF mutations are particularly frequent in malignant melanoma (70%), thyroid cancer (40%), and colorectal cancer (10%) (mutation frequencies are based on COSMIC (Catalogue of Somatic Mutations in Cancer; Wellcome Trust Sanger Institute) v95, November 24, 2021).

[0005] RAS proteins are small GTPases that transmit extracellular growth signals to intracellular effectors to control important processes such as cell differentiation, proliferation, and survival (Nat. Rev. Cancer 2003, 3, 459). Upon RTK stimulation, physiological activation of RAS occurs on the plasma membrane, leading to GTP loading of GTPases and their activation. Activated RAS interact and activate a series of effector molecules, among which RAF kinase is the most critical RAS interactor in cancer development (Nature Rev. Drug Discov. 2014, 13, 828). Oncogenic mutations in glycine 12, glycine 13, or glutamine 61 in RAS isotypes result in aberrant and constitutive signaling in human cancer (Nat. Rev. Cancer 2003, 3, 459) (COSMIC Release v95, November 24, 2021).

[0006] Downstream of the RAS, mammalian cells express three paralogs of RAF (ARAF, BRAF, and CRAF), which share a conserved C-terminal kinase domain (KD) (Nat. Rev. Mol. Cell Biol. 2015, 16, 281) and an N-terminal regulatory region (NTR) containing the RAS-binding domain (RBD). In unstimulated cells, RAF proteins are chelated in the cytoplasm as monomers. Activation of RAS binding by GTP induces membrane anchoring of the RAF kinase (Nat. Rev. Mol. Cell Biol. 2015, 16, 281). Simultaneously, the RAF protein undergoes side-to-side dimerization of the kinase domain and catalytic activation (Nature 2009, 461, 542). Activated RAF protein transmits signals through a phosphorylation cascade from RAF to MEK and then from MEK to ERK, leading to a series of substrates being phosphorylated by ERK, thereby triggering a cell-specific response (Nat. Rev. Mol. Cell Biol. Oct 2020; 21(10), 607).

[0007] To date, activating mutations in the RAF isotype have been primarily confined to the BRAF gene, although rare variants have been observed in ARAF and CRAF, highlighting the functional importance of this isotype (COSMIC Release v95, November 24, 2021). The most common cancerous mutation in BRAF is the substitution of valine for glutamate at position 600 (called BRAF). V600EThe BRAF enhances its activity by stabilizing its active form (Cell 2004, 116, 855). Besides the V600E allele, various mutations have occurred at other residues (e.g., G466V, D594G, etc.), leading to enhanced RAF signaling through multiple mechanisms (Nat. Rev. Mol. Cell Biol. 2015, 16, 281). Based on their dependence on RAS activity and RAF dimerization, they are divided into three main categories (Category 1 to Category 3) (Nature, August 10, 2017; 548(7666), 234-238). The key roles of wild-type BRAF and CRAF in mediating RAS-driven tumorigenesis by stimulating ERK signaling have been widely validated (Cancer Cell 2011, 19, 652; Cancer Discov. 2012, 2, 685; Nat. Commun. 2017, 8, 15262). Therefore, tumor cell activation via RAS and RAF depends on elevated and sustained signaling via the RAS-ERK pathway, which strongly supports the concept of targeting RAF family kinases in cancer.

[0008] To address existing medical needs, a broad range of ATP-competitive RAF inhibitors have been developed over the past decade (Nat. Rev. Cancer 2017, 17, 676). Efforts have primarily focused on the most common non-RAS-dependent BRAF mutations (BRAF). V600E This led to the development and FDA approval of sulfonamide derivatives (such as vemurafenib and dabrafenib). Some of these RAF inhibitors have been shown to be effective for patients with recurrent BRAF. V600E Allele-mediated metastatic melanoma has shown impressive efficacy and has been approved for the treatment of this patient population (N. Engl. J. Med. 2011, 364, 2507; Lancet 2012, 380, 358). BRAF-mediated metastatic melanoma has shown promising efficacy and has been approved for the treatment of this patient population (N. Engl. J. Med. 2011, 364, 2507; Lancet 2012, 380, 358). V600E The clinical response to BRAF-dependent melanoma stems from effective ATP-competitive inhibition of the monomeric form of this specific dimerization-independent mutant BRAF protein (Cancer Cell 2015, 28, 370). Unfortunately, acquired resistance to these agents always develops, primarily due to reactivation of the RAS-ERK pathway (partly through mechanisms stimulating RAF dimerization). These include upregulation of RTK signaling, RAS mutations, and BRAF... V600E Amplification or truncation (Sci.Signal.2010,3,ra84; Nature 2010,468,973; Nature 2011,480,387; Nature Commun.2012,3,724).

[0009] Meanwhile, tumors exhibiting RAS activity—due to RAS mutation activation or elevated RTK signaling, but otherwise BRAF wild-type—showed resistance to BRAF. V600E Major resistance to inhibitors (Nature 2010, 464, 431). Conversely, RAF inhibitors have been found to induce ERK signaling in the presence of elevated RAS activity, thereby enhancing tumor cell proliferation (Nature 2010, 464, 431). This paradoxical phenomenon, known as the paradoxical effect, has also been observed in normal tissues dependent on physiological RAS activity and underlies some of the adverse effects of RAF inhibitors observed in melanoma patients, such as the development of new secondary tumors (e.g., squamous cell carcinoma and keratoacanthoma) (Nat. Rev. Cancer 2014, 14, 455). Therefore, BRAF... V600E Inhibitors are ineffective against RAS-driven cancers and are even contraindicated. The potential mechanism stems from the ability of compounds to promote the dimerization of RAF kinase domains in the presence of active RAS (Nature 2010, 464, 431). This event is not limited to BRAF but also involves other members of the RAF family and is determined by the compound's binding mode and affinity (Nat. Chem. Biol. 2013, 9, 428).

[0010] Two strategies have recently been employed to circumvent the limitations of first-generation RAF inhibitors in RAS-mutated cancers. The first strategy relies on the observation that paradoxical ERK activation is a dose-dependent phenomenon, meaning that induction occurs at subsaturated inhibitor concentrations, but the pathway is inhibited at saturated concentrations when the compound occupies both protomers of the RAF dimer. Therefore, this first strategy focuses on developing molecules with higher binding affinity to all RAF paralogs in order to saturate RAF proteins at lower drug concentrations, thereby reducing paradoxical pathway induction (Bioorg. Med. Chem. Lett. 2012, 22, 6237; Cancer Res. 2013, 73, 7043; J. Med. Chem. 2015, 58, 4165; Cancer Cell 2017, 31, 466; J Med Chem. 2020, 63, 2013; Clin Cancer Res. 2021, 27, 2061; Nature 2021, 594, 418). However, these compounds retain strong RAF dimer induction capabilities, thus paradoxically stimulating RAS-ERK signaling, albeit at a lower magnitude than previous generations of RAF inhibitors. Although such compounds have shown improved properties, recent studies have shown that most do not affect ARAF isotypes, leading to paradoxical pathway activation and primary resistance, as well as acquired resistance in in vitro and clinical settings (Clin Cancer Res. 2021, 27, 2061; Nature 2021, 594, 418). A second strategy involves designing compounds that conformationally bias the BRAF kinase domain in its inactive state and thus do not paradoxically induce ERK signaling. This has given rise to the "paradox breaker" (PB) molecule PLX8394, a derivative of PLX4032 / vemurafenib (Nature 2015, 526, 583). These molecules retain resistance to BRAF. V600E Its high efficacy should therefore be proven to be suitable for treating BRAF. V600E --dependent melanoma. However, although PLX8394 does not induce ERK signaling in the tested RAS-mutant cell lines, it remains ineffective and has no effect on RAS-mutant tumors.

[0011] There remains a need for inhibitors that effectively and consistently block RAS-ERK signaling and cell proliferation in human tumor cells carrying multiple RAS and RAF genotypes. Importantly, it is highly desirable to develop such inhibitors that do not induce conflicting pathways in multiple RAS-mutant tumor cell lines. Summary of the Invention

[0012] According to one aspect, the present invention relates to compounds of formula I, or pharmaceutically acceptable salts or solvates thereof:

[0013]

[0014] in:

[0015] R 1 Selected from substituted or unsubstituted OR 3 SR 3 NH2, NHR 3 、N(R 3 2. C 3-8 cycloalkyl, C 4-8 Heterocyclic alkyl, C 6-10 Aryl and C 5-10 Mixed aromatics;

[0016] R 2 Selected from substituted C6 aryl and C 5-10 heteroaryl, substituted or unsubstituted C 4-8 Heterocyclic alkyl groups and N(R) 3 )2;

[0017] R 3 Each occurrence is independently selected from substituted or unsubstituted C. 1-8 Alkyl, C 3-8 cycloalkyl, C 4-8 Heterocyclic alkyl, C 6-10 Aryl and C 5-10 Mixed aromatics;

[0018] X 1 It is a halogen or an electron-withdrawing group;

[0019] X 2 Selected from H, halogens, and electron-withdrawing groups;

[0020] X 3 and X 4 Each is selected from H, halogen, electron-withdrawing group, C 1-3 Alkyl, C 3-4 cycloalkyl and OC 1-3 alkyl;

[0021] Y is selected from H, halogen, CN, OH, OC. 1-8 Alkyl, NH2, NHC 1-8 Alkyl, N(C) 1-8 Alkyl groups 2 and substituted or unsubstituted C 1-8 alkyl;

[0022] The condition is that the compound is not:

[0023]

[0024] The compounds of Formula I are also defined according to any implementation method and the examples described throughout this specification.

[0025] According to another aspect, the present invention relates to pharmaceutical compositions for use as defined in any of the foregoing embodiments, the compositions comprising a compound as defined herein together with a pharmaceutically acceptable carrier, diluent or excipient.

[0026] On the other hand, the present invention relates to the use of compounds as defined herein for the treatment of diseases or conditions selected from: proliferative diseases or conditions, developmental abnormalities caused by RAS-ERK signaling cascade dysregulation (RAS pathway disorders), or inflammatory diseases or immune system disorders.

[0027] The present invention further relates to methods for treating diseases or conditions selected from: proliferative diseases or conditions, developmental abnormalities caused by RAS-ERK signaling cascade dysregulation (RAS pathway disorders), or inflammatory diseases or immune system disorders, including administering a compound as defined herein to a subject in need. Methods for inhibiting abnormal cell proliferation are also contemplated, including contacting cells with a compound as defined herein.

[0028] In one embodiment of the above uses and methods, the disease or condition is selected from tumors and developmental abnormalities, such as diseases or conditions associated with RAF gene mutations (e.g., ARAF, BRAF, or CRAF), diseases or conditions associated with RAS gene mutations (e.g., KRAS), or diseases or conditions associated with both RAF and RAS gene mutations. In one embodiment, the disease or condition is associated with receptor tyrosine kinase mutations or amplifications (e.g., EGFR, HER2) or mutations in regulators of RAS downstream of the receptor (e.g., gain of SOS1 function, loss of NF1 function).

[0029] For example, the disease or condition is a tumor, such as those selected from melanoma, thyroid cancer (e.g., papillary thyroid carcinoma), colorectal cancer, ovarian cancer, breast cancer, endometrial cancer, liver cancer, sarcoma, gastric cancer, pancreatic cancer, Barrett's adenocarcinoma, glioma (e.g., ependymoma), lung cancer (e.g., non-small cell lung cancer), head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, and hairy cell leukemia. For example, the tumor is selected from colon or colorectal cancer, lung cancer, pancreatic cancer, thyroid cancer, breast cancer, and melanoma. For example, any of the uses and methods of the present invention includes inhibiting the RAS-ERK signaling pathway without significantly inducing the paradoxical pathway.

[0030] Further objects and features of the compounds, compositions, methods, and uses of the present invention will become more apparent after reading the following non-limiting description of exemplary embodiments and examples, which should not be construed as limiting the scope of the invention. Attached Figure Description

[0031] Figure 1 This demonstrates the paradoxical induction of pERK signaling in RAS-mutant HCT116 cells (Y). MIN >-20%) of the compounds described herein (Examples 80 and 81) and compounds that strongly induce this pathway in the same cell lines (Y MIN Representative IC50 values ​​of the compound (PLX4720; CAS#918505-84-7) with a concentration of ~-600% 50 Inhibitor response curve.

[0032] Figure 2 The results of immunoblotting analysis of RAS-mutant HCT-116 cells treated with a representative compound that induces paradoxical signaling without inducing pERK or pMEK (Example 80; top) and a compound that induces the pathway in the same cell line (PLX4720; bottom) are shown. Detailed Implementation

[0033] All technical and scientific terms and expressions used herein have the same definitions as those commonly understood by one of ordinary skill in the art to which this invention pertains. However, definitions of some terms and expressions used are provided below. If a term definition in publications, patents, and patent applications incorporated herein by reference contradicts the definition set forth in this specification, the definition in this specification shall prevail. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter disclosed.

[0034] i. definition

[0035] The chemical structures described in this article were drawn according to standard procedures. Additionally, when drawn atoms (e.g., carbon atoms) appear to have incomplete valences, it is assumed that the valence is satisfied by one or more hydrogen atoms, even if these are not explicitly drawn. Hydrogen atoms should be inferred as part of the compound.

[0036] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that the singular forms “a,” “an,” and “the” also include the plural forms unless the context clearly indicates otherwise. Thus, for example, a reference to a composition containing “one compound” also covers mixtures of two or more compounds. It should also be noted that the term “or” is generally used in its meaning as including “and / or” unless the context clearly indicates otherwise. Furthermore, with regard to the terms “comprising,” “including,” “having,” “having,” “having,” or variations thereof used in the detailed description and / or claims, such terms are intended to be included in a manner similar to the term “comprising / including.”

[0037] The term "about" or "approximately" means within an acceptable range of error for a particular value as determined by a person skilled in the art, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within one or more standard deviations. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1%, of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude of a value, preferably within 5 times, and more preferably within 2 times. When a particular value is described in this application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable range of error for the particular value.

[0038] As used herein, the terms “compound,” “compound described herein,” “compound of this application,” “pyrimido[5,4-d]pyrimidine compound,” “pyrimidopyrimidine compound,” and equivalent expressions refer to compounds described herein, such as those covered by structural formula I, optionally with reference to any applicable embodiments, and also include exemplary compounds, such as the compounds of Examples 1 through 114, and their pharmaceutically acceptable salts, solvates, esters, and prodrugs (where applicable). When a zwitterionic form is possible, the compound may be drawn as its neutral form for practical purposes, but the compound is understood to also include its zwitterionic form. Embodiments herein may also exclude one or more compounds. Compounds can be identified by their chemical structure or their chemical name. In the event of a conflict between chemical structure and chemical name, the chemical structure shall prevail.

[0039] Unless otherwise stated, the structures described herein are also intended to include all isomers of the structure (e.g., enantiomers, diastereomers, and geometric (or conformations)) (where applicable); for example, the R and S configurations of each asymmetry center. Therefore, single stereochemical isomers of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers, and geometric (or conformations), are within the scope of this specification. Unless otherwise stated, therapeutic compounds also encompass all possible tautomers of the compounds shown (if any). The term also includes isotopically labeled compounds, wherein one or more atoms have atomic masses different from the most abundant atomic masses in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include, but are not limited to, those listed below. 2 H(D), 3 H(T), 11 C 13 C 14 C 15 N、 18 O、 17 O, any isotope of sulfur, etc. The compound can also exist in unsolvable and solvated forms, including hydrated forms. The compound can exist in various crystalline or amorphous forms. Generally, all physical forms are equivalent to the intended uses herein and are intended to fall within the scope of this invention.

[0040] When a particular enantiomer is preferred, in some embodiments it may be substantially free of the corresponding enantiomer and may also be enantiomer-enriched. "Enantiomer enrichment" means that the compound consists of a significantly larger proportion of one enantiomer. In some embodiments, the compound is made of at least about 90 wt% of the preferred enantiomer. In other embodiments, the compound consists of at least about 95 wt%, 98 wt%, or 99 wt% of the preferred enantiomer. The preferred enantiomer can be separated from the racemic mixture by any method known to those skilled in the art, including high-performance liquid chromatography (HPLC) on a chiral support and the formation and crystallization of chiral salts, or prepared by asymmetric synthesis.

[0041] The expression "pharmaceutically acceptable salt" refers to those salts of the compounds of the present invention that, within reasonable medical judgment, are suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). These salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or individually by reacting the free base functional group of the compound with a suitable organic or inorganic acid (acid addition salt) or by reacting the acid functional group of the compound with a suitable organic or inorganic base (base addition salt).

[0042] The term "solvent" refers to the physical association of one of the compounds of this invention with one or more solvent molecules, including water and non-aqueous solvent molecules. Such physical association may include hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The term "solvent" encompasses both solution-phase solvates and separable solvates. Exemplary solvates include, but are not limited to, hydrates, hemihydrates, ethanolates, semi-ethanolates, n-propanolates, isopropanolates, 1-butanolates, 2-butanolates, and solvates of other physiologically acceptable solvents, such as the three classes of solvents described in the International Conference on Harmonization (ICH), Guide for Industry, Q3CImpurities: Residual Solvents (1997). Therefore, the compounds described herein also include each of their solvates and mixtures thereof.

[0043] As used herein, the term "pharmaceutically acceptable ester" means an ester of a compound formed by the methods described herein that is hydrolyzable in vivo, and includes those esters that readily decompose in the human body to leave behind a parent compound or its salts. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanes, alkenes, cycloalkanes, and alkanediacids, wherein each alkyl or alkenyl moiety advantageously has no more than six carbon atoms. Examples of specific esters include, but are not limited to, formate, acetate, propionate, butyrate, acrylate, and ethyl succinate with hydroxyl groups, and alkyl esters with acidic groups. Other ester groups include sulfonates or sulfates.

[0044] As used herein, "pharmaceuticalally acceptable prodrug" refers to those prodrugs of compounds formed by the methods of the present invention that, within the limits of reasonable medical judgment, are suitable for contact with human and lower animal tissues and have excessive toxicity, irritation, allergic reactions, etc., in proportion to a reasonable benefit / risk ratio, and are effective for their intended use. As used herein, "prodrug" means a compound that can be converted in vivo by metabolic pathways (e.g., by hydrolysis) to provide any compound described in this specification.

[0045] Abbreviations may also be used throughout the application, unless otherwise stated, and are intended to have the meaning commonly understood in the art. Examples of such abbreviations include Me (methyl), Et (ethyl), Pr (propyl), i-Pr (isopropyl), Bu (butyl), t-Bu (tert-butyl), i-Bu (isobutyl), s-Bu (sec-butyl), c-Bu (cyclobutyl), Ph (phenyl), Bn (benzyl), Bz (benzoyl), CBz or Cbz or Z (benzyloxycarbonyl), Boc or BOC (tert-butoxycarbonyl), and Su or Suc (succinimide). For further clarification, additional definitions of the specific abbreviations are also included in the description in the Examples section.

[0046] The number of carbon atoms in a hydrocarbon substituent can be determined by the prefix "C". x -C y "or "C x-y "" indicates that x is the minimum number of carbon atoms in the substituent and y is the maximum number of carbon atoms in the substituent. However, when the prefix "C" is used... x -C y "or "C x-y "When related to a group (e.g., heterocyclic alkyl, heteroaryl, etc.) that incorporates one or more heteroatoms by definition, x and y define the minimum and maximum number of atoms in the ring, including carbon atoms and one or more heteroatoms, respectively."

[0047] As used herein, the term "alkyl" refers to a saturated, straight-chain, or branched hydrocarbon group that typically contains 1 to 20 carbon atoms. For example, "C 1-8 "Alkyl" contains 1 to 8 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, octyl, etc.

[0048] As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing one or more double bonds and typically containing 2 to 20 carbon atoms. For example, "C 2-8 "Alkenyl" contains 2 to 8 carbon atoms. Alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl, etc.

[0049] As used herein, the term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing one or more triple bonds and typically containing 2 to 20 carbon atoms. For example, "C 2-8 The alkynyl group contains 2 to 8 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octyynyl, etc.

[0050] The terms "cycloalkyl," "alicyclic," "carbocyclic," "carbocyclic," and equivalent expressions refer to groups that contain saturated or partially unsaturated (non-aromatic) carbocyclic rings in monocyclic or polycyclic systems, including spirocyclic (sharing one atom), fused (sharing at least one bond), or bridged (sharing two or more bonds) carbocyclic systems, having three to fifteen ring members. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopenten-1-yl, cyclopenten-2-yl, cyclopenten-3-yl, cyclohexyl, cyclohexen-1-yl, cyclohexen-2-yl, cyclohexen-3-yl, cycloheptyl, bicyclo[4,3,0]nonyl, norbornyl, etc. The term cycloalkyl includes both unsubstituted and substituted cycloalkyl groups. For example, the term "C 3-n "Cycloalkyl" refers to a cycloalkyl group having 3 to a specified number of "n" carbon atoms in its ring structure. Unless the number of carbons is otherwise specified, "lower cycloalkyl" as used herein has at least 3 and equal to or less than 8 carbon atoms in its ring structure.

[0051] As used herein, the terms “heterocycle,” “heterocyclic alkyl,” “heterocyclic group,” “heterocyclic ring” are used interchangeably and refer to a chemically stable 3- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more, preferably one to four, heteroatoms as defined above, in addition to a carbon atom. When used to refer to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having one to three heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen can be N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolealkyl), or NR (as in N-substituted pyrrolealkyl). Heterocyclic rings may be attached to their dangling (side chain) groups at any heteroatom or carbon atom that produces a chemically stable structure, and any ring atom may optionally be substituted. Examples of heterocyclic alkyl groups include, but are not limited to, 1,3-dioxopentane, pyrrolylalkyl, pyrrolidone, pyrazolinyl, pyrazolylalkyl, imidazolinyl, imidazolinyl, piperidinyl, piperazinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolinyl, isothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiaranyl, tetrahydrodithiophenyl, tetrahydrothiaphenyl, thiomorpholinyl, thiazolinyl, azacyclic butyl, oxacyclic butyl, thiocyclic butyl, high-piperidinyl, oxacyclic heptyl, thiacyclic heptyl, and oxazolyl. basalt, diazoxide The heterocyclic group includes groups such as thiazolinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, 2H-pyranyl, 4H-pyranyl, dioxane, dithiazyl, dithiamonyl, dithiamonyl, dihydropyranyl, dihydrothiopheneyl, dihydrofuranyl, 3-azabicyclo[3,1,0]hexane, 3-azabicyclo[4,1,0]heptane, quinazinyl, quininecycloyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, etc. Heterocyclic groups also include groups in which the heterocycle is fused with one or more aryl, heteroaryl, or alicyclic rings, such as indolyl, 3H-indolyl, benzodihydropyranyl, benzopyranyl, phenanthridineyl, 2-azabicyclo[2.2.1]heptane, octahydroindolyl, or tetrahydroquinolinyl, wherein the attachment group or dot is located on the heterocyclic ring. The heterocyclic group can be monocyclic or bicyclic. The term "heterocyclic alkyl" refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl and heterocyclic moieties are optionally substituted independently. For example, the term "C 3-n "Heterocyclic alkyl" refers to a heterocyclic alkyl group having 3 to a specified number of atoms (including carbon atoms and heteroatoms) in a ring structure.

[0052] As used herein, the term "partially unsaturated" refers to a ring moiety that contains at least one double or triple bond between ring atoms but is not aromatic. The term "partially unsaturated" is intended to cover rings with multiple unsaturated sites, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0053] The term "aryl," used alone or as part of a larger portion (e.g., "arylalkyl," "arylalkoxy," "aryloxy," or "aryloxyalkyl"), refers to an aromatic group having 4n+2 conjugated π (pi) electrons, where n is an integer from 1 to 3, in a monocyclic portion or bicyclic or tricyclic fused ring system having a total of 6 to 15 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In some embodiments of this specification, "aryl" refers to an aromatic ring system, including but not limited to phenyl, biphenyl, naphthyl, azulel, anthracene, etc., which may have one or more substituents. The term "arylalkyl" or "arylalkyl" refers to an alkyl residue attached to an aromatic ring. Examples of arylalkyl groups include, but are not limited to, benzyl, phenethyl, etc. As used herein, the term "aryl" also includes groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, indenyl, phthalimide, naphthimide, fluorenyl, phenanthridine, or tetrahydronaphthyl. For example, the term "C 6-n "Aryl" refers to an aryl group having 6 to a specified number of "n" atoms in a ring structure.

[0054] The term "heteroaryl," used alone or as part of a larger portion (e.g., "heteroarylalkyl" or "heteroarylalkoxy"), refers to an aromatic group having 4n+2 conjugated π (pi) electrons, where n is an integer from 1 to 3 (e.g., having 5 to 18 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in a cyclic array); and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" includes, but is not limited to, nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of basic nitrogen. Heteroaryl groups can be monocyclic or have two or more fused rings. The term "heteroaryl," as used herein, also includes groups in which a heteroaryl ring is fused to one or more aryl, alicyclic, or heterocyclic rings, wherein the attachment group or point is located on the heteroaryl ring. Non-limiting examples of heteroaryl groups include thiophene, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiazolyl, pyridyl, pyrazinyl, triazinyl, indole, 3H-indole, isoindole, indazinyl, benzothiophene (benzothiophene), benzofuranyl, dibenzofuranyl, inzolyl, benzimidazolyl, benzooxazolyl, benzothiazolyl, benzotriazolyl, pyrrolopyridyl (e.g., pyrrolo[3,2-b]pyridyl or pyrrolo[3 [2,2-c]pyridinyl), pyrazolopyridinyl (e.g., pyrazolo[1,5-a]pyridinyl), furano[pyridinyl], purinyl, imidazo[pyrazinyl] (e.g., imidazo[4,5-b]pyrazinyl), quinolinyl (quinolinyl), isoquinolinyl (isoquinolinyl), quinolone, isoquinolone, cenolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinazinyl, naphridinyl and pteridinyl, carbazoyl, acridineyl, phenanthidyl, phenazinyl, phenothiazinyl, phenotoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido[2,3-b]-l,4-oxazin-3(4H)-one. The heteroaryl group can be monocyclic or bicyclic. The heteroaryl group includes optionally substituted rings. The term "heteroaryl" refers to an alkyl group substituted with a heteroaryl group, wherein the alkyl and heteroaryl portions are optionally substituted independently. Examples include, but are not limited to, pyridylmethyl, pyrimidinylethyl, etc. For example, the term "C 5-n "Heteroaryl" refers to a heteroaryl group having 5 to a specified number of "n" atoms (including carbon atoms and heteroatoms) in its ring structure.

[0055] As described herein, the compounds of the present invention may contain an "optionally substituted" portion. Generally, the term "substituted," whether or not it is preceded by the term "optionally," means that one or more hydrogens of the specified portion are replaced by suitable substituents. Unless otherwise stated, the "optionally substituted" group may have suitable substituents at each substituted position of the group, and when more than one position in any given structure can be replaced by more than one substituent selected from a particular group, the substituents may be the same or different at each position. Combinations of substituents contemplated herein are preferably those that result in the formation of chemically stable or chemically viable compounds. The term "chemically stable" as used herein means a compound that is substantially unchanging when subjected to conditions that allow it to be generated, detected, and, in some embodiments, recovered, purified, and used for one or more of the purposes disclosed herein.

[0056] The term "halogen" refers to a halogen atom, namely a fluorine, chlorine, bromine or iodine atom, preferably fluorine or chlorine.

[0057] The term "optionally substituted" refers to a group that is substituted or unsubstituted by a substituent that independently replaces one, two, three, or more hydrogen atoms thereon. The substituents include, but are not limited to, F, Cl, Br, I, OH, CO2H, alkoxy, oxo, thiooxy, NO2, CN, CF3, NH2, NHalkyl, NHalkenyl, NHynyl, NHcycloalkyl, NHaryl, NH heteroaryl, NH heterocyclic, dialkylamino, diarylamino, diheteroarylamino, O-alkyl, O-alkenyl, O-ynyl, O-cycloalkyl, O-aryl, O-heteroaryl, O-haloalkyl, O-heterocyclic, C(O)alkyl, C(O)alkenyl, C(O)ynyl, C(O)cycloalkyl, C(O)aryl, C(O)heterocyclic. C(O) heterocyclic alkyl, CO2 alkyl, CO2 alkenyl, CO2 ynyl, CO2 cycloalkyl, CO2 aryl, CO2 heteroaryl, CO2 heterocyclic alkyl, OC(O) alkyl, OC(O) alkenyl, OC(O) ynyl, OC(O) cycloalkyl, OC(O) aryl, OC(O) heteroaryl, OC(O) heterocyclic alkyl, C(O)NH2, C(O)NH alkyl, C(O)NH alkenyl, C(O)NH ynyl, C(O)NH cycloalkyl, C(O)NH aryl, C(O)NH heteroaryl, C(O)NH heterocyclic alkyl, OCO2 alkyl, OCO2 alkenyl, OCO2 ynyl, OCO2 cycloalkyl, OCO2 aryl, OCO2 heteroaryl, OCO2 heterocyclic alkyl, OC(O) )NH2, OC(O)NHalkyl, OC(O)NHalkenyl, OC(O)NHkynyl, OC(O)NHcycloalkyl, OC(O)NHaryl, OC(O)NHhearyl, OC(O)NHhecycloalkyl, NHC(O)alkyl, NHC(O)alkenyl, NHC(O)kynyl, NHC(O)cycloalkyl, NHC(O)aryl, NHC(O)hearyl, NHC(O)hearycycloalkyl, NHCO2alkyl, NHCO2alkenyl, NHCO2kynyl, NHCO2cycloalkyl, NHCO2aryl, NHCO2hearyl, NHCO2hearycycloalkyl, NHC(O)NH2, NHC(O)NHalkyl, NHC(O)NHalkenyl, NHC(O)NHalkenyl , NHC(O)NH cycloalkyl, NHC(O)NH aryl, NHC(O)NH heteroaryl, NHC(O)NH heterocycloalkyl, NHC(S)NH2, NHC(S)NH alkyl, NHC(S)NH alkenyl, NHC(S)NH alkynyl, NHC(S)NH cycloalkyl, NHC(S)NH aryl, NHC(S)NH heteroaryl, NHC(S)NH heterocycloalkyl, NHC(NH)NH2, NHC(NH)NH alkyl, NHC(NH)NH alkenyl, NHC(NH)NH alkenyl, NHC(NH)NH cycloalkyl, NHC(NH)NH aryl, NHC(NH)NH heteroaryl, NHC(NH)NH heterocycloalkyl, NHC(NH) alkyl,NHC(NH)alkenyl, NHC(NH)alkenyl, NHC(NH)cycloalkyl, NHC(NH)aryl, NHC(NH)heteroaryl, NHC(NH)heterocycloalkyl, C(NH)NHalkyl, C(NH)NHalkenyl, C(NH)NHkynyl, C(NH)NHcycloalkyl, C(NH)NHaryl, C(NH)NHheteroaryl, C(NH)NHheterocycloalkyl, P(O)(alkyl)2, P(O)(alkenyl)2, P(O)(kynyl) S(O)alkyl, P(O)alkyl, P(O)aryl, P(O)heteroaryl, P(O)heterocyclic alkyl, P(O)alkyl, P(O)OH, P(O)alkenyl, P(O)ynyl, P(O)cycloalkyl, P(O)aryl, P(O)heteroaryl, P(O)alkyl, S(O)alkyl, S(O)alkenyl, S(O)ynyl, S(O)cycloalkyl Alkyl, S(O)aryl, S(O)2alkyl, S(O)2alkenyl, S(O)2ynyl, S(O)2cycloalkyl, S(O)2aryl, S(O)heteroaryl, S(O)heterocyclic alkyl, SO2NH2, SO2NHalkyl, SO2NHalkenyl, SO2NHynyl, SO2NHcycloalkyl, SO2NHaryl, SO2NHheteroaryl, SO2NHheterocyclic alkyl, NHSO2alkyl, NHSO2alkenyl, NHSO2ynyl, NHSO2 Cycloalkyl, NHSO2 aryl, NHSO2 heteroaryl, NHSO2 heterocycloalkyl, CH2NH2, CH2SO2CH3, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, cycloalkyl, carbocyclic, heterocyclic, polyalkoxyalkyl, polyalkoxy, methoxymethoxy, methoxyethoxy, SH, S-alkyl, S-alkenyl, S-alkynyl, S-cycloalkyl, S-aryl, S-heteroaryl, S-heterocycloalkyl or methylthiomethyl.

[0058] ii. compound

[0059] The enumeration of chemical groups in any definition of a variable herein includes the definition of the variable as any single group or combination of the listed groups. The description of embodiments of the variables herein includes the embodiment as any single embodiment or combination with any other embodiment or part thereof. Therefore, the following embodiments may exist alone or in combination, if applicable.

[0060] The compounds of the present invention exhibit a pyrimido[5,4-d]pyrimidine core structure with defined substituents attached thereto to achieve the beneficial activity of the product. Examples of pyrimido[5,4-d]pyrimidine compounds as defined herein are shown by general formula I:

[0061]

[0062] in:

[0063] R 1 Selected from substituted or unsubstituted OR 3 SR 3 NH2, NHR 3 、N(R 3 2. C 3-8 cycloalkyl, C 4-8 Heterocyclic alkyl, C 6-10 Aryl and C 5-10 Mixed aromatics;

[0064] R 2 Selected from substituted C6 aryl and C 5-10 heteroaryl, substituted or unsubstituted C 4-8 Heterocyclic alkyl groups and N(R) 3 )2;

[0065] R 3 Each occurrence is independently selected from substituted or unsubstituted C. 1-8 Alkyl, C 3-8 cycloalkyl, C 4-8 Heterocyclic alkyl, C 6-10 Aryl and C 5-10 Mixed aromatics;

[0066] X 1 It is a halogen or an electron-withdrawing group;

[0067] X 2 Selected from H, halogens, and electron-withdrawing groups;

[0068] X 3 and X 4 Each is selected from H, halogen, electron-withdrawing group, C 1-3 Alkyl, C 3-4 cycloalkyl and OC 1-3 alkyl;

[0069] Y is selected from H, halogen, CN, OH, OC. 1-8 Alkyl, NH2, NHC 1-8 Alkyl, N(C) 1-8 Alkyl)2 and substituted or unsubstituted C 1-8 alkyl;

[0070] Or its pharmaceutically acceptable salts or solvates;

[0071] The condition is that the compound is not:

[0072]

[0073] For example, the electron-withdrawing group is selected from perhaloalkyl (e.g., CF3 or CCl3), CN, NO2, sulfonates, alkylsulfonyl (e.g., SO2Me or SO2CF3), alkyl carbonyl (e.g., C(O)Me), carboxylic esters, alkoxy carbonyl (e.g., C(O)OMe), and amino carbonyl (e.g., C(O)NH2). In one embodiment, X 1 It is Cl and X 2 Is it F, or X? 1 It is F and X 2 Is it H, or X? 1 and X 2 Both are F. In another implementation, X 3 and X 4 Each is H. In yet another implementation, X... 3 It is F and X 4 It is H.

[0074] According to one embodiment, Y is H and all other groups are as defined herein. According to another embodiment, Y is NH2 and all other groups are as defined herein.

[0075] For example, the aminoarylsulfonamide moiety in Formula I is designated as L and selected from:

[0076]

[0077] In this context, the dashed line (---) represents a key.

[0078] In yet another implementation, R 2 It is a substituted C6 aryl or C 5-10 heteroaryl, such as R 2 It is a C6 aryl group substituted with at least one of the following groups: F, Cl, Br, CN, NO2, and substituted or unsubstituted C6 aryl groups. 1-3 Alkyl, C 3-4 cycloalkyl or OC 1-3 Alkyl group. For example, R 2 It is a group of the following formula:

[0079]

[0080] in:

[0081] R 4 Selected from H, F, Cl, Br, CN, and substituted or unsubstituted C 1-3 Alkyl, C 3-4 cycloalkyl or OC 1-3 Alkyl groups, such as R 4 Selected from H, F, Cl, Br, Me, Et, CN, CHF2 and CF3;

[0082] R 5 Selected from H, F, Cl, CN, and substituted or unsubstituted C 1-3 Alkyl, C 3-4 cycloalkyl or OC 1-3 Alkyl groups, such as R 5 Selected from H, F, Me, CF3, CN, and Cl;

[0083] R 6 Selected from H, F, Cl, Br, NO2, NH2, and substituted or unsubstituted C 1-3 Alkyl, C 3-4 cycloalkyl or OC 1-3 Alkyl groups, such as R 6 Selected from H, F, Cl, Br, and substituted or unsubstituted C 1-3 Alkyl, C 3-4 cycloalkyl or OC 1-3 Alkyl, or R 6 Selected from H, F, Cl, Me, Et, and OMe;

[0084] R 7 Selected from H, F, Cl, and substituted or unsubstituted C 1-3 Alkyl groups, such as R 7 Selected from H, Me, F and Cl;

[0085] R 8 Selected from H, F, and substituted or unsubstituted C 1-3 Alkyl groups, such as R 8 Selected from H, Me, and F;

[0086] Or, R 4 and R 5 、or R 5 and R 6 Together with the adjacent carbon atoms, they form substituted or unsubstituted carbon rings or heterocycles, provided that the heterocycle (R) 2 It is not benzoxazolinone; and

[0087] (---) represents a key;

[0088] Where R 4 When it is H or F, then R 5 R 6 R 7 or R 8 At least one of them is not H or F; and

[0089] Where R 5 When it is CN, then R 4 R 6 R 7 or R 8At least one of them is not H.

[0090] In one implementation, R 8 It is H. In another implementation, R 4 Selected from F, Cl, Et and Me, R 5 R 7 and R 8 Each is H, and R 6 Selected from H, Cl, Me, and OMe. In another embodiment, R 4 Selected from F, Cl, and Me, R 6 R 7 and R 8 Each is H, and R 5 Selected from F and Cl.

[0091] In another implementation, R 4 Selected from C1 and substituted or unsubstituted C1 1-3 Alkyl (e.g., Me); preferably R 4 Is it Cl or Me; R 5 Selected from H, F, Cl, and substituted or unsubstituted C 1-3 Alkyl (e.g., Me); R 6 Selected from H, and substituted or unsubstituted OC 1-3 Alkyl groups (e.g., OCH3); and R 7 and R 8 Each is represented by H.

[0092] In another implementation, R 4 Selected from H, Cl, Br and methyl, R 5 Selected from H, F and Cl, R 6 Selected from H, F, Cl, Me, and OMe, and R 7 and R 8 Each is represented by H.

[0093] In another implementation, R 4 Selected from C1 and substituted or unsubstituted C1 1-3 Alkyl groups (e.g., Me), preferably R 4 Is it Cl or Me; R 5 Selected from H, F, Cl, and substituted or unsubstituted C 1-3 Alkyl groups (e.g., Me), preferably R 5 It is F, Cl, or Me; R 6 Selected from H, F, Cl, and substituted or unsubstituted C 1-3 Alkyl groups (e.g., Me), and substituted or unsubstituted OCs 1-3 Alkyl groups (e.g., OCH3), preferably R 6 Is it H or F, or R?6 It is Cl, or substituted or unsubstituted C. 1-3 Alkyl, or substituted or unsubstituted OC 1-3 Alkyl, or CH3, or OCH3; and R 7 and R 8 Each is H. In yet another implementation, R... 6 It is the replaced C 1-3 alkyl.

[0094] In another instance, R 2 It is a substituted C5 heteroaryl group, such as the group of the following formula:

[0095]

[0096] in:

[0097] X 5 Selected from NH, NC 1-3 Alkyl, NC 3-4 Cycloalkyl, O, and S;

[0098] R 9 R 10 R 11 Each is independently selected from H, F, Cl, CN, and substituted or unsubstituted C. 1-3 Alkyl, C 3-4 Cycloalkyl, C(O)OC 1-3 Alkyl or OC 1-3 Alkyl, condition R 9 and R 11 One of them is H, and the other is not H; and

[0099] (---) represents a key.

[0100] Alternative location, R 2 It is a group of the following formula:

[0101]

[0102] in:

[0103] X 5 Selected from NH, NC 1-3 Alkyl, NC 3-4 Cycloalkyl, O, and S;

[0104] R 9 Selected from F, Cl, CN, and substituted or unsubstituted C 1-3 Alkyl, C 3-4 Cycloalkyl, C(O)OC 1-3 Alkyl or OC 1-3 alkyl;

[0105] R 10 and R 12 Each is independently selected from H, F, Cl, CN, and substituted or unsubstituted C. 1-3 Alkyl, C 3-4 Cycloalkyl, C(O)OC 1-3 Alkyl or OC 1-3 Alkyl; and

[0106] (---) represents a key.

[0107] In a preferred embodiment, R 9 and R 10 Each is independently selected from F, Cl, CN, and substituted or unsubstituted C. 1-3 Alkyl, C 3-4 Cycloalkyl, C(O)OC 1-3 Alkyl or OC 1-3 Alkyl groups, preferably Cl and substituted or unsubstituted C 1-3 Alkyl, more preferably R 9 and R 10 Both are Cl. In another embodiment, X 5 It can be O or S, with S being preferred.

[0108] In another implementation, R 2 It is the replaced C 5-10 Heteroaryl groups, such as those with the following formula:

[0109]

[0110] in:

[0111] X 9 X 10 X 11 X 12 and X 13 Independently selected from N and C, where X 9 X 10 X 11 X 12 and X 13 At least one and at most two of them are N; and

[0112] R 19 R 20 R 21 R 22 and R 23 Selected from H, F, Cl, Br, CN, NO2, NH2, and substituted or unsubstituted C 1-3 Alkyl, C 3-4 cycloalkyl or OC 1-3 Alkyl groups, or when they are attached to X 9 X 10X 11 X 12 or X 13 When it is N, R 19 R 20 R 21 R 22 and R 23 It does not exist;

[0113] Where X 9 and X 13 At least one of them is not N; and

[0114] Where X 9 and X 13 If one of them is N, then the other is neither N nor CH.

[0115] In another instance, R 2 It is a C5 heterocyclic alkyl group. For example, R 2 It is a group of the following formula:

[0116]

[0117] in:

[0118] R 13 Each occurrence is independently selected from F, Cl, and substituted or unsubstituted C. 1-3 Alkyl, C 3-4 cycloalkyl or C 1-3 Alkoxy;

[0119] n is an integer selected from 0 to 8; or

[0120] n is between 2 and 8, and both R 13 Together with the adjacent carbon atoms, they form C 3-4 cycloalkyl; and

[0121] (---) represents a key.

[0122] In one implementation, R 13 It is in position 3. In another implementation, R 13 The choice is selected from F, Me, OMe, and CH2OMe, and n is 1 or 2. For example, R 13 It is a methoxy group at the 3-position, and n is 1.

[0123] In another instance, R 2 It is N(R) 3 2. For example, R 2 It is N(R) 3 )2, and R 3 Selected from substituted or unsubstituted C 1-8 Alkyl or C 3-8 Cycloalkyl.

[0124] In another embodiment, the compound of formula I is a compound of formula II, or a pharmaceutically acceptable salt or solvate thereof:

[0125]

[0126] Where R 1 R 4 R 5 and R 6 Each independently, as defined herein, preferably, R 4 Selected from Cl, Br and methyl; R 5 Selected from H, F, Cl and methyl; and R 6 Selected from H, F, Cl, Me and OMe.

[0127] In yet another embodiment, the compound of formula I is a compound of formula III, or a pharmaceutically acceptable salt or solvate thereof:

[0128]

[0129] Where R 1 R 9 R 10 R 12 and X 5 Each is independent as defined in this article.

[0130] Exemplary R 2 The groups are shown from group B1 to group B77 as defined below:

[0131]

[0132]

[0133]

[0134] (---) represents a key.

[0135] In one implementation, R 2 Selected from groups B1 to B37, B41 to B44, B49, B51 to B55, B57, B59, B62 to B67, B71 to B74, B76 and B77, or preferably R 2 Selected from groups B1 to B33, B36, B41, B42, B51 to B54, B59, B65, B73 and B77, or more preferably R 2 Selected from groups B1, B2, B6, B8, B11, B12, B15, B20, B21, B36, B41, B42, B53, B54, B59, B65 and B73, or most preferably R.2 Selected from groups B21, B36, B41, B42, B52, B53, B54, B59, B65 and B73.

[0136] In one embodiment of the compound of formula I, R 1 Is it OR 3 or SR 3 For example, R 1 It is SR 3 In various implementations, R 3 Is it substituted or unsubstituted C? 1-8 Alkyl (e.g., C10) 1-3 alkyl).

[0137] In another implementation, R 1 It is a substituted or unsubstituted C6 aryl group. In another embodiment, R 1 Is it substituted or unsubstituted C? 4-6 Heterocyclic alkyl groups. For example, R 1 It is optional to be selected from one or two halogens, OH, C 1-6 Alkyl and OC 1-6 C-substituted alkyl groups 4-5 Heterocyclic alkyl groups. For example, R 1 It is an N-pyrrolidinyl group substituted with one or two groups selected from F and OH.

[0138] In another implementation, R 1 Is it substituted or unsubstituted C? 5-6 A heteroaryl group, or a substituted or unsubstituted C9 heteroaryl group. In yet another embodiment, R 1 It is selected from the following substituted or unsubstituted groups: thiophene, imidazolyl, pyrazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, indole, indazole, benzimidazolyl, benzotriazolyl, pyrrolopyridinyl (e.g., pyrrolo[3,2-b]pyridinyl, or pyrrolo[3,2-c]pyridinyl), pyrazolopyridinyl (e.g., pyrazolo[1,5-a]pyridinyl), purine, imidazopyrazinyl (e.g., imidazo[4,5-b]pyrazinyl), and quinolinyl (quinolinyl), preferably R. 1 It is selected from the following substituted or unsubstituted groups: imidazolyl, pyrazolyl, triazolyl, indole, indazole, benzimidazolyl, benzotriazolyl, pyrrolopyridyl (e.g., pyrrolo[3,2-b]pyridyl, or pyrrolo[3,2-c]pyridyl), pyrazolopyridyl (e.g., pyrazolo[1,5-a]pyridyl), purine, and imidazopyrazinyl (e.g., imidazo[4,5-b]pyrazinyl), more preferably R 1 The nitrogen atom is attached to the pyrimidine nucleus.

[0139] R 1 Examples include groups selected from the following:

[0140]

[0141] Wherein (---) represents a bond, and the group is optionally further substituted.

[0142] For example, R 1 It is selected from the following substituted or unsubstituted groups:

[0143]

[0144] (---) represents a key.

[0145] In one implementation, R 1 It is further substituted by at least one of the following substituents: OH, halogen, CN, NO2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, OC 1-6 Alkyl, C 5-10 heteroaryl, C 3-10 cycloalkyl, C 4-10 Heterocyclic alkyl, C(O)R 15 C(O)N(R) 14 2. SO2R 15 SO2N(R) 14 )2、N(R 16 )C(O)R 15 、N(R 16 SO2R 15 、N(R 16 )C(O)N(R 14 )2、N(R 16 SO2N(R) 14 )2、N(R 14 )2、P(O)(R 15 )2、CH2C(O)R 15 CH2C(O)N(R) 14 2. CH2SO2R 15 CH2SO2N(R) 14 )2、CH2N(R 16 )C(O)R 15 CH2N(R) 16 SO2R 15 CH2N(R) 16 )C(O)N(R 14 )2、CH2N(R 16 SO2N(R)14 )2 and CH2N(R 14 )2;

[0146] in:

[0147] R 14 Each time it appears, it is independently selected from H and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Heterocyclic alkyl, C6 aryl and C 5-10 heteroaryl, or two R 14 Together with the nitrogen atoms adjacent to them, they form C 4-10 Heterocyclic alkyl groups;

[0148] R 15 Each time it appears, it is independently selected from C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C6 aryl and C 5-10 heteroaryl; and

[0149] R 16 Each time it appears, it is independently selected from H and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C6 aryl and C 5-10 Mixed aromatics;

[0150] This includes in R 1 In China (including R) 14 R 15 and R 16 The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups (as defined in the definition) may optionally be further substituted.

[0151] In another implementation, R 1 It is a group of the following formula:

[0152]

[0153] in:

[0154] R 17 Selected from H, OH, halogens, CN, NO2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, OC 1-6 Alkyl, C 5-10 heteroaryl, C 3-10 cycloalkyl, C4-10 Heterocyclic alkyl, C(O)R 15 C(O)N(R) 14 2. SO2R 15 SO2N(R) 14 )2、N(R 16 )C(O)R 15 、N(R 16 SO2R 15 、N(R 16 )C(O)N(R 14 )2、N(R 16 SO2N(R) 14 )2、N(R 14 )2、P(O)(R 15 )2、CH2C(O)R 15 CH2C(O)N(R) 14 2. CH2SO2R 15 CH2SO2N(R) 14 )2、CH2N(R 16 )C(O)R 15 CH2N(R) 16 SO2R 15 CH2N(R) 16 )C(O)N(R 14 )2、CH2N(R 16 SO2N(R) 14 )2 and CH2N(R 14 )2;

[0155] X 6 It is N or CH; and

[0156] X 7 It is N and R 18 Does not exist; or

[0157] X 7 It is C and R 18 Selected from C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, OC 1-6 Alkyl, C 5-10 heteroaryl, C 3-10 cycloalkyl, C 4-10 Heterocyclic alkyl, C(O)R 15 C(O)N(R) 14 2. SO2R 15 SO2N(R) 14 )2、N(R 16 )C(O)R 15 、N(R 16 SO2R15 、N(R 16 )C(O)N(R 14 )2、N(R 16 SO2N(R) 14 )2、N(R 14 )2、P(O)(R 15 )2、CH2C(O)R 15 CH2C(O)N(R) 14 2. CH2SO2R 15 CH2SO2N(R) 14 )2、CH2N(R 16 )C(O)R 15 CH2N(R) 16 SO2R 15 CH2N(R) 16 )C(O)N(R 14 )2、CH2N(R 16 SO2N(R) 14 )2 and CH2N(R 14 )2;

[0158] Where R 14 R 15 and R 16 As defined above;

[0159] This includes in R 1 In China (including R) 14 R 15 R 16 R 17 and R 18 The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, or heteroaryl groups (as defined in the definition) may optionally be further substituted; and

[0160] (---) represents a key.

[0161] In another implementation, R 1 It is a group of the following formula:

[0162]

[0163] in:

[0164] X 15 X 16 X 17 and X 18 Independently selected from O, N, S and CR 17 , where R 17 As defined above;

[0165] Where X 15 X16 X 17 and X 18 At most two of them are O, N, or S.

[0166] In one embodiment, the compound of formula I is a compound of formula IV or V, or a pharmaceutically acceptable salt or solvate thereof:

[0167]

[0168] Where R 4 R 5 R 6 R 17 R 18 X 6 X 7 X 15 X 16 X 17 and X 18 Each independently as defined herein, preferably R 4 Selected from Cl, Br and methyl; R 5 Selected from H, F, Cl and methyl; and R 6 Selected from H, F, Cl, Me and OMe.

[0169] In another embodiment, the compound of formula I is a compound of formula VI or VII, or a pharmaceutically acceptable salt or solvate thereof:

[0170]

[0171] Where R 9 R 10 R 12 R 17 R 18 X 5 X 6 X 7 X 15 X 16 X 17 and X 18 Each is independent as defined in this article.

[0172] In one embodiment of the above formula, X 6 It is N. In another implementation, X 6 It is CH.

[0173] In another implementation, X 7 It is N, R 17 Selected from H, OH, CN, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, OC1-6 Alkyl, C 5-10 heteroaryl, C 3-10 cycloalkyl, C 4-10 Heterocyclic alkyl, C(O)R 15 C(O)N(R) 14 2. SO2R 15 SO2N(R) 14 )2、N(R 16 )C(O)R 15 、N(R 16 SO2R 15 、N(R 16 )C(O)N(R 14 )2、N(R 16 SO2N(R) 14 )2、N(R 14 )2、P(O)(R 15 )2、CH2C(O)R 15 CH2C(O)N(R) 14 2. CH2SO2R 15 CH2SO2N(R) 14 )2、CH2N(R 16 )C(O)R 15 CH2N(R) 16 SO2R 15 CH2N(R) 16 )C(O)N(R 14 )2、CH2N(R 16 SO2N(R) 14 )2 and CH2N(R 14 )2, and R 18 It does not exist, where in R 14 R 15 R 16 or R 17 The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, or heteroaryl groups are optionally further substituted, preferably R 17 Selected from C 1-6 Alkyl, C 5-10 heteroaryl, C 4-10 Heterocyclic alkyl, N(R) 14 )2、N(R 16 )C(O)R 15 、N(R 16 SO2R 15 C(O)N(R) 14 )2 and SO2N(R 14 )2, where in R 14 R 15 R 16 or R17 The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, or heteroaryl groups may optionally be further substituted. For example, R 17 Selected from H, NH2, and optionally substituted C 5-10 heteroaryl or C 4-10 Heterocyclic alkyl groups, preferably R 17 C is an optional substitute 5-10 heteroaryl or C 4-10 Heterocyclic alkyl groups.

[0174] In another implementation, R 17 C is an optional substitute 4-10 Heterocyclic alkyl groups, wherein the heterocyclic alkyl group may be monocyclic or bicyclic and comprises 1 to 3 heteroatoms, preferably wherein X 7 It is N. In a preferred embodiment, the heterocyclic alkyl group is, for example, selected from at least one of F, OH, oxo, CN, C. 1-4 Alkyl and OC 1-4 Alkyl group substitution, wherein the C 1-4 Alkyl groups may optionally be further substituted (e.g., by F, OH, OC). 1-3 Alkyl groups may be substituted. For example, heterocyclic alkyl groups may be selected from optionally substituted piperidine, piperazine, thiomorpholine, and morpholine groups, or bicyclic structures (bridging or spirocyclic) containing piperidine, piperazine, thiomorpholine, or morpholine rings.

[0175] In another implementation, X 7 It is C, for example, X 7 It is C and R 18 Selected from C 1-6 Alkyl, C 5-10 heteroaryl, C 3-10 cycloalkyl, C 4-10 Heterocyclic alkyl, C(O)R 15 C(O)N(R) 14 2. SO2R 15 SO2N(R) 14 )2、N(R 16 )C(O)R 15 、N(R 16 SO2R 15 、N(R 16 )C(O)N(R 14 )2、N(R 16 SO2N(R) 14 )2、N(R 14 )2、P(O)(R 15 )2、CH2C(O)R 15 CH2C(O)N(R) 14 2. CH2SO2R 15CH2SO2N(R) 14 )2、CH2N(R 16 )C(O)R 15 CH2N(R) 16 SO2R 15 CH2N(R) 16 )C(O)N(R 14 )2、CH2N(R 16 SO2N(R) 14 )2 and CH2N(R 14 )2, where in R 14 R 15 R 16 or R 18 The alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, or heteroaryl groups are optionally further substituted, preferably R 18 Selected from C(O)N(R) 14 2. SO2R 15 and SO2N(R) 14 )2. Among the subclasses of these implementation methods, R 17 Selected from H, OH, C 1-6 Alkyl, N(R) 14 2. and optional substitution of C 5-10 heteroaryl. For example, R 17 Selected from H, NH2, and optionally substituted C 5-10 Heteroaryl groups, preferably H or NH2.

[0176] In yet another implementation, R 14 Each occurrence is independently selected from H, or optionally substituted C. 1-6 Alkyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 4-10 Heterocyclic alkyl groups, and optionally substituted C 5-6 heteroaryl, or two R 14 Together with the nitrogen atoms adjacent to them, they form C 4-10 Heterocyclic alkyl groups.

[0177] In another implementation, R 17 It is N(R) 14 )2, wherein R 14 Together with the nitrogen atoms adjacent to them, they form C 4-10 Heterocyclic alkyl groups, wherein the heterocyclic alkyl group may be monocyclic or bicyclic, and comprises 1 to 3 heteroatoms, preferably wherein X 7 It is N. In a preferred embodiment, the heterocyclic alkyl group is, for example, selected from at least one of F, OH, oxo, CN, C. 1-4 Alkyl and OC 1-4 Alkyl group substitution, wherein the C1-4 Alkyl groups may optionally be further substituted (e.g., by F, OH, OC). 1-3 Alkyl groups may be substituted. For example, heterocyclic alkyl groups may be selected from optionally substituted piperidine, piperazine, thiomorpholine, and morpholine groups, or bicyclic structures (bridging or spirocyclic) containing piperidine, piperazine, thiomorpholine, or morpholine rings.

[0178] In another embodiment, R 1 Selected from:

[0179]

[0180] Where R 14 As defined in this article, and (---) represents a key.

[0181] In yet another embodiment, R 1 Selected from:

[0182]

[0183] Where R 14 As defined in this article, and (---) represents a key.

[0184] The Examples section also provides additional sub-general embodiments, wherein each substituent group R 1 (C group), R 2 (B group), Y, and L are as defined. Examples of combinations are further illustrated below and in Tables 2 through 5. Representative preferred compounds from Examples 1 through 163 are also described herein.

[0185] More specifically, exemplary R 1 The groups are defined as C1 to C493 as follows:

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210] (---) represents a key.

[0211] In one implementation, R 1 Selected from groups C1 to C493, or R 1 Selected from groups C1 to C23, C27, C60, C69, C71 to C73, C81 to C83, C88, C114, C182 to C184, C196, C220, C223 to C226, C275, C292, C310, C312, C313, C323, C346, C376, C402, C404, C414, C418, C419, C434, C435, C438, C440, C441, C472, C483, C488, and C490, for example, R 1The group is selected from C1, C3, C5, C7, C22, C23, C27, C60, C69, C73, C81 to C83, C88, C182 to C184, C196, C224 to C226, C313, C323, C376, C402, C404, C414, C418, C419, C438 and C488, for example selected from C7, C22, C23 and C60, or selected from C183, C323, C376, C414, C418, C419, C438 and C488.

[0212] The following embodiments describe R 1 (C1 to C493), R 2 Combinations of (B1 to B77) and L (L1 to L4) groups can be used to produce compounds of formula I, where Y is H or NH2:

[0213] C1-L-B1; C1-L-B2; C1-L-B3; C1-L-B4 to B70; C1-L-B71; C1-L-B72; C1-L-B73; C1-L-B74; C1-L-B75 to B77;

[0214] C2-L-B1; C2-L-B2; C2-L-B3; C2-L-B4 to B70; C2-L-B71; C2-L-B72; C2-L-B73; C2-L-B74; C2-L-B75 to B77;

[0215] C3-L-B1; C3-L-B2; C3-L-B3; C3-L-B4 to B70; C3-L-B71; C3-L-B72; C3-L-B73; C3-L-B74; C3-L-B75 to B77;

[0216] C4 to C488-L-B1; C4 to C488-L-B2; C4 to C488-L-B3; C4 to C488-L-B4 to B70; C4 to C488-L-B71; C4 to C488-L-B72; C4 to C488-L-B73; C4 to C488-L-B74; C4 to C488-L-B75 to B77;

[0217] C489-L-B1; C489-L-B2; C489-L-B3; C489-L-B4 to B70; C489-L-B71; C489-L-B72; C489-L-B73; C489-L-B74; C489-L-B75 to B77;

[0218] C490-L-B1; C490-L-B2; C490-L-B3; C490-L-B4 to B70; C490-L-B71; C490-L-B72; C490-L-B73; C490-L-B74; C490-L-B75 to B77;

[0219] C491 to C493-L-B1; C491 to C493-L-B2; C491 to C493-L-B3; C491 to C493-L-B4 to B70; C491 to C493-L-B71; C491 to C493-L-B72; C491 to C493-L-B73; C491 to C493-L-B74; or C491 to C493-L-B75 to B77.

[0220] In one embodiment, the compound is as defined in Formula I, wherein:

[0221] -R 1 Selected from groups C1 to C493, or R 1 Selected from groups C1 to C23, C27, C60, C69, C71 to C73, C81 to C83, C88, C114, C182 to C184, C196, C220, C223 to C226, C275, C292, C310, C312, C313, C323, C346, C376, C402, C404, C414, C418, C419, C434, C435, C438, C440, C441, C472, C483, C488, and C490, for example, R 1 Selected from groups C1, C3, C5, C7, C22, C23, C27, C60, C69, C73, C81 to C83, C88, C182 to C184, C196, C224 to C226, C313, C323, C376, C402, C404, C414, C418, C419, C438 and C488;

[0222] -R 2 Selected from B1, B2, B8, B11, B12, B20 to B23, B34 to B37, B41 to B44, B49, B51 to B54, B57, B59, B62 to B67, B71 to B74, and B77; and

[0223] -L is a group selected from L1 to L4, and Y is H or NH2, preferably Y is H.

[0224] In another embodiment, the compound is as defined in Formula I, wherein R 1Selected from C7, C22, C23, C60, C73, C81, C83, C183, C376, C404, C414, C418, C419, C438 and C488, R 2 The group is selected from B12, B21, B36, B41, B42, B52 to B54, B59, B65 and B73, where L is a group selected from L1 to L4, and Y is H or NH2, preferably Y is H.

[0225] The exemplary compounds as defined herein include each individual compound covered in Examples 1 through 163 in Tables 2, 3, 4, and 5.

[0226] Examples of preferred compounds are those listed in Tables 3, 4, and 5: 31, 36, 40, 51, 55 to 60, 69, 72, 80 to 83, 88, 93, 94, 96 to 122, 124 to 147, 149, 151 to 160, 162, and 163. Examples of more preferred compounds include those listed in Tables 3 and 5: 80 to 83, 93, 94, 96, 98 to 101, 104, 106, 111, 112, 114 to 116, 119, 120, 122, 125, 128 to 134, 139, 142, 144 to 146, 153, 155, 157, 159, and 162.

[0227] It should be understood that any of the above-described compounds may be in any amorphous, crystalline, or polymorphic form, including any salt or solvate form, or mixtures thereof. The compounds of this specification may be further modified by attaching various functional groups via any synthetic means described herein to enhance selective biological properties. Such modifications are known in the art and include those that increase biopermeability to a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral bioavailability, increase solubility to allow administration by injection, alter metabolism, and change excretion rates.

[0228] These compounds can be prepared by conventional chemical synthesis, such as those exemplified in the embodiments and examples of this invention. Other methods for synthesizing the compounds of the chemical formulas herein will be apparent to those skilled in the art, as will be appreciated by those skilled in the art. Furthermore, various synthetic steps can be performed in an alternating order or sequence to obtain the desired compounds.

[0229] iii. Methods, uses, formulations and application

[0230] As used herein, the term "effective amount" means the amount of a drug or agent that will elicit a biological or medical response in a tissue, system, animal, or human, as sought by, for example, a researcher or clinician. Furthermore, the term "therapeuticly effective amount" means any amount that results in the treatment, cure, prevention, or improvement of a disease, condition, or its symptoms, or a reduction in the rate of progression of a disease or condition, compared to a corresponding subject who did not receive such an amount. The term also includes, within its scope, amounts that effectively enhance normal physiological function.

[0231] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing or alleviating a disease or condition or one or more symptoms thereof as described herein, delaying its onset, or inhibiting its progression. In some embodiments, treatment may be administered after one or more proliferations have occurred. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to susceptible individuals before symptoms appear (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms have subsided, for example, to prevent or delay their recurrence.

[0232] In one implementation, the disease or condition to be treated is a proliferative disease or symptom, or a kinase-mediated disease or symptom. More specifically, the disease or condition to be treated includes proliferative diseases or symptom, developmental abnormalities caused by RAS-ERK signaling cascade dysregulation (RAS pathway disorders), inflammatory diseases, or immune system disorders.

[0233] Based on some examples, the proliferative disorders or conditions to be treated are tumors, inflammatory diseases or ailments, or developmental abnormalities involving constitutive activating mutations in the RAS and / or RAF genes (e.g., KRAS and / or ARAF, BRAF, or CRAF mutations). The disease or condition may also be further associated with receptor tyrosine kinase mutations or amplifications (e.g., EGFR, HER2) or mutations in regulators of RAS downstream of the receptor (e.g., SOS1 gain-of-function, NF1 loss-of-function). For example, compounds as defined herein are inhibitors of signaling enzymes (e.g., B- and CRAF), which not only affect those carrying RAF mutations (e.g., BRAF) V600E These enzymes are involved in controlling cell proliferation in tumors, and importantly, they are also involved in controlling cell proliferation in the context of mutated RAS-driven cancers. Therefore, the compounds of the present invention can be used, for example, to treat diseases associated with the activity of these signaling enzymes and characterized by excessive or abnormal cell proliferation.

[0234] According to one implementation, the disease or symptom is characterized by uncontrolled cell proliferation, i.e., a “proliferative symptom” or “proliferative disease.” More specifically, these diseases and symptoms involve cells with autonomous growth capacity, i.e., an abnormal state of disorder characterized by rapidly proliferating cell growth, which typically forms distinct clumps and exhibits partial or complete lack of structural organization and functional coordination with normal tissue.

[0235] For example, proliferative conditions or diseases are defined as “neoplasm,” “neoplastic condition,” “tumor,” “cancer,” and “tumor,” terms collectively intended to encompass hematopoietic system tumors (such as lymphoma or leukemia) and solid tumors (such as sarcoma or carcinoma), including all types of precancerous and cancerous growth or carcinogenic processes, metastatic tissues, or malignant transformations of cells, tissues, or organs, regardless of histopathological type or stage of invasion. Hematopoietic system tumors are malignant tumors affecting hematopoietic structures (structures associated with blood cell formation) and components of the immune system, including leukemia (related to white blood cells (leukocytes) and their precursors in the blood and bone marrow) originating from the bone marrow, lymphoid, or erythroid lineages, and lymphoma (related to lymphocytes). Solid tumors include sarcomas, which are malignant tumors originating from connective tissues such as muscle, cartilage, blood vessels, fibrous tissue, fat, or bone. Solid tumors also include carcinomas, which are malignant tumors arising from epithelial structures, including the external epithelium (e.g., the skin and endometrium, lungs, and cervix) and the internal epithelium lining various glands (e.g., breast, pancreas, thyroid). Examples of tumors include leukemia and hepatocellular carcinoma, sarcoma, angioendothelial carcinoma, breast cancer, central nervous system cancers (e.g., astrocytoma, glioma, neuroblastoma, oligodendroglioma, and glioblastoma), prostate cancer, lung cancer and bronchial cancer, laryngeal cancer, esophageal cancer, colon cancer, colorectal cancer, gastrointestinal cancer, melanoma, ovarian cancer and endometrial cancer, kidney cancer and bladder cancer, liver cancer, endocrine cancers (e.g., thyroid cancer), and pancreatic cancer. For example, the diseases or conditions mentioned are selected from colon cancer, lung cancer, pancreatic cancer, thyroid cancer, breast cancer, and skin cancer. Examples of tumors include melanoma, papillary thyroid carcinoma, colorectal cancer, ovarian cancer, breast cancer, endometrial cancer, liver cancer, sarcoma, gastric cancer, Barrett's adenocarcinoma, glioma (including ependymoma), lung cancer (including non-small cell lung cancer), head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, and hairy cell leukemia.

[0236] In this implementation, the patient presenting with one of the aforementioned hematopoietic system or solid tumors has previously received treatment with a RAS-ERK pathway-targeting inhibitor (including RTK, RAF, MEK, or ERK inhibitors) but has developed resistance to said inhibitor. Inhibitors include standard of care treatments such as vemurafenib, dabrafenib, cobimetinib, trametinib, YERVOY, OPDIVO, or any combination of these agents.

[0237] In the implementation, the disease to be treated is defined as a developmental abnormality caused by RAS-ERK signaling cascade dysregulation (RAS pathway disorders: for example, Noonan syndrome, Costello syndrome, LEOPARD syndrome, cardiofacial skin syndrome, and hypertrophic cardiomyopathy).

[0238] In this implementation, the disease to be treated is defined as an inflammatory disease or an immune system disorder. Examples of such inflammatory diseases or immune system disorders include inflammatory bowel disease, Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis, thyroiditis, type 1 diabetes, sarcoidosis, psoriasis, allergic rhinitis, asthma, and COPD (chronic obstructive pulmonary disease).

[0239] In one embodiment, the compound as defined herein is an inhibitor of RAS-ERK signaling and cell proliferation in tumor cells carrying at least one mutated RAS or RAF genotype, which does not induce or substantially does not induce paradoxical pathways.

[0240] As used herein, the term "patient or subject" refers to an animal, such as a mammal. Therefore, a subject can refer to, for example, a mouse, rat, dog, cat, horse, cow, pig, guinea pig, primate, including humans, etc. Preferably, the subject is a human.

[0241] Therefore, this specification also relates to methods for treating subjects (e.g., human subjects) with proliferative diseases or conditions (e.g., RAF-mutated and / or RAS-driven cancers with mutations). The method involves administering a therapeutically effective amount of a compound as defined herein to a subject in need of such treatment.

[0242] In some embodiments, this specification provides a method for treating a subject's condition (as described herein), which includes administering a compound of this specification to a subject identified as needing it. The identification of those patients needing treatment for the aforementioned condition is entirely within the competence and knowledge of those skilled in the art. The medical field recognizes certain methods for identifying patients at risk of developing the aforementioned condition, which can be treated by the subject's methods, such as the subject patient's family history and the presence of risk factors associated with developing the disease state. Those skilled in the art can readily identify such candidate patients using, for example, clinical tests, physical examinations, medical / family history, and genetic testing.

[0243] Methods for assessing the efficacy of treatment in subjects include determining pre-treatment symptoms of the disease using methods well-known in the art, and then administering a therapeutically effective amount of the compound of the present invention to the subject. Symptoms of the disease are reassessed at appropriate time points after compound administration (e.g., 1 week, 2 weeks, 1 month, 6 months). Moderation (e.g., reduction) of symptoms and / or biomarkers (e.g., pERK or pMEK) indicates efficacy of treatment. Symptoms and / or biomarkers of the disease can be determined periodically throughout the treatment course. For example, symptoms and / or biomarkers of the disease can be examined every few days, weeks, or months to assess further efficacy of treatment. A reduction in symptoms and / or biomarkers of the disease indicates that the treatment is effective.

[0244] In some implementations, a therapeutically effective amount of a compound as defined herein may be administered to the patient alone or in the form of a composition mixed with a pharmaceutically acceptable carrier, adjuvant, or mediator.

[0245] The terms "pharmaceutically acceptable carrier, adjuvant, or mediator" and equivalent expressions refer to non-toxic carriers, adjuvants, or mediators that do not impair the pharmacological activity of the compound with which they are formulated. Pharmaceutically acceptable carriers, adjuvants, or excipients that can be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffering substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, mixtures of saturated vegetable fatty acids in the form of glycerides, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0246] The compositions described herein can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, or via implanted receptacle. As used herein, the term “parenterally” includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Other routes of administration include intradermal or transdermal administration.

[0247] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitol fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, surfactants, sweeteners, flavoring agents, and aromatizers.

[0248] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable media and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are commonly used as solvents or suspending media. For this purpose, any mild, non-volatile oil, including synthetic monoglycerides or diglycerides, can be used. Additionally, fatty acids such as oleic acid are also used in the preparation of injectable formulations.

[0249] Injectable formulations can be sterilized, for example, by filtering through a bacterial trap filter, or by incorporating a sterilizing agent in the form of a sterile solid composition, which may be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0250] To prolong the effect of the administered compound, it is generally desirable to slow the absorption of the compound administered subcutaneously or intramuscularly. This can be achieved by using liquid suspensions of poorly water-soluble crystalline or amorphous materials. The absorption rate of the compound depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of parenteral-administered compounds can be achieved by dissolving or suspending the compound in an oil-based medium. Injectable reservoir forms are prepared by forming microcapsule matrices of the compound within a biodegradable polymer (e.g., polylactide-polyglycolic acid). The rate of compound release can be controlled depending on the compound-to-polymer ratio and the properties of the specific polymer used.

[0251] Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by encapsulating the compound in liposomes or microemulsions that are compatible with body tissues.

[0252] The composition for rectal administration is preferably a suppository, which can be prepared by mixing the compound of this specification with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or suppository wax, which is solid at ambient temperature but liquid at body temperature and thus melts in the rectum and releases the active compound.

[0253] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and / or silicate, b) binders such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone (PVP), sucrose, and gum arabic, c) humectants such as glycerin, d) disintegrants such as agar, calcium carbonate, potato or cassava starch, alginic acid, certain silicates, and sodium carbonate, e) solution blockers such as paraffin, f) absorption enhancers such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glyceryl monostearate, h) absorbents such as kaolin and bentonite, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer.

[0254] Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, using excipients such as lactose or toffee and high molecular weight polyethylene glycol. Solid dosage forms of tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells, such as enteric coatings and other coatings well known in the field of pharmaceutical formulation. They may optionally contain light-blocking agents and may also have compositions in which one or more active ingredients are optionally released in a delayed manner, either only or preferably in a specific portion of the intestine. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, using excipients such as lactose or toffee and high molecular weight polyethylene glycol.

[0255] The composition can also be in the form of microcapsules having one or more excipients as described above. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared by coating and shelling, such as enteric coating, controlled-release coating, and other coatings well known in the field of pharmaceutical formulation. In such solid dosage forms, the active compound can be mixed with at least one inert diluent, such as sucrose, lactose, or starch. In accordance with normal practice, such dosage forms may also contain substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage forms may also contain buffers. They may optionally contain light-blocking agents and may also have compositions in which they optionally release one or more active ingredients in a delayed manner only or preferably in a specific part of the intestine. Examples of encapsulation compositions that can be used include polymers and waxes.

[0256] Dosage forms of the compounds described in this specification for topical or transdermal application include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, or patches. The active ingredient is mixed under aseptic conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers. Ophthalmic preparations, ear drops, and eye drops are also covered within the scope of this specification. Additionally, the use of transdermal patches is considered, which has the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0257] The pharmaceutically acceptable compositions provided herein can also be administered via nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the field of pharmaceutical formulation and can be prepared as saline solutions using benzyl alcohol or other suitable preservatives, bioavailability enhancers, fluorocarbons, and / or other conventional solubilizers or dispersants.

[0258] The pharmaceutically acceptable compositions provided herein can be formulated for oral administration. Such products can be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0259] The amount of compounds that can be combined with carrier materials to produce compositions in a single dosage form will vary depending on the patient to be treated and the specific mode of administration. The provided compositions can be formulated to allow administration of inhibitory doses ranging from 0.01 mg / kg body weight / day to 100 mg / kg body weight / day to patients receiving these compositions.

[0260] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including age, weight, general health condition, sex, diet, timing of administration, excretion rate, drug combination, the judgment of the treating physician, and the severity of symptoms associated with proliferative disorders or conditions. The amount of compounds provided in the composition will also depend on the specific compounds in the composition.

[0261] The compounds or compositions described herein may be administered in any amount and via any route of administration to effectively treat or alleviate the severity of the symptoms considered herein. The exact amount required will vary from subject to subject, depending on the subject’s species, age and general condition, severity of infection, specific agent, mode of administration, etc. The compounds provided are preferably formulated in unit dosage forms to facilitate administration and uniformity of dosage. As used herein, “unit dosage form” means a physically discrete unit of pharmaceutical preparation suitable for the patient to be treated. However, it should be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the bounds of reasonable medical judgment.

[0262] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracerebrospinally, intraperitoneally, topically (e.g., by powder, ointment, or drops), buccally, as oral or nasal sprays, etc., depending on the severity of the infection being treated. In some embodiments, the provided compounds can be administered orally or parenterally at dose levels of about 0.01 mg / kg to about 50 mg / kg, preferably about 1 mg / kg to about 25 mg / kg, of the subject's body weight once or more daily to achieve the desired therapeutic effect.

[0263] It should be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within a reasonable medical judgment. The total daily inhibitory amount of the compounds of the present invention administered to a subject in a single or divided dose may be, for example, from 0.01 mg / kg body weight to 50 mg / kg body weight, or more typically from 0.1 mg / kg body weight to 25 mg / kg body weight. A single-dose composition may contain such an amount or approximation thereof to constitute a daily dose. In one embodiment, a treatment regimen according to the present invention comprises administering about 10 mg to about 1000 mg of one or more compounds of the present invention daily in a single or multiple dose to a patient requiring such treatment.

[0264] Depending on the disease or condition to be treated, additional therapeutic agents may also be present in the compositions of the present invention or administered alone as part of a dosage regimen, such as additional chemotherapeutic agents. Non-limiting examples of other therapeutic agents that may be used in combination with the compounds of the present invention include antiproliferative compounds, such as aromatase inhibitors; anti-estrogens; anti-androgens; gonadorelin agonists; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule activators; alkylating agents; retinoids, carotenoids, tocopherols; cyclooxygenase inhibitors; MMP inhibitors; antimetabolites; platinum compounds; methionine aminopeptidase inhibitors; bisphosphonates; antiproliferative antibodies; heparanase inhibitors; Ras oncogenic isotype inhibitors; telomerase inhibitors; proteasome inhibitors; compounds for the treatment of hematologic malignancies; kinin spindle protein inhibitors; Hsp90 inhibitors; mTOR. Inhibitors; PI3K inhibitors; Flt-3 inhibitors; CDK4 / 6 inhibitors; HER2 inhibitors (Herceptin, Trastuzumab); EGFR inhibitors (Iressa, Tarceva, Heliotropin, Tagrisso, Erbitux); RAS inhibitors; MEK inhibitors (Trametinib, Bemetinib, Cobitinib); ERK inhibitors (Uritinib); anti-PD-1 antibodies (Opdivo, Keytruda); anti-CTLA4 antibodies (Yervoy); antitumor antibiotics; nitrosoureas; compounds that target / reduce the activity of protein or lipid kinases, compounds that target / reduce the activity of protein or lipid phosphatases, or any other anti-angiogenic compounds.

[0265] This treatment can also be combined with other treatments or interventions, such as surgery, radiation therapy (e.g., gamma radiation, neutron beam therapy, electron beam therapy, proton therapy, brachytherapy, and whole-body radioisotopes), biological response modulators (e.g., interferon, interleukin, tumor necrosis factor (TNF), and drugs to mitigate adverse effects).

[0266] The description of the implementation of the variables in this document includes the implementation as any single implementation or in combination with any other implementation or part thereof.

[0267] Example

[0268] List of abbreviations:

[0269] Ac: Acetyl group

[0270] AcOEt: Ethyl acetate

[0271] AcOH: Acetic acid

[0272] Aryl

[0273] ATCC: American Culture Collection

[0274] ATP: adenosine triphosphate

[0275] BINOL: [1,1'-Bidinaphthalene]-2,2'-diol

[0276] Boc: tert-butoxycarbonyl

[0277] BOP: (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

[0278] br: width

[0279] BSA: Bovine serum albumin

[0280] CCL: Cancer Cell Line

[0281] DCE: 1,2-Dichloroethane

[0282] DCM: Dichloromethane

[0283] DIEA (or DIPEA): N,N-diisopropylethylamine (Huenig base)

[0284] DME: 1,2-Dimethoxyethane

[0285] DMF: N,N-dimethylformamide

[0286] DMSO: Dimethyl sulfoxide

[0287] DTT: Dithiothreitol

[0288] EA: Ethyl acetate

[0289] EC 50 Half-maximum effective concentration

[0290] ECL: Enhanced Chemiluminescence

[0291] EDTA: Ethylenediaminetetraacetic acid

[0292] Et2O: Diethyl ether

[0293] EtOH: Ethanol

[0294] Eu: Europium

[0295] FBS: Fetal bovine serum

[0296] GST: Glutathione S-transferase

[0297] HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N',-tetramethylureonium hexafluorophosphate

[0298] HEPES: 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid

[0299] Het: heterocyclic ring

[0300] Hex: Hexane

[0301] HRMS: High Resolution Mass Spectrometry

[0302] HPLC: High Performance Liquid Chromatography

[0303] HRP: Horseradish peroxidase

[0304] IC 50 Half-maximum inhibition concentration

[0305] IPA or iPrOH: Isopropanol

[0306] LCMS: Liquid Chromatography-Mass Spectrometry

[0307] MeCN: Acetonitrile

[0308] MS: Mass Spectrometry

[0309] NMP: N-methylpyrrolidone

[0310] NMR: Nuclear Magnetic Resonance

[0311] ON: overnight

[0312] PBS: Phosphate-buffered saline

[0313] pERK: Phosphorylated extracellular signal-regulated kinase

[0314] PMB: p-Methoxybenzyl

[0315] PMSF: Phenylmethylsulfonyl fluoride

[0316] Rf: Retention factor

[0317] RPMI-1640: Roswell Park Memorial Institute Culture Medium

[0318] RT: Room temperature

[0319] SDS: Sodium dodecyl sulfate

[0320] SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

[0321] SEM: Trimethylsilylethoxymethyl

[0322] SNAr: Nucleophilic Aryl Substitution

[0323] TBST: Tris-buffered saline containing 0.2% Tween-20

[0324] TBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylureonium tetrafluoroborate

[0325] TEV: Tobacco Etching Viral Protease

[0326] TFA: Trifluoroacetic acid

[0327] THF: Tetrahydrofuran

[0328] TLC: Silica gel thin-layer chromatography

[0329] TR-FRET: Time-resolved fluorescence resonance energy transfer

[0330] Ts: p-Toluenesulfonate

[0331] Y MIN Minimum data point of the dose-activity curve

[0332] The following non-limiting embodiments are illustrative and should not be construed as further limiting the scope of the invention. These embodiments will be better understood with reference to the accompanying drawings.

[0333] The examples presented below provide synthesis and experimental results for certain exemplary compounds. As is well known to those skilled in the art, reactions were carried out under an inert atmosphere (nitrogen or argon) where necessary to protect the reactants from air and moisture. Temperatures are given in degrees Celsius (°C). Unless otherwise stated, solution percentages and ratios express volume-to-volume relationships. The reactants used in the examples below can be obtained as described herein, or, if not described herein, are commercially available or can be prepared from commercially available materials by methods known in the art. Rapid chromatography was performed at 254 nm on silica (SiO2) using a Teledyne Isco Rf Combiflash instrument using commercial normal-phase silica. Mass spectrometry analysis was recorded using electrospray mass spectrometry. NMR was recorded on a 400 MHz Varian instrument.

[0334] Preparative HPLC was performed using an Agilent instrument with a Phenomenex-Kinetex C18 (21 x 100 mm, 5 μm) column at a flow rate of 20 mL / min (RT) and UV detection at 220 nm and 254 nm. Unless otherwise specified, the mobile phase consisted of solvent A (5% MeOH, 95% water + 0.1% formic acid) and solvent B (95% MeOH, 5% water + 0.1% formic acid). Occasionally, 0.05% TFA or 0.1% AcOH or other additives were used in place of 0.1% formic acid in both solvents, as noted herein. MeCN was also used in place of MeOH in both mobile phases to achieve the more challenging separations specified herein. Specific gradient conditions are provided in the examples, but the following are representative: isocratic T(0) → T(3 min) using 10% to 50% solvent B, depending on the compound polarity, followed by a 12-minute gradient to 100% solvent B. The final 5 minutes used 100% solvent B.

[0335] LCMS analysis was performed on an Agilent instrument. The column was a Phenomenex Kinetex C18 column (2.6 μm). Liquid chromatography was performed on a 3x30 mm (3 x 10 mm) plate at a flow rate of 1.5 mL / min (RT) with UV detection at 220 nm and 254 nm. The mobile phase consisted of solvent A (95% H₂O / 5% MeOH / 0.1% AcOH) and solvent B (95% MeOH / 5% H₂O / 0.1% AcOH) using the following gradient: T(0) 100% A → T(0.5 min) 100% B → isocratic 100% B to T(2 min). MS detection was performed in parallel in both positive and negative modes using APCI detection.

[0336] Unless otherwise stated, all figures used in the specification and claims to represent amounts of components, reaction conditions, concentrations, properties, stability, etc., should be understood to be modified by the term "about" in all cases. At a minimum, each numerical parameter should be interpreted according to the number of significant figures reported and by applying ordinary rounding techniques. Therefore, unless otherwise indicated, the numerical parameters set forth in this specification and the appended claims are approximate values ​​that may vary depending on the properties sought to be obtained. Although the numerical ranges and parameters illustrating a broad range of embodiments are approximate, the values ​​described in the specific embodiments are reported as accurately as possible. However, any numerical value inherently contains some error due to variations in experiments, test measurements, statistical analyses, etc.

[0337] Synthesis, bioactivity, and characterization of the examples:

[0338] All compounds described herein were prepared according to the methods shown in Tables 2 through 5. Characterization data obtained by mass spectrometry and NMR are provided for each example. The compounds were tested in the assays described in the Biological Experiments section. Conventions for reporting biological data are provided as footnotes in the respective tables.

[0339] General synthesis method A:

[0340] Commercially available 2,6-difluoro-3-nitrobenzoic acid A-1 (Scheme A) can be converted to urethane A-2 via the Curtius reaction according to the procedure described in J. Med. Chem. 2003, 46, 1905. The nitroaromatic hydrocarbon A-2 is then catalytically hydrogenated using hydrogen and a catalyst (e.g., carbon-supported palladium metal or carbon-supported palladium hydroxide (Pearlman catalyst)) to yield aniline A-3. Aniline A-3 reacts with a sulfonating agent, such as sulfonyl chloride, in the presence of an organic base that can be used as a solvent, such as pyridine, with or without a catalyst, such as 4-dimethylaminopyridine, and with or without an additional solvent, such as dichloromethane or tetrahydrofuran, to produce a sulfonamide intermediate A-4, which can be deprotected using a strong acid (e.g., a dioxane solution of anhydrous hydrochloric acid) to an aniline salt, such as A-5. Alternatively, 2,6-difluoroaniline A-6 can be converted to its acetaniline A-7 and then to the monoprotected diphenylamine A-8 using an acetylation agent, such as acetic anhydride, as described in WO 2012 / 101238A1. Sulfonation to sulfonamide A-9 is achieved using a sulfonating agent under similar conditions to those used for the conversion of urethane A-3 to sulfonamide A-4, in the presence of an organic base (e.g., pyridine), with or without a catalyst (e.g., 4-dimethylaminopyridine) and a solvent (e.g., dichloromethane or tetrahydrofuran). Treatment of acetaniline A-9 with an aqueous solution of hydrochloric acid in the presence of a co-solvent, such as an alcohol, yields aniline salt A-5.

[0341] Option A

[0342]

[0343] 8-Chloro-2-(methylthio)pyrimidine-10 is commercially available or can be prepared as described in WO 2012 / 101238A1. Inhibitors of general formula I are prepared by nucleophilic substitution between A-10 and aniline derivative A-5, following a procedure similar to that described in WO 2012 / 101238A1. Inhibitors of general formula II are prepared from inhibitors of general formula I via a two-step procedure, which involves first oxidizing the thiomethyl group generally to a mixture of the corresponding methyl sulfoxide and methyl sulfone, which is then reacted with a nucleophile (e.g., a 1° or 2° amine, an alcohol, phenol, or an NH-containing heterocycle, etc.), following a procedure similar to that described in WO 2012 / 101238A1. The latter step is typically carried out in the presence of a base (e.g., an organic base, such as DIEA, trimethylamine, pyridine, etc.) in a solvent (e.g., DMSO or NMP) at a temperature range of 70°C to 140°C.

[0344] General synthesis method B:

[0345] An alternative method for preparing the inhibitor of the present invention is described in Scheme B. As shown in Scheme B, intermediate A-2 (described in Scheme A and prepared as described in J. Med. Chem. 2003, 46, 1905) is converted to nitroaniline hydrochloride B-1 by cleaving the urethane protecting group under acidic conditions (e.g., HCl in dioxane or TFA). Chloroprene-10 (prepared as described in Scheme A (WO 2012 / 101238A1)) is nucleophilically substituted with an aniline salt (e.g., B-1) or preferably with a free aniline base under conditions similar to those described in WO 2012 / 102138A1 to provide intermediate B-2. Then, under acidic conditions, at a temperature range of 50°C to 100°C, in a solvent (e.g., MeOH, EtOH, or EtOAc, etc.), using methods well-established and known to those skilled in the art, such as tin(II) chloride, iron, or zinc powder, the nitro functional group of intermediate B-2 is reduced to the corresponding aniline B-3. Then, aniline B-3 is sulfonated under alkaline conditions using sulfonyl chloride as described in Scheme A to provide an inhibitor of general formula I. Then, the inhibitor of general formula I is converted to an inhibitor of general formula II as described in Scheme A.

[0346] Option B

[0347]

[0348] General synthesis method C:

[0349] Alternative methods for preparing the inhibitors of the present invention having general formulas III and IV are described in Scheme C. As shown in Scheme C, intermediate B3 prepared as described in Scheme B can be converted to chlorosulfonylaniline C-1 by treatment with sulfonyl chloride in the presence of an organic base such as a 3° amine (e.g., trimethylamine, DIEA, etc.). Intermediate C-1 is reacted with a 1° or 2° amine, such as a pyrrolidine derivative, to provide an inhibitor of general formula III. An inhibitor of general formula IV is then obtained by a two-step procedure involving the oxidation of a thiomethyl group to a mixture of sulfoxides and sulfones, followed by reaction with a nucleophile as described in Scheme A.

[0350] Option C

[0351]

[0352] General synthesis method D:

[0353] Examples of preparing inhibitors of general formula V according to the order shown in scheme D.

[0354] Option D

[0355]

[0356] 2,4,8-Trichloropyrimidine D-1 was prepared by the method described in ACS Med. Chem. Lett. 2011, 2,538, and converted to 4-amino-2,8-dichloropyrimidine D-2 by treatment with ammonia as described in WO 2010 / 026262A1. Dichloropyrimidine D-2 was subjected to a regioselective nucleophilic substitution with an aniline salt of general formula A-5 under the general conditions described in Scheme A to provide 8-chloropyrimidine intermediate D-3. Chloropyrimidine D-3 underwent a second substitution with a nucleophile, such as a 2° amine or an NH-containing heterocycle, following a similar scheme as described in WO 2012 / 101238A1, to provide an inhibitor of general formula V. This latter step was typically carried out in the presence of a base (e.g., an organic base, such as DIEA, trimethylamine, pyridine, etc.) in a solvent (e.g., DMSO or NMP) at a temperature range of 70°C to 140°C. Alternatively, intermediate D-3 reacts with a nucleophile, such as a 2° amine or an NH-containing heteroaryl group (e.g., imidazole, benzimidazole, etc.), under copper-catalyzed cross-coupling conditions in the presence of an organic ligand (e.g., racemic BINOL) and an inorganic base (e.g., cesium carbonate). These reactions are typically carried out in solvents (e.g., DMSO or NMP) at temperatures ranging from 80°C to 140°C. Other methods involving the coupling of chloropyrimidine to such nucleophiles through metal-catalyzed processes are well known to those skilled in the art and can be used to obtain inhibitors of general formula V.

[0357] General synthesis method E:

[0358] Inhibitors of general formula VI are prepared as described in scheme E. The intermediate of general formula I is first prepared according to general method A or B, and then oxidized to a mixture of methyl sulfoxide and methyl sulfone, as described in general method A. Then, intermediate I is coupled to a 3-indole carboxylic acid ester (e.g., methyl ester, X=CH) or a 3-indazole carboxylic acid ester (e.g., methyl ester, X=N) according to a scheme similar to that described in WO 2012 / 101238A1. This latter step is typically carried out in the presence of a base (e.g., organic or inorganic base, such as Cs₂CO₃, KOtBu, DIEA, trimethylamine, pyridine, etc.), in a solvent (e.g., THF, DMSO, or NMP), at ambient temperatures up to 140°C. Within a temperature range from ambient temperature to 100°C, the ester protecting group is deprotected using an inorganic base (e.g., NaOH or KOH) in a mixture of water and a miscible organic solvent (e.g., methanol, ethanol, THF, dioxane, etc.), followed by acidification with a mineral (e.g., aqueous hydrochloric acid or sulfuric acid), an inorganic salt solution (e.g., aqueous NH4Cl or KHSO4), or an organic acid (e.g., aqueous citric acid or acetic acid). Intermediate E-1 is then coupled to an amine using standard amide coupling reagents (e.g., TBTU, HATU, DCC, EDC, etc.) to provide an amide derivative of general formula VI.

[0359] Option E

[0360]

[0361] General synthesis method F:

[0362] Following the general procedure described in Scheme F, in step 1, the thiomethyl intermediate I prepared according to general method A or B is oxidized to a mixture of methyl sulfoxide and methyl sulfone, as described in general method A. In step 2, 3-indolesulfonyl chloride is prepared according to Org. Lett. 2011, 13, 3588, and condensed with an amine in a solvent (e.g., THF) in the presence of an organic base (e.g., DIEA, trimethylamine, etc.) to provide intermediate 3-indolesulfonamide intermediate F-1. Alternatively, after removing the toluenesulfonyl protecting group by treatment with an aqueous solution of an inorganic base (e.g., KOH), N-toluenesulfonyl-protected indole-3-sulfonyl chloride (prepared by the method described in Chemical and Pharmaceutical Bulletin 2009, 57, 591) is reacted with a primary or secondary amine in a solvent (e.g., THF) in the presence of a tertiary base (e.g., DIEA or triethylamine) to provide intermediate sulfonamide F-1. Then, following a similar scheme as described in WO 2012 / 101238A1, intermediate F-1 is condensed with an oxidized mixture from intermediate I of step 1. This latter step is typically carried out in the presence of a base (e.g., an organic base such as DIEA, trimethylamine, pyridine, etc.) in a solvent (e.g., DMSO or NMP) at a temperature ranging from 70°C to 140°C to provide an inhibitor of general formula VII.

[0363]

[0364] X = H or T s

[0365] General synthesis method G:

[0366] 3-Indolethiocyanate G-1 (prepared according to the procedure described in Phosphorus, Sulfur and Silicon and the Related Elements 2014, 189, 1378) is reduced to the corresponding sulfide salt using a reducing agent, such as sodium sulfide nonahydrate, and directly alkylated without separation from the alkyl halide to provide sulfide intermediate G-2. Sulfide intermediate G-2 is then converted to sulfone intermediate G-3 using an oxidizing agent, such as 3-chloroperoxybenzoic acid. The final inhibitor of general structure VIII is then obtained in the usual manner described above.

[0367] Option G

[0368]

[0369] General synthesis method H:

[0370] A commercially available bright red solution of 3-fluoro-2-nitroaniline H-1 is reacted with a primary or secondary amine in a solvent (e.g., MeCN, DMSO, or NMP) and in the presence of an inorganic base (e.g., potassium carbonate) or an organic base (e.g., DIEA), heated under thermal or microwave conditions in a temperature range of 40°C to 120°C to provide intermediate H-2. The reduction of the nitro group in intermediate H-2 can be achieved using a metal (e.g., Fe or Zn) in the presence of ammonium chloride in an alcoholic solvent (e.g., isopropanol) in a temperature range of 40°C to 80°C. The 1,2-phenylenediamine intermediate is then directly converted to the desired benzimidazole intermediate H-3 upon heating with formic acid at a temperature of 40°C to 80°C. The final inhibitor of the universal structure IX is then obtained from intermediate benzimidazole H-3 and I under the general conditions described above.

[0371] Option H

[0372]

[0373] General Synthesis Method I:

[0374] In the presence of ammonium chloride, at a temperature range of 40°C to 80°C, in an alcoholic solvent (e.g., isopropanol), nitroaniline H-2 obtained according to general synthetic method H is reduced to 1,2-phenylenediamine I-1 using a metal (e.g., Fe or Zn). The 1,2-phenylenediamine intermediate I-1 is then converted to the desired benzotriazole intermediate I-2 after treatment with an inorganic nitrite, such as sodium nitrite, under acidic conditions (e.g., AcOH). Then, under the general conditions described above, the final inhibitor of the universal structure X is obtained from intermediates I-2 and I.

[0375] Option I

[0376]

[0377] sulfonyl chloride:

[0378] The following sulfonyl chlorides are obtained from commercial sources and used as is: 4-methoxybenzenesulfonyl chloride, 2,4-dichlorobenzenesulfonyl chloride, 2,4-dimethylbenzenesulfonyl chloride, 2-chlorobenzenesulfonyl chloride, 2-methylbenzenesulfonyl chloride, 4-ethylbenzenesulfonyl chloride, 2-cyanobenzenesulfonyl chloride, 2,4-dimethoxybenzenesulfonyl chloride, 2-trifluoromethylbenzenesulfonyl chloride, 3-chlorobenzenesulfonyl chloride, 3-methylbenzenesulfonyl chloride, 2,3-dichlorobenzenesulfonyl chloride, 3-chloro-2-methylbenzenesulfonyl chloride, 2-bromobenzenesulfonyl chloride. Sulfonyl chloride, 2-chloro-4-fluorobenzenesulfonyl chloride, 2-chloro-6-fluorobenzenesulfonyl chloride, 2,5-dichlorobenzenesulfonyl chloride, 2,5-dimethylbenzenesulfonyl chloride, 2-chloro-6-methylbenzenesulfonyl chloride, 3-fluoro-2-methylbenzenesulfonyl chloride, 2-chloro-4-methylbenzenesulfonyl chloride, 1,3-benzodioxane-5-sulfonyl chloride, 2-chloro-4-(trifluoromethyl)benzenesulfonyl chloride, 2-methyl-4-nitrobenzenesulfonyl chloride, 2-(difluoromethyl)benzenesulfonyl chloride.

[0379] Other sulfonyl chlorides are prepared by using or following the procedures described in the literature.

[0380] 2-Fluoro-4-methoxybenzenesulfonyl chloride:

[0381]

[0382] Following the procedure described in EP2752410A1, 2-fluoro-4-methoxyaniline (1.00 g, 7.1 mmol) was dissolved in acetonitrile (25 mL), and concentrated HCl (10 mL) was added. The mixture was cooled to 0 °C in an ice-salt bath. Then, a solution of NaNO2 (0.59 g, 8.5 mmol) in water (1 mL) was added in portions, and the mixture was stirred at 0 °C for 1.5 h (a light brown solution with a small amount of white solid suspended). AcOH (12 mL) was added to the resulting mixture, and after stirring at 0 °C for 10 min, NaHSO3 (7.37 g, 10 equivalents, 71 mmol) was added. After stirring for 5 min, copper(II) chloride (0.96 g, 1 equivalent) and CuCl (70 mg, 0.1 equivalents) were added, and the green suspension was stirred in an ice bath to allow the temperature to rise to room temperature within 1 h, after which it was stirred at room temperature for another 18 h (TLC R). f :0.45, in 2:1 hexane / EtOAc). The reaction mixture was then poured into water (100 mL) and extracted with EtOAc. The extract was washed with water, dried over MgSO4, and filtered through a silica gel pad (15 mL) using 1:1 hexane / EA as the eluent. Volatiles were removed under reduced pressure to give 1.22 g of clear, light brown oil (TLC showed the presence of more polar, unidentified impurities after aqueous post-treatment). 1¹H NMR (CDCl₃) δ: 7.88 (t, J = 8.6 Hz, 1H), 6.76–6.93 (m, 2H), 3.93 (s, 3H). Homogeneity ~70%, obtained through… 1 H NMR.

[0383] The following sulfonyl chlorides were prepared using a similar procedure:

[0384] 2-Cyano-4-fluorobenzenesulfonyl chloride: Prepared using 2-cyano-4-fluoroaniline as the starting material. The crude material is highly impure, but has been successfully used in sulfonation reactions.

[0385] 4-Methoxy-2-trifluoromethylbenzenesulfonyl chloride: Prepared using 4-methoxy-2-trifluoromethylaniline: 1 H NMR (CDCl3) δ: 8.30 (d, J = 9.0 Hz, 1H), 7.42 (d, J = 2.3 Hz, 1H), 7.19 (dd, J = 9.0, 2.7 Hz, 1H), 3.99 (s, 3H).

[0386] 4-Chloro-2-methylbenzenesulfonyl chloride:

[0387]

[0388] The product was prepared by chlorosulfonation of m-chlorotoluene according to the procedure described in Acta Crystallographica Section E 2009, 65(4), o800. m-Chlorotoluene (1 mL) was dissolved in CHCl3 (4 mL), and the solution was cooled in an ice bath. Chlorosulfonic acid (2.5 mL) was added dropwise over 15 minutes as HCl gas was slowly released. Afterward, the reaction mixture was warmed to room temperature. TLC showed no more starting material (Rf = 0.8, in 8:2 hexane / EA) and the formation of a new spot with a slight tailing (Rf = 0.7, in 8:2 hexane / EA). The reaction mixture was poured onto ice (50 mL), DCM (15 mL) was added, and the organic phase of the product was separated, washed with cold water, dried (MgSO4), and concentrated to a colorless oil (0.87 g), which was used without further purification. 1 H NMR (CDCl3) δ: 8.01 (d, J = 8.6 Hz, 1H), 7.43 (s, 1H), 7.40 (dd, J = 8.6, 2.0 Hz, 1H), 2.78 (s, 3H).

[0389] The following sulfonyl chlorides were prepared using a similar procedure with some modifications, as described below:

[0390] 4-Methoxy-2-methylbenzenesulfonyl chloride:

[0391]

[0392] 3-Methoxytoluene (6.00 g) was dissolved in CHCl3 (30 mL) and the solution was cooled to -35 °C (bath temperature). Chlorosulfonic acid (15 mL) was added dropwise over 20 minutes (no significant HCl / SO2 gas escape was observed). The clear solution was then stirred for 15 minutes at -30 °C to -25 °C (no gas escape was noted). The reaction mixture was carefully poured onto ice (50 mL), DCM (50 mL) was added, and the slightly emulsified organic phase of the product was separated, washed with cold water, dried (MgSO4), and concentrated to a colorless oil, which was then dried under vacuum (8.28 g, 76% yield). NMR showed the presence of a single isomer: 1 H NMR (CDCl3) δ: 8.01 (d, J = 8.6 Hz, 1H), 6.72-6.95 (m, 2H), 3.90 (s, 3H), 2.75 (s, 3H).

[0393] 2-Chloro-4-methoxybenzenesulfonyl chloride:

[0394]

[0395] 3-Chloroanisole (1.00 g) was dissolved in CHCl3 (4 mL) and the solution was cooled to approximately -35 °C. Chlorosulfonic acid (2.5 mL) was added dropwise to CHCl3 (2 mL) over 15 minutes. After completion, only the baseline material was observed by TLC (SM Rf = 0.8, in 8:2 hex / EA). Upon heating the reaction mixture to room temperature, gas was observed to escape, and isomers were identified by TLC (Rf = 0.30 and 0.25, in 8:2 hex / EA). After stirring at room temperature for 30 minutes, a white precipitate began to form. The reaction mixture was poured onto ice (50 mL), DCM (15 mL) was added, and the organic phase of the product was separated, washed with cold water, dried (MgSO4), and concentrated to a colorless oil that crystallized into white needle-like substances (0.89 g) upon standing. 1 1H NMR showed a mixture of two isomers in a 60:40 ratio, which were separated by silica gel rapid chromatography using 8:2 hexane / EtOAc as the eluent. Desired isomer (more polar): 1 H NMR (CDCl3) δ: 7.90 (d, J = 8.6 Hz, 1H), 7.04-7.19 (m, 2H), 4.08 (s, 3H).

[0396] 2,3-Dimethylbenzenesulfonyl chloride:

[0397]

[0398] As described in WO2003 / 055478, it is separated as a minor isomer after chlorosulfonation of o-xylene. 1 H NMR (CDCl3) δ: 7.96 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 7.4 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 2.71 (s, 3H), 2.41 (s, 3H).

[0399] 3-Fluoro-2-methyl-4-methoxybenzenesulfonyl chloride:

[0400]

[0401] Step 1: Potassium carbonate (6.58 g, 47.6 mmol) was added to a solution of 2-fluoro-3-methylphenol (4.32 mL, 39.7 mmol) in acetone (50 mL), followed by iodomethane (2.75 mL, 43.7 mmol). The reaction mixture was then refluxed overnight at 60 °C. The reaction mixture was then cooled to room temperature, filtered (washed with 2 x 10 mL acetone), and concentrated under reduced pressure. The crude product was extracted from water (30 mL) and EtOAc (2 x 50 mL). The organic layer was then separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel rapid chromatography using 0 to 5% EtOAc / hexane to give 2-fluoro-3-methylanisole as a clear, colorless liquid (5.30 g, 95% yield). 1 H NMR (CDCl3) δ: 6.95 (td, J = 8.0, 1.4 Hz, 1H), 6.85-6.71 (m, 2H), 3.87 (s, 3H), 2.28 (d, J = 2.3 Hz, 3H).

[0402] Step 2: Over a 5-minute period, add a solution of chlorosulfonic acid (1.13 mL, 16.5 mmol) in DCM (5.6 mL) to a solution of 2-fluoro-3-methylanisole (1.00 g, 7.13 mmol) from Step 1 in DCM (5.6 mL). Stir the light brown reaction mixture containing the viscous liquid layer at room temperature for 10 minutes, then quench by pouring into a mixture of water (10 mL) and ice (5 g). Extract the aqueous phase with DCM (2 × 10 mL), dry on Na₂SO₄, filter, and concentrate under reduced pressure to give the desired sulfonyl chloride as a colorless liquid (1.70 g, 100% yield). The material is used without further purification. 1 H NMR (CDCl3) δ: 7.87 (dd, J = 9.0, 1.8 Hz, 1H), 6.97-6.86 (m, 1H), 3.97 (s, 3H), 2.66 (d, J = 2.8 Hz, 3H).

[0403] 3-Chloro-2-methyl-4-methoxybenzenesulfonyl chloride:

[0404]

[0405] Following a procedure similar to that for 3-fluoro-2-methyl-4-methoxybenzenesulfonyl chloride (step 1) and starting with 2-chloro-3-methylphenol, 2-chloro-3-methylanisole, as a colorless liquid, was obtained in quantitative yield: 1 H NMR (CDCl3) δ: 7.12 (t, J = 7.9 Hz, 1H), 6.87-6.83 (m, 1H), 6.79 (d, J = 8.2 Hz, 1H), 3.89 (s, 3H), 2.38 (s, 3H).

[0406] Treatment with chlorosulfonic acid (step 2) as described for 3-fluoro-2-methyl-4-methoxybenzenesulfonyl chloride yielded the desired 3-chloro-2-methyl-4-methoxybenzenesulfonyl chloride as a colorless liquid in 96% yield. 1 H NMR (CDCl3) δ: 8.02 (d, J = 9.1 Hz, 1H), 6.90 (d, J = 9.1 Hz, 1H), 4.00 (s, 3H), 2.83 (s, 3H).

[0407] 2-Ethylbenzenesulfonyl chloride:

[0408]

[0409] Dissolve 2-ethylthiophenol (1.46 mL, 10.3 mmol) and KCl (776 mg, 10.3 mmol) in water (38 mL) and add in small portions. (15.8 g, 25.8 mmol). After stirring at room temperature for 1 hour, the reaction was considered complete by LCMS analysis, and the reaction mixture was extracted with EtOAc (4 x 5 mL). The extract was dried (Na2SO4) and concentrated under reduced pressure to give a white crystalline solid (1.43 g, 68% yield), which was used as follows: 1 H NMR (CDCl3) δ: 8.07 (dd, J=8.1, 1.3Hz, 1H), 7.66 (td, J=7.6, 1.3Hz, 1H), 7.49 (d, J=7.7Hz, 1H), 7.45-7.38 (m, 1H), 3.20 (q, J=7.5Hz), 1.36 (t, J=7.5Hz).

[0410] 3-Fluoro-2-ethylbenzenesulfonyl chloride:

[0411]

[0412] Step 1: Dissolve 2-bromo-6-fluorobenzaldehyde (6.00 g, 29.5 mmol) in anhydrous THF (60 mL) and cool the solution to -78 °C under an argon atmosphere. Add methyl magnesium bromide (3.0 M solution in diethyl ether, 13.4 mL, 40.3 mmol) dropwise and stir the mixture at -78 °C for 30 minutes. Then quench the reaction mixture with 10% hydrochloric acid (50 mL) and extract the product into diethyl ether (2 × 50 mL). Dry the extract (MgSO4) and concentrate it, and pass the residue through... Purification was performed on silica gel using 10%–30% EtOAc / hexane as the eluent to give the desired alcohol derivative as a colorless oil (6.20 g, 96% yield): 1 H NMR (CDCl3) δ: 7.35 (ddd, J = 7.9, 3.1, 2.0 Hz, 1H), 7.16-6.99 (m, 2H), 5.35 (q, J = 6.7 Hz, 1H), 1.61 (dd, J = 6.8, 1.1 Hz, 3H).

[0413] Step 2: Indium(III) chloride (412 mg, 1.83 mmol) was suspended in DCM (40 mL) and diisopropylsilane (8.42 mL, 49.3 mmol) was added. The alcohol from Step 1 (4.00 g, 18.3 mmol) was added to DCM (8 mL), and the mixture was stirred at RT for 3 h to obtain a clear solution. The reaction mixture was quenched with water (50 mL), extracted with diethyl ether (3 × 20 mL), washed with brine, and dried (MgSO4). The silyl ether of the starting alcohol was obtained by rapid chromatographic concentration and purification using hexane as the eluent.

[0414] The substance was dissolved in DCE (41 mL), and dichloroisopropylsilane (0.78 mL, 4.6 mmol) and indium(III) chloride (103 mg, 4.6 mmol) were added. The mixture was stirred at 80 °C for 3 hours. After cooling to ambient temperature, the reaction mixture was diluted with hexane (100 mL), washed with water (100 mL), and the aqueous phase was back-extracted with hexane (2 x 50 mL). The combined organic phases were dried (Na₂SO₄) and concentrated to give a colorless oil, which was purified by silica gel rapid chromatography using hexane as the eluent to give 2-bromo-6-fluoroethylbenzene (3.71 g, 100%) as a colorless oil. 1 H-NMR (CDCl3) δ: 7.32 (d, J = 7.8 Hz, 1H), 7.11-6.91 (m, 2H), 2.82 (dq, J = 7.5, 2.2 Hz, 2H), 1.20-1.15 (m, 3H).

[0415] Step 3: The aryl bromide (3.71 g, 18.3 mmol) from Step 2 was dissolved in toluene (60 mL) and N,N-diisopropylethylamine (6.40 mL, 36.5 mmol) was added. The solution was then degassed by three cycles of evacuation and nitrogen backfilling. Tris(dibenzylacetone)dipalladium(0) (836 mg, 0.9 mmol), 4,4-bis(diphenylphosphino)-9,9-dimethylxanthene (1.08 g, 1.83 mmol), and 2-ethylhexyl-3-mercaptopropionate (4.59 mL, 19.2 mmol) were added, and the mixture was degassed twice more and then refluxed overnight under nitrogen. The reaction mixture was then cooled to room temperature, quenched with water (50 mL), and extracted with EtOAc (2 × 50 mL). The combined organic phases were washed with 10% HCl aqueous solution (75 mL) and dried (Na2SO4). The residue was concentrated under reduced pressure and purified by rapid chromatography using 0–15% EtOAc / hexane as eluent to give the desired sulfide intermediate (4.00 g), which was an orange oily substance contaminated with some unreacted starting thiol. This material was used as is for the next step.

[0416] Step 4: Dissolve the crude sulfide derivative from Step 3 (4.00 g, assumed 11.7 mmol) in THF (41 mL) and add potassium tert-butoxide (1.0 M in THF, 14.1 mL, 14.1 mmol) dropwise. Stir the resulting solution at room temperature for 1 hour. Then quench the reaction mixture by adding saturated NH4Cl aqueous solution (40 mL) and extract with EtOAc (2 x 30 mL). Concentrate the combined organic phases and wash the deep orange residue through a small silica gel pad with hexane to give a mixture of thiols and disulfides (1.50 g), which is used as is for the next step (Note: foul odor).

[0417] Step 5: Suspend the crude mixture of thiophenol and disulfide from Step 4 (1.50 g, assumed to be 9.6 mmol) and KCl (723 mg (9.6 mmol)) in water (40 mL) and add in batches. (14.8 g, 24 mmol). After stirring at room temperature for 1 hour, the reaction mixture was extracted with EtOAc (2 × 20 mL), and the extract was dried (Na2SO4) and concentrated under reduced pressure to give crude sulfonyl chloride, which was used as is to prepare the corresponding fragments A-5 and aniline A-8 (see Table 1).

[0418] 3-Chloro-2-ethylbenzenesulfonyl chloride:

[0419]

[0420] The sulfonyl chloride was prepared using the same procedure as for 3-fluoro-2-ethylbenzenesulfonyl chloride, but starting with 2-bromo-6-chlorobenzaldehyde:

[0421] Step 1 (98% yield, white solid): 1 H NMR (CDCl3) δ: 7.49 (dd, J = 8.0, 1.2 Hz, 1H), 7.33 (dd, J = 8.0, 1.2 Hz, 1H), 7.05 (t, J = 8.0 Hz, 1H), 5.58 (q, J = 6.9 Hz, 1H), 1.64 (d, J = 6.9 Hz, 3H).

[0422] Step 2 (100% yield, colorless oily substance): 1 H-NMR (CDCl3) δ: 7.43 (dd, J = 8.0, 1.2 Hz, 1H), 7.29 (dd, J = 8.0, 1.2 Hz, 1H), 6.96 (t, J = 8.0 Hz, 1H), 2.97 (q, J = 7.5 Hz, 2H), 1.17 (t, J = 7.5 Hz, 3H).

[0423] Step 3 (81% yield, orange oily substance): 1 H-NMR(CDCl3)δ:7.24-7.18(m,2H),7.08(t,J=7.9Hz,1H),4.07-3.96(m,2H),3.17(t,J=7.4Hz,2H),2.96(q,J=7.5Hz,2 H), 2.64 (t, J = 7.4Hz, 2H), 1.56 (dd, J = 11.9, 5.8Hz, 2H), 1.40-1.21 (m, 9H), 1.16 (t, J = 7.5Hz, 3H), 0.89 (t, J = 7.4Hz, 6H).

[0424] Step 4 (99% yield, colorless liquid): 1 H-NMR (CDCl3) δ: 7.15 (d, J = 7.9Hz, 2H), 6.97-6.92 (m, 1H), 3.41 (s, 1H), 2.87 (q, J = 7.5Hz, 2H), 1.18 (t, J = 7.5Hz, 3H).

[0425] Step 5 (crude material used without purification): 1 H-NMR (CDCl3) δ: 8.03 (dd, J = 8.2, 1.3Hz, 1H), 7.73 (dd, J = 8.0.1.3Hz, 1H), 7.37 (t, J = 8.1Hz, 1H), 3.30 (q, J = 7.4Hz, 2H), 1.33 (t, J = 7.4Hz, 3H).

[0426] 2-Methyl-3-(trifluoromethyl)benzenesulfonyl chloride:

[0427]

[0428] The sulfonyl chloride was prepared using the same procedure as for 3-fluoro-2-ethylbenzenesulfonyl chloride, but starting with commercially available 2-methyl-3-(trifluoromethyl)bromobenzene:

[0429] Step 3 (100% yield, orange oily substance): 1 H-NMR(CDCl3)δ:7.49(d,J=7.9Hz,2H),7.26-7.19(m,1H),4.06-4.00(m,2H),3.18(dd,J=9.1,5.6Hz,2 H), 2.65 (dd, J = 9.2, 5.6Hz, 2H), 2.50 (d, J = 1.3Hz, 3H), 1.33-1.23 (m, 11H), 0.88 (td, J = 7.4, 2.3Hz, 6H).

[0430] Step 4 (quantitative yield, colorless oily substance): 1 H-NMR (CDCl3) δ: 7.43 (dd, J = 7.9, 2.2 Hz, 2H), 7.12 (t, J = 7.8 Hz, 1H), 3.42 (s, 1H), 2.43 (s, 3H).

[0431] Step 5 (quantitative yield, white solid): 1 H-NMR (CDCl3) δ: 8.31 (d, J = 8.1 Hz, 1H), 8.00 (t, J = 7.7 Hz, 1H), 7.55 (dd, J = 15.6, 7.6 Hz, 1H), 2.93 (s, 3H).

[0432] General procedure for preparing sulfonyl chloride from aryl bromide:

[0433]

[0434] Step 1: Dilute aryl bromide 1 (1.00 mmol) in toluene (1.70 mL). Degas the mixture by bubbling it through the solution with nitrogen for 5 minutes. Add tris(dibenzylacetone)-dipalladium(0) (0.02 mmol), 4,4-bis(diphenylphosphino)-9,9-dimethylxanthene (0.04 mmol), and N,N-diisopropylethylamine (2.0 mmol), and then degas the mixture again for 5 minutes. Then add benzyl mercaptan (1 mmol) and heat the resulting mixture under reflux overnight (oil bath T = 115 °C). After completion, cool the reaction mixture to room temperature, dilute with EtOAc (20 mL), and quench with H2O (20 mL). Extract the aqueous layer with EtOAc (2 × 20 mL). Wash the combined organic layers with brine (50.0 mL), dry (Na2SO4), filter, and concentrate under reduced pressure. The crude product was further purified by rapid chromatography (0-10% EtOAc / hexane, 35 mL / min, product eluted in 100% hexane). The target fraction was collected and concentrated under reduced pressure to give title compound 2.

[0435] Step 2: Compound 2 (1.00 mmol) was dissolved in acetic acid (1.90 mL) and H₂O (0.65 mL) was added to provide a heterogeneous solution. N-chlorosuccinimide (4.00 mmol) was added in portions. The reaction mixture was stirred and monitored by LCMS (sample quenched with N-methylpiperazine). After the reaction was complete, the mixture was concentrated under reduced pressure. The resulting mixture was slowly poured into a saturated aqueous solution of NaHCO₃, resulting in gas release. The mixture was extracted with EtOAc (2 x 75 mL). The combined organic layers were washed with brine, dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The crude compound was further purified by normal-phase chromatography (0–40% EtOAc / hexane, 60 mL / min, product appears in 100% hexane). The target fraction was collected and concentrated under reduced pressure to give title compound 3.

[0436] 2-Chloro-3-methylbenzenesulfonyl chloride:

[0437]

[0438] Sulfonyl chlorides are prepared from commercially available 1-bromo-2-chloro-3-methylbenzene using a standard procedure.

[0439] Benzyl (2-chloro-3-methylphenyl) sulfide: yellow solid, 51% yield, 95% purity (at 220 nm). (ES) - MH = 247.2; 1H NMR (400MHz, CDCl3) δ7.39-7.35(m,2H),7.33-7.28(m,2H),7.28-7.23(m,1H+CDCl3),7.11-7.03(m,3H),4.15(s,2H),2.38(s,3H).

[0440] 2-Chloro-3-methylbenzenesulfonyl chloride: Pale yellow oil, 70% yield, 60% purity (at 254 nm). LCMS: LCMS sample was quenched with N-methylpiperazine (resulting in sulfonamide MW = 288.8), (ES... + M+H=289.2. Used as a crude product.

[0441] 3-Fluoro-2-(trifluoromethyl)benzenesulfonyl chloride:

[0442]

[0443] Sulfonyl chlorides are prepared from commercially available 1-bromo-3-fluoro-2-(trifluoromethyl)benzene using a standard procedure:

[0444] Benzyl (3-fluoro-2-(trifluoromethyl)phenyl) sulfide: yellow oil, 66% yield, 98% purity (at 254 nm). (ES) - MH = 285.2.

[0445] 3-Fluoro-2-(trifluoromethyl)benzenesulfonyl chloride: pale yellow oil, 93% yield, 98% purity (at 254 nm). The LCMS sample was quenched with N-methylpiperazine (resulting in a sulfonamide MW = 326.1), (ES... + M+H=327.1.

[0446] 3-Chloro-2-(trifluoromethyl)benzenesulfonyl chloride:

[0447]

[0448] Sulfonyl chlorides are prepared from commercially available 1-bromo-3-chloro-2-(trifluoromethyl)benzene according to a general procedure:

[0449] Benzyl (3-chloro-2-(trifluoromethyl)phenyl) sulfide: white solid, 59% yield, 90% purity (at 220 nm). (ES) - MH = 301.2; 1 H NMR (400MHz, CDCl3) δ7.46-7.14 (m, 8H+CDCl3), 4.16 (s, 2H).

[0450] 3-Chloro-2-(trifluoromethyl)benzenesulfonyl chloride: colorless oil, 66% yield. LCMS: LCMS sample quenched with N-methylpiperazine (resulting in sulfonamide MW = 343.5), (ES + M+H=343.2. Used as a crude product.

[0451] 3,4-Difluoro-2-methylbenzenesulfonyl chloride:

[0452]

[0453] Sulfonyl chlorides are prepared from commercially available 1-bromo-3-chloro-2-(trifluoromethyl)benzene according to a general procedure:

[0454] Benzyl (3,4-difluoro-2-methylphenyl) sulfide: orange oil, 96% yield, 96% purity (at 254 nm). (ES) - MH = 249.2; 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 7.32–7.17 (m, 7H), 4.16 (s, 2H), 2.18 (d, J = 2.7 Hz, 3H). 3,4-Difluoro-2-methylbenzenesulfonyl chloride: pale yellow oil, 49% yield. LCMS: LCMS sample quenched with N-methylpiperazine (resulting sulfonamide MW = 290.3), (ES⁻) + M + H = 291.2; 1 H NMR (400MHz, DMSO-d6) δ7.56 (ddd, J=8.4, 5.5, 1.8Hz, 1H), 7.15 (dd, J=18.4, 8.4Hz, 1H), 2.46 (d, J=2.8Hz, 3H).

[0455] 2,4-Dimethyl-3-fluorobenzenesulfonyl chloride:

[0456]

[0457] Sulfonyl chlorides are prepared from commercially available 1-bromo-2,4-dimethyl-3-fluorobenzene using standard procedures:

[0458] Benzyl (3-fluoro-2,4-dimethylphenyl) sulfide: Orange oil, crude product with a 95% yield and 80% purity (at 254 nm), used as crude product.

[0459] 3-Fluoro-2,4-Dimethylbenzene-1-sulfonyl chloride: Orange oil, crude product in 89% yield, 74% purity (at 254 nm). LCMS: LCMS sample quenched with N-methylpiperazine (resulting in sulfonamide MW = 286.4), (ES... +M+H=287.1. Used as a crude product.

[0460] 2-Methylpyridine-3-sulfonyl chloride:

[0461]

[0462] The preparation of sulfonyl chlorides begins with commercially available 3-bromo-2-methylpyridine following a standard procedure:

[0463] 3-(benzylthio)-2-methylpyridine: orange liquid, 88% yield, 94% purity (at 220 nm). (ES) + M+H=215.8,(ES) - MH = 214.1. 1 H NMR (400MHz, DMSO-d6) δ8.30(dd,J=4.9,1.5Hz,1H),7.57-7.45(m,1H),7.31-7.27(m,5H),7.07(dd,J=7.6,5.1Hz,1H),4.09(s,2H),2.58(s,3H).

[0464] 2-Methylpyridine-3-sulfonyl chloride: pale yellow oil, 100% yield, 95% purity (at 254 nm). The LCMS sample was diluted with H₂O (resulting in sulfonic acid MW = 173.1) (ES). - MH = 171.9; 1 H NMR (400MHz, CDCl3) δ8.80 (dd, J=4.8, 1.6Hz, 1H), 8.33 (dd, J=8.1, 1.7Hz, 1H), 7.43-7.36 (m, 1H), 3.02 (s, 3H).

[0465] 6-Methoxy-4-methylpyridine-3-sulfonyl chloride:

[0466]

[0467] Preparation of 2-ethylhexyl 3-((6-methoxy-4-methylpyridin-3-yl)thio)propionate (2): 5-bromo-2-methoxy-4-methylpyridine (6.00 g, 29.7 mmol) was dissolved in toluene (100 mL), and N,N-diisopropylethylamine (10.4 mL, 59.4 mmol) was added. The mixture was degassed by bubbling the solution through nitrogen for 5 minutes. Tris(dibenzylacetone)dipalladium (0) (1.36 g, 1.49 mmol), 4,4-bis(diphenylphosphino)-9,9-dimethylxanthene (1.75 g, 2.97 mmol), and 2-ethylhexyl-3-mercaptopropionate (7.47 mL, 31.2 mmol) were added. The mixture was degassed again for 5 minutes. The mixture was heated under reflux overnight (oil bath T = 117 °C). The reactants were cooled to room temperature, diluted with EtOAc (100 mL), and quenched with H2O (100 mL). The organic and aqueous layers were separated, and the aqueous layer was extracted with EtOAc (2 x 50.0 mL). The combined organic layers were washed with HCl (10% in H2O, 50.0 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude product was purified by rapid chromatography (330 g silica gel column, EtOAc-hexane, 0-20%) to give the title compound as an orange oil (10.0 g, 99% yield). (ES) + M + H = 340.2; 1 HNMR(400MHz, CDCl3)δ8.19(s,1H),6.64(s,1H),3.99(dd,J=5.9,2.4Hz,2H),3.93(s,3H),2.96(t,J=7.3Hz,2 H), 2.55 (t, J = 7.3Hz, 2H), 2.42 (d, J = 0.5Hz, 3H), 1.56 (dt, J = 12.1, 6.0Hz, 1H), 1.39-1.22 (m, 8H), 0.88 (m, 6H).

[0468] Preparation of 6-methoxy-4-methylpyridine-3-thiol (3): Potassium tert-butoxide (1.00 M in THF, 35.3 mL, 35.3 mmol) was added dropwise to a solution of 2 (10.0 g, 29.5 mmol) in THF (105 mL) at -78 °C, and a precipitate was formed. The resulting suspension was stirred at -78 °C for 30 min. The reaction mixture was quenched by adding NH4Cl (50.0 mL) and the mixture was extracted with CH2Cl2 (2 x 50.0 mL). The combined organic layers were dried (Na2SO4), filtered, and concentrated under reduced pressure to give a deep orange liquid. The crude product was purified by rapid chromatography (100% hexane) to give a mixture (4.56 g) of the title compound and its disulfide, which was used in the next step without further purification. LCMS: Thiol 3: (ES +M+H=156.9 and disulfides: (ES) + M+H=309.0.

[0469] Preparation of 6-methoxy-4-methylpyridine-3-sulfonyl chloride (4): Potassium chloride (2.21 g, 29.3 mmol) was added to a mixture of thiol 3 (4.56 g, 29.4 mmol) in H2O (123 mL), followed by addition in batches. (45.2 g, 73.5 mmol). After the reaction was complete (1 h), the mixture was extracted with EtOAc (2 × 20.0 mL), and the combined organic layers were dried (Na2SO4) and concentrated under reduced pressure. The crude product obtained was used without any further purification.

[0470] 3-Methylpyridine-4-sulfonyl chloride:

[0471]

[0472] Step 1: Dissolve 4-bromopyridine (1.00 mmol) in toluene (1.70 mL) and add N,N-diisopropylethylamine (2.00 mmol). Degas the mixture by bubbling it through the solution with nitrogen for 5 minutes. Add tris(dibenzylacetone)-dipalladium(0) (0.02 mmol), 4,4-bis(diphenylphosphino)-9,9-dimethylxanthione (0.04 mmol), and phenylmethanethiol / benzylthiol (1.00 mmol). Degas the mixture again for 5 minutes. Heat the mixture under reflux for 18 h (oil bath T = 115 °C). Cool the reactants to room temperature, dilute with EtOAc (10.0 mL), and quench with H2O (10.0 mL). The aqueous and organic layers were separated, and the aqueous layer was extracted with EtOAc (2 x 10.0 mL). The combined organic phases were washed with HCl (10% in H2O, 10.0 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude product was purified by rapid chromatography (EtOAc-hexane, 10% to 35%) to give sulfide 2 (90% yield). (ES) + M+H=216.1; 1 H NMR (400MHz, CDCl3) δ8.28 (d, J = 4.6, 1H), 8.24 (s, 1H), 7.43-7.27 (m, 5H), 7.20 (t, J = 5.4Hz, 1H), 4.25 (s, 2H), 2.28 (s, 3H).

[0473] Step 2: Dissolve compound 2 (1.00 mmol) in CH2Cl2 (11.5 mL) and cool to -10 °C. Add HCl (1.00 M in H2O, 5.70 mL) and stir at -10 °C for 5 minutes. Add sodium hypochlorite (10% solution in H2O, 3.00 mmol) over 10 minutes, keeping the temperature below 0 °C. Stir the mixture at 0 °C for 10 minutes. Separate the organic and aqueous layers. Dry the organic layer (Na2SO4). The crude sulfonyl chloride can be used directly for the next step without further purification or evaporation: Quench the LCMS sample with N-methylpiperazine (resulting in sulfonamide MW = 255.3); (ES + M+H + =256.2.

[0474] 2,3-Dimethylpyridine-4-sulfonyl chloride:

[0475]

[0476] Using the same procedure as with 3-methylpyridine-4-sulfonyl chloride, begin with 2,3-dimethyl-4-bromopyridine:

[0477] Step 1: 4-(benzylthio)-2,3-dimethylpyridine (2): (92% yield). LCMS: (ES + M + H = 230.2; 1 HNMR (400MHz, CDCl3) δ: 8.17 (d, J = 5.5 Hz, 1H), 7.42-7.28 (m, 5H), 6.99 (d, J = 5.5 Hz, 1H), 4.18 (s, 2H), 2.53 (s, 3H), 2.24 (s, 3H).

[0478] Step 2: 2,3-Dimethylpyridine-4-sulfonyl chloride (3): The LCMS sample was quenched with N-methylpiperazine (the resulting sulfonamide MW = 269.3); (ES + M+H + =270.2.

[0479] Protected sulfonyl chloride fragment B69:

[0480]

[0481] Step 1: Add 20 mL of saturated NaHCO3 aqueous solution to a solution of commercially available aminopyridine (1.00 g, 4.27 mmol) and N-benzylcarbamoyl chloride (0.9421 g, 5.5546 mmol) in EtOAc (20 mL). Stir the solution at room temperature for 16 h. After stirring, add EtOAc to the reaction mixture, separate the organic layer, wash with brine, dry with MgSO4, filter, and concentrate. Adsorb the residue onto SiO2, then purify it on SiO2 with EtOAc in hexane to obtain the desired protected aminopyridine (1.00 g, 2.72 mmol, 64%). 1 H NMR (400MHz, DMSO-d6) δ: 10.35 (s, 1H), 8.09 (d, J = 8.61Hz, 1H), 7.47 (d, J = 9.00Hz, 1H), 7.23-7.44 (m, 4H), 5.16 (s, 2H), 2.53 (s, 3H) MS m / z 369.2 (MH) + ).

[0482] Step 2: The degassed solution of iodopyridine (0.61 g, 1.66 mmol), tris(dibenzylacetone)-dipalladium(O)chloroform adduct (86 mg, 0.0828 mmol), 9,9-dimethyl-9h-xanthon-4,5-diyl)bis(diphenylphosphine) (96 mg, 0.166 mmol), DIPEA (0.576 mL, 3.31 mmol), and benzyl mercaptan (0.233 mL, 1.99 mmol) from Step 1 in toluene (15 mL) was stirred at 115 °C for 3 h under N2. After completion, SiO2 was added to the reaction mixture and the mixture was concentrated under vacuum. The residue was purified on a SiO2 column using EtOAc in hexane to provide the desired sulfide (560 mg, 93%). 1 H NMR(400MHz, CDCl3)δ:7.71(d,J=8.61Hz,1H),7.54(d,J=8.61Hz,1H),7.46(br.s.,1H),7.3 1-7.43(m,5H),7.19-7.26(m,2H),7.12-7.19(m,2H),5.22(s,2H),3.96(s,2H),2.41(s,3H). MS m / z365.2(MH + ).

[0483] Step 3: Add N-chlorosuccinimide (330 mg, 2.47 mmol) to a solution of the sulfide (300 mg, 0.823 mmol) from Step 2 in 16 mL of water containing 90% AcOH. Stir the reaction mixture at room temperature for 3 hours. Evaporate the reaction mixture to dryness, then dilute in EtOAc and wash with water, followed by washing with brine. Dry the organic layer over MgSO4, filter, and concentrate under vacuum to obtain the desired sulfonyl chloride group B49, which is used as is (282 mg, 99%). 1 H NMR (400MHz, CDCl3) δ: 8.28 (d, J = 9.00Hz, 1H), 8.02 (d, J = 9.00Hz, 1H), 7.72 (br s, H), 7.34-7.60 (m, 5H), 5.27 (s, 2H), 2.86 (s, 3H). MS m / z 341.2(MH + ).

[0484] 2,2-Difluorobenzo[d][1,3]m-dioxacyclopentene-4-sulfonyl chloride:

[0485]

[0486] Thionyl chloride (5.96 mL) was added dropwise to water (30 mL) over 20 minutes, and the mixture was stirred at room temperature for 48 hours to produce a solution containing sulfur dioxide. In a separate container, 2,2,-difluorobenzo[d][1,3]-m-dioxane-4-amine (1.00 g, 5.78 mmol) was added dropwise to ice-cooled concentrated HCl (7 mL) to produce a white precipitate. A solution of sodium nitrite (523 mg, 7.5 mmol) in water (2 mL) was added dropwise to aniline hydrochloride over 5 minutes to produce an orange reaction mixture. The orange suspension was then gradually added at 5 °C to a solution of sulfur dioxide from the above mixture, to which 10 mg of cuprous chloride had previously been added. The mixture was stirred in an ice bath for another 2 hours (gas was observed to escape and orange liquid was observed to settle at the bottom of the flask). After confirmation by LCMS analysis, the reaction mixture was extracted with DCM (2 x 20 mL), dried (Na2SO4), filtered, and concentrated to give the desired sulfonyl chloride as an orange oil, which could be used directly without further purification (100% crude yield): 1H-NMR (400 MHz, CDCl3) δ 7.65 (dd, J = 8.4, 1.1 Hz, 1H), 7.44 (dd, J = 8.1, 1.1 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H).

[0487] 4-Chloro-3-fluoro-2-methylbenzenesulfonyl chloride:

[0488]

[0489] Preparation of N-(3-fluoro-2-methylphenyl)neopentamide (2): Triethylamine (14.9 mL, 106 mmol) was added to a solution of 3-fluoro-2-methylaniline (10.9 mL, 93.0 mmol) in THF (240 mL) at 0 °C over 10 minutes, followed by neopentanoyl chloride (13.1 mL, 105 mmol). The mixture was heated to room temperature and stirred for 2 hours. The evaporation was evaporated under reduced pressure, and the residue was partitioned between H2O (250 mL) and EtOAc (150 mL). The organic and aqueous layers were separated. The aqueous layer was extracted with EtOAc (4 x 60 mL). The combined organic layers were washed with brine (60 mL), dried (Na2SO4), and concentrated under reduced pressure to give title compound 2 as a solid (18.5 g, 95% yield). (ES) + M+H=210.2.

[0490] Preparation of N-(4-chloro-3-fluoro-2-methylphenyl)neopentamide (3): N-chlorosuccinimide (2.76 g, 20.1 mmol) was added to a DMF (50.0 mL) solution of compound 2 (4.20 g, 20.1 mmol) over 10 minutes at room temperature (in 3 fractions). The mixture was heated at 80 °C for 90 minutes. Additional N-chlorosuccinimide (541 mg, 4.01 mmol) was added and stirred at 80 °C for 45 minutes. The mixture was cooled to room temperature and diluted with EtOAc (30 mL) and water (60 mL). The organic and aqueous layers were separated. The aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with H2O (3 x 30 mL) and brine (20.0 mL), dried (Na2SO4), and concentrated under reduced pressure to give the crude compound (5.20 g). The crude product was dissolved in cyclohexane (30 mL) and heated at 45–50 °C until all solids dissolved. The solution was cooled to room temperature. The precipitated white solid was filtered off and washed with cyclohexane (3 x 5 mL) to give title compound 3 as a solid (1.95 g, 40% yield). (ES) + M+H=244.1.

[0491] Preparation of 4-chloro-3-fluoro-2-methylaniline (4): HCl (6.00 M in water, 23 mL, 138 mmol) was added to a solution of compound 3 (1.60 g, 6.57 mmol) in dioxane (18 mL) for 5 min at room temperature. The mixture was heated at 100 °C for 20 h. The mixture was cooled to room temperature. Solid K₂CO₃ was added in portions (exothermic) until pH 8-9 was obtained. The mixture was extracted with EtOAc (4 x 20 mL). The combined organic layers were washed with brine (20 mL), dried (Na₂SO₄), and concentrated under reduced pressure to give 1.6 g of crude product, which was dried under vacuum for 24 h to give title compound 4 (850 mg, 81% yield) as an oil. It was used in the next reaction without further purification. 1 H NMR (400MHz, CDCl3) δ6.99 (t, J = 8.3Hz, 1H), 6.40 (d, J = 8.6Hz, 1H), 2.22-2.06 (m, 3H).

[0492] Preparation of 4-chloro-3-fluoro-2-methylbenzene-1-sulfonyl chloride (5): Thionyl chloride (29.1 mL, 395 mmol) was added dropwise to H2O (92.1 mL) over 20 minutes under ice-cooling. The sulfur dioxide-containing solution was stirred at 0 °C for 2 hours and then at room temperature for 18 hours. Separately, concentrated HCl (23 mL) was added in portions to compound 4 (3.00 g, 18.8 mmol) at 0 °C to give a beige precipitate. This was stirred at 0 °C for 5 minutes. A solution of sodium nitrite (1.70 g, 24.4 mmol) in H2O (2 mL) was added dropwise over approximately 10 minutes. The sulfur dioxide solution containing copper chloride (I) (38.4 mg, 376 μmol) mentioned above was gradually added to the reaction mixture over 40 minutes at 5 °C. The mixture was further stirred for 2 hours under ice-cooling and then at room temperature for 4 days. Dilute the mixture with CH2Cl2 (20 mL). Separate the aqueous and organic layers. Extract the aqueous layer with CH2Cl2 (3 x 20 mL). Dry the combined organic layers (Na2SO4), filter, and concentrate to give crude compound 5 as an oil (1.95 g, 30% yield, 70% purity). Crude compound 5 was used as is without further purification. LCMS: Quench the LCMS sample with N-methylpiperazine (resulting sulfonamide MW = 306.784); (ES + M + H = 307.1; 1 H NMR (400MHz, CDCl3) δ7.81 (d, J = 8.4Hz, 1H), 7.46 (t, J = 7.5Hz, 1H), 2.69 (s, 3H).

[0493] 3-Methyl-2-phenylthiosulfonyl chloride:

[0494]

[0495] As described in U.S. Patent 3,991,081, it is prepared by chlorosulfonation of 3-methylthiophene. 1 H NMR (CDCl3) δ: 7.67 (d, J = 5.1 Hz, 1H), 7.03 (d, J = 5.1 Hz, 1H), 2.63 (s, 3H).

[0496] 3-Chloro-2-phenylthiosulfonyl chloride:

[0497]

[0498] 3-Chlorothiophene (1.00 g) was dissolved in CHCl3 (10 mL), and the solution was cooled to -30 °C. Chlorosulfonic acid (2.4 mL) was added dropwise over 5 minutes (no noticeable gas escape). The orange-brown solution was then stirred for 30 minutes, the temperature was raised to -10 °C, and then to room temperature over another 30 minutes. The reaction mixture was then stirred at room temperature for 2 hours (no gas escape was observed, and TLC showed product formation (Rf = 0.4, in 8:2 hex / EA)). The reaction mixture was poured onto ice (50 mL), DCM (25 mL) was added, and the organic phase of the emulsion was separated, washed with cold water, dried (MgSO4), and concentrated to a yellow oil, which was then dried under vacuum without further purification and used (0.55 g). 1 H NMR (CDCl3) δ: 7.76 (d, J = 5.7 Hz, 1H), 7.16 (d, J = 5.7 Hz, 1H).

[0499] 4-Chloro-3-methylthiophene-2-sulfonyl chloride:

[0500]

[0501] Preparation of 3-chloro-4-methylthiophene: In a 100 mL flask, 5.76 g (56.5 mmol) of copper chloride (I) was added to 3-bromo-4-methylthiophene (3.16 mL, 28.2 mmol) in DMF (20.1 mL) at room temperature. The mixture was heated in an oil bath at 160 °C for 24 hours. The crude product was poured onto H2O (50 mL). The resulting mixture was stirred at room temperature for 10 minutes. The resulting brownish-green solid was filtered and washed with water (3 x 10.0 mL) and Et2O (4 x 10.0 mL). The filtrate was extracted with Et2O (3 x 25.0 mL). The combined organic layers were washed with H2O (2 x 20.0 mL) and brine (20.0 mL), dried (Na2SO4), and concentrated under reduced pressure to give an orange oil (0.890 g, 59% yield of crude product). 1 H NMR (400MHz, CDCl3) δ7.09 (d, J = 3.5Hz, 1H), 6.99-6.95 (m, 1H), 2.22-2.21 (m, 3H).

[0502] Preparation of 4-chloro-3-methylthiophene-2-sulfonyl chloride: 3-chloro-4-methylthiophene (1.00 g, 7.54 mmol) was dissolved in CHCl3 (4.43 mL), and a solution of chlorosulfonic acid (1.19 mL, 17.3 mmol) in CHCl3 (1.48 mL) was added over 5 minutes at room temperature. The mixture was stirred for 10 minutes. Phosphorus pentachloride (4.13 g, 18.9 mmol) was added to the reaction mixture, followed by CHCl3 (7.50 mL). The mixture was heated at 50 °C for 1 hour. The reaction mixture was slowly added to an aqueous solution of NaHCO3 + ice (30 mL). The mixture was stirred for 10 minutes. Extraction was performed with CH2Cl2 (4 x 10 mL). The combined organic layers were dried (Na2SO4) and concentrated under reduced pressure to give the title compound as an oil (1.30 g, 30% yield, 40% purity). It was used in the next reaction without further purification. LCMS: The LCMS sample was quenched with N-methylpiperazine; (ES) + M+H=295.1; 1 H NMR (400MHz, CDCl3) δ7.57 (s, 1H), 2.57 (s, 3H).

[0503] 3,4-Dichlorothiophene-2-sulfonyl chloride:

[0504]

[0505] Preparation of 3,4-dichlorothiophene: In a 100 mL flask, 13.9 g (137 mmol) of copper chloride (I) was added to 3,4-dibromothiophene (5.03 mL, 45.5 mmol) in 32 mL of DMF at room temperature. The mixture was heated in an oil bath at 160 °C for 24 hours. The crude product was poured onto 100 mL of H₂O and diluted with 60 mL of Et₂O. The mixture was stirred at room temperature for 10 minutes. The resulting brownish-green solid was filtered, washed with 3 x 20 mL of H₂O, and then with 4 x 20 mL of Et₂O. The filtrate was extracted with 4 x 30 mL of Et₂O. The combined organic layers were washed with 2 x 30 mL of H₂O and 30 mL of brine, dried (MgSO₄), and concentrated under reduced pressure to give title compound 2 (5.50 g, 79% yield) as a red oil. 1 H NMR (400MHz, CDCl3) δ7.21 (s, 2H).

[0506] Preparation of 3,4-dichlorothiophene-2-sulfonyl chloride: A solution of chlorosulfonic acid (757 μL, 11.0 mmol) in 2 mL of CHCl3 was added to a solution of compound 2 (1.61 g, 10.5 mmol) in 5.98 mL of CHCl3 over 5 minutes. The mixture was stirred at room temperature for 20 minutes. Phosphorus pentachloride (5.77 g, 26.3 mmol) was added to the mixture in four portions. The mixture was heated at 50 °C for 18 hours. Volatiles were removed under reduced pressure, and the residue was dissolved in CH2Cl2 (25 mL) and washed with saturated aqueous NaHCO3 solution (3 x 15 mL), H2O (3 x 10 mL), and brine (10 mL). The organic layer was dried (Na2SO4) and concentrated under reduced pressure to give title compound 3 (2.32 g, 88% yield). LCMS: The LCMS sample was quenched with N-methylpiperazine (the resulting sulfonamide MW = 315.240); (ES + M+H=315.1.

[0507] (3R)-3-methoxy-1-pyrrolidinesulfonyl chloride:

[0508]

[0509] Following the procedure described in US Patent Application US2011 / 0311474A1, (R)-3-methoxypyrrolidine hydrochloride (0.30 g, 2.1 mmol) was suspended in a mixture of 4 mL toluene and 2 mL DCM. Triethylamine (0.64 mL, 4.6 mmol) was added, and the mixture was sonicated for 4–5 min to provide a fine white suspension. In a separate flask, 4 mL of toluene was cooled to -40 °C in an acetonitrile / dry ice bath. Thionyl chloride (0.71 mL, 8.7 mmol) was added, and the solution was stirred for 5 min. The pyrrolidine suspension was then added dropwise to the cold (-40 °C) thioyl chloride solution over 10 min. The resulting suspension was stirred at the same temperature for 1 h, and then warmed to room temperature. The solid was filtered off and washed with toluene. The filtrate was concentrated to give 400 mg of the desired product as a light brown oil, which could be used directly without further purification (0.40 g). 1 H NMR (CDCl3) δ: 4.07 (tt, J=4.6, 2.1Hz, 1H), 3.48-3.69 (m, 4H), 3.36 (s, 3H), 2.12-2.23 (m, 1H), 1.98-2.12 (m, 1H).

[0510] (3S)-3-methoxy-1-pyrrolidinesulfonyl chloride:

[0511]

[0512] Prepared in a similar manner to the (R)-isomer, but starting with (S)-3-methoxypyrrolidine hydrochloride.

[0513] Other aminosulfonyl chlorides prepared in a similar manner using commercially available 2° amines and the procedure described in US2011 / 0311474A1 include:

[0514] (S)-3-fluoropyrrolidine-1-sulfonyl chloride: A white solid obtained from (S)-3-fluoropyrrolidine hydrochloride: 1 H NMR (DMSO-d6) δ: 4.54 (dt, J = 52.6, 3.2 Hz, 1H), 2.72-3.03 (m, 5H), 1.34-1.58 (m, 2H).

[0515] 3,3-Difluoropyrrolidine-1-sulfonyl chloride: A white solid obtained from 3,3-difluoropyrrolidine hydrochloride. 1 H NMR (CDCl3) δ: 3.81 (t, J = 12.3 Hz, 2H), 3.74 (t, J = 7.4 Hz, 2H), 2.53 (tt, J = 13.0, 7.5 Hz, 2H).

[0516] 3-Methoxyazacyclobutane-1-sulfonyl chloride: Obtained from 3-methoxyazacyclobutane hydrochloride. 1 H NMR(CDCl3)δ:4.22-4.33(m,3H),3.97-4.09(m,2H),3.33(s,3H).

[0517] Preparation of (R)-3-(chloromethyl)pyrrolidine-1-sulfonyl chloride:

[0518]

[0519] A solution of (R)-3-(hydroxymethyl)pyrrolidine (200 mg, 2 mmol) and triethylamine (0.61 mL, 4.35 mmol) in anhydrous DCM (15 mL) was added dropwise at -78 °C to a stirred solution of thioyl chloride (0.48 mL, 5.9 mmol) in DCM (5 mL). The reaction mixture was stirred at -78 °C for 30 minutes, and then heated to room temperature over 1 hour. The reaction mixture was then washed with 1 M hydrochloric acid aqueous solution (5 mL) and brine (5 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound as a colorless oil, which was used as is in the preparation of Example 28.

[0520] Difluoroaniline hydrochloride intermediate A-5 (Ar=4-methoxyphenyl) was prepared from tert-butyl carbamate A-2.

[0521]

[0522] Step 1 - Preparation of aniline intermediate A-3: Nitroaromatic A-2 (5.00 g, 18 mmol, prepared according to the procedure described in J. Med. Chem. 2003, 46, 1905) and 20% Pd(OH)2 / C (130 mg) were suspended in MeOH (50 mL), and the mixture was stirred under a hydrogen balloon for 18 hours. TLC analysis showed that the reduction was complete (Rf = 0.45, in 2:1 hexane / EtOAc). The suspension was then passed through... The solution was filtered to remove the catalyst, washed with MeOH, and the solvent was evaporated under reduced pressure. Upon exposure to air, the initially colorless solution quickly turned a very deep greenish-blue. The crude intermediate aniline A-3, exhibiting a deep greenish-purple foam, was obtained and immediately used in the next step without further purification. 1 H NMR (DMSO-d6) δ: 8.58 (s, 1H), 6.77 (td, J = 9.4, 2.0 Hz, 1H), 6.60 (td, J = 9.4, 5.5 Hz, 1H), 4.97 (s, 2H), 1.43 (s, 9H).

[0523] Step 2 - Preparation of sulfonamide A-4 (Ar = 4-methoxyphenyl): Crude aniline A-3 from Step 1 (assumed to be 18 mmol) was dissolved in THF (30 mL) and excess 4-methoxyphenylsulfonyl chloride (7.53 g, 36 mmol) was added, followed by the addition of pyridine (6 mL). The mixture was stirred at 50 °C for 18 h. THF was removed under reduced pressure, and the residue was partitioned between EtOAc and water. The extract was washed with saturated NaHCO3 aqueous solution and brine and dried over MgSO4. The desiccant slurry was passed through a 75 mL silica gel pad and washed with EtOAc to remove the desiccant and baseline material. The solvent was removed, giving a brown oily substance, which was purified by rapid chromatography on silica (~250 mL) using 20%–50% EtOAc / hexane as the eluent. After vacuum drying, product A-4 was obtained as a brown foam (8.16 g), which was purified by... 1 The ¹H NMR was contaminated with unreacted sulfonyl chloride at a 2:1 ratio. The material was used directly as is for the next step: 1 H NMR (CDCl3) δ: 7.68 (d, J = 9.0Hz, 2H), 7.41 (td, J = 8.8, 5.5Hz, 1H), 6.85-6.98 (m, 3H), 6.57 (br s, 1H), 5.85 (br s, 1H), 3.85 (s, 3H), 1.46 (s, 9H). MS m / z413.0(MH), m / z 313.0(MH-Boc).

[0524] Step 3 – Preparation of aniline hydrochloride A-5 (Ar = 4-methoxyphenyl): Crude carbamate A-4 (8.16 g) from Step 3 was stirred for 1.5 h at room temperature in a dioxane solution of 4N HCl (25 mL), during which a beige solid gradually precipitated. After 1.5 h, another 10 mL of the dioxane solution of 4N HCl was added, and stirring was continued for another 1 h. The reaction mixture was then diluted with 50 mL of diethyl ether, and the beige precipitate was collected by filtration, washed with ether, and dried under vacuum. Aniline salt A-5 (4.38 g) in pure form was obtained from nitroaromatic A-2, with an overall yield of 68%. 1 H NMR(DMSO-d6)δ:9.73(s,1H),7.62(d,J=8.6Hz,2H),7.06(d,J=9.0Hz,2H),6.69-6.88(m,1H),6.30(td,J=8.6,5.5Hz,1H),3.81(s,3H).MS m / z 313.0(MH).

[0525] Preparation of difluoroaniline hydrochloride intermediate A-5 (Ar = 2,3-dichlorophenyl) from acetaniline A-8.

[0526]

[0527] Preparation of acetanilide A-8: Acetanilide A-7 can be prepared by acetylation of 2,6-difluoroaniline A-6 with acetic anhydride, following the procedure described in Bioorg. Med. Chem. 2016, 24, 2215. As described in WO 2012 / 101238A1, intermediate A-7 is converted to acetanilide A-8 by sequential nitration followed by reduction of the nitro group to aniline.

[0528] Step 1 - Preparation of sulfonamide A-9 (Ar = 2,3-dichlorophenyl): Aniline A-8 (8.50 g, 45.5 mmol) was dissolved in THF (145 mL), and pyridine (4 eq., 14.7 mL) was added to the brown solution, followed by 2,3-dichlorobenzenesulfonyl chloride (1.2 eq., 13.45 g). The resulting reaction mixture was stirred at 45 °C for 3.5 h, and the conversion was determined to be complete by LCMS monitoring. The reaction mixture was cooled to room temperature and then partitioned between EtOAc and 2-Me-THF (1:1) and water. 1 N HCl solution was added until a weakly acidic pH was obtained. A large amount of grayish-white solid was present in the two-phase mixture, which was filtered off (first batch). The filtrate layer was separated, and the aqueous layer was extracted with EtOAc more than twice. The combined organic extracts were washed once with water, then washed with brine, dried over MgSO4, filtered, and concentrated to ~20 mL. The resulting suspension was sonicated, and the solid was collected by filtration and washed with EtOH (batch 2). The two batches were combined and dried under reduced pressure. A-9 (15.3 g, 85% yield) was obtained as a beige solid, which could be used directly without further purification. 1 H NMR(DMSO-d6)δ:10.61(s,1H),9.67(s,1H),7.95(dd,J=8.0,1.4Hz,1H),7.85 (dd,J=8.0,1.4Hz,1H),7.51(t,J=8.0Hz,1H),7.05-7.18(m,2H),2.00(s,3H). MS m / z 395.0(MH + ).

[0529] Step 2 – Preparation of aniline hydrochloride A-5 (Ar = 2,3-dichlorophenyl): In a 500 mL round-bottom flask, acetanilide A-9 (7.00 g, 17.7 mmol) was suspended in ethanol (65 mL), and a 1:1 mixture of concentrated HCl and water (65 mL) was added. The flask was fitted with a stoppered reflux condenser and heated at 80 °C with stirring. After 24 hours, the conversion was judged to be ~70% according to LCMS monitoring. Additional EtOH (65 mL) and 6N HCl (65 mL) were added to the suspension, and stirring was continued at 80 °C for at least 7 hours. LCMS then indicated complete conversion to the desired aniline. The reaction mixture was diluted with 50 mL of water while hot and filtered through a cotton plug to remove any remaining insoluble material. It was then concentrated to dryness under reduced pressure. The residue was azeotropically dried by evaporating toluene three times under reduced pressure, followed by drying under vacuum to give 7.2 g of the desired product A-5 as a yellow solid in the form of an HCl salt. 1 H NMR(DMSO-d6)δ:10.30(s,1H),7.93(dd,J=8.2,1.2Hz,1H),7.83(dd,J=8.0,1. 4Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 6.68-6.96 (m, 1H), 6.31 (td, J = 8.6, 5.5 Hz, 1H). MS m / z350.9(MH).

[0530] Table 1

[0531]

[0532]

[0533]

[0534] General synthetic method A - Preparation of inhibitor I and inhibitor II from intermediate A-5 (Examples 1 and 36):

[0535]

[0536] Step 1 (Preparation of Example 1): Chlorpyrimethamine A-10 (0.546 g, 1.25 equivalents) and aniline hydrochloride A-5 (Ar = 4-methoxyphenyl; 0.750 g, 1 equivalent) were dissolved in AcOH (10 mL), and the brown solution was stirred at 50 °C for 1 h. LCMS showed complete conversion to the desired crude product (Example 1). The reaction mixture was cooled to RT and diluted with water (50 mL), producing a gray precipitate collected by filtration, which was washed with water and dried under vacuum. 100 mg of the crude material sample was purified by passing it through a silica gel pad (3 mL) using a 1:1 hex / EA eluent to remove the colored baseline material. After removing volatiles from the light purple-red solution, the material was lyophilized from MeCN-water to provide the inhibitor of Example 1 (73 mg).

[0537] Steps 2 and 3 (Preparation of Example 36): m-CPBA (185 mg, 1.07 mmol, 1.5 equivalent) was added to a solution of crude thiomethyl derivative (Example 1; 355 mg, 0.72 mmol, 1 equivalent) in 7 mL of DCM at room temperature. The mixture was stirred at this temperature for 1 h. LCMS showed complete conversion to an 80:20 mixture of sulfoxide and sulfone. The mixture was concentrated to remove most of the DCM and then placed in EtOAc and washed three times with NaHCO3 solution. The combined aqueous layers were back-extracted with EtOAc, and the combined organic layers were washed once with water and then with brine. The organic layers were then dried with MgSO4, filtered, and concentrated to give 356 mg of yellow foam, which was used as is without further purification: MS m / z 507 and 523 (MH). + ).

[0538] The crude mixture of sulfoxide and sulfone (25 mg, 0.05 mmol) and benzimidazole (12 mg, 0.1 mmol, 2 equivalents) were placed in a 4 mL vial and dissolved in NMP (1 mL). Then, DIEA (43 μL, 0.25 mmol, 5 equivalents) was added, and the resulting mixture was stirred at 60 °C for 19 hours (LCMS showed complete reaction). The reaction was quenched by adding 0.2 mL of AcOH, and then diluted to 2 mL with methanol. The solution was filtered and then purified by preparative HPLC (MeOH / H₂O / 0.1% HCO₂H conditions, 10%–100% methanol gradient). The fraction containing the main peak was pooled and partially concentrated to remove methanol. The resulting suspension was dissolved by adding a few mL of acetonitrile, and then the solution was frozen and lyophilized. 8.1 mg of the desired product as a pink solid was obtained (Example 36).

[0539] Other examples of inhibitors of general formula I and general formula II prepared in a similar manner, along with characterization data, are listed in Tables 1 and 2.

[0540] Inhibitor I and Inhibitor II were synthesized using general synthetic method B (Examples 12 and 56):

[0541]

[0542] Step 1: Dissolve 1.50 g of carbamate A-2 in 5 mL of DCM and add 2 mL of TFA. After stirring at room temperature for 2 hours, deprotection was completed (LCMS). The reaction mixture was concentrated and dried under reduced pressure.

[0543] Step 2: Although the crude TFA salt from Step 1 (above) can be used directly in Step 2, the desired intermediate B-2 is contaminated with varying amounts of 8-hydroxy-2-thiomethylpyrimidine obtained from the decomposition of A-10 in the solvent. This side reaction can be minimized and a cleaner intermediate B-2 obtained if the aniline TFA salt is neutralized to free aniline before reacting with A-10 as follows. The crude TFA salt from Step 1 is dissolved in DCM and the solution is washed with NaHCO3. After drying (MgSO4), the volatiles are removed to obtain a brown, sticky solid of free aniline B-1 (0.85 g): 1 H NMR(DMSO-d6)δ:7.22-7.38(m,1H),7.06-7.19(m,1H),5.92(br s,2H).

[0544] Chloroprene-10 (550 mg, 2.6 mmol) and B-1 aniline free base (0.39 g, 2.25 mmol) were dissolved in acetic acid (7 mL), and the mixture was stirred at 55 °C for 1 hour. LC-MS showed complete conversion to the desired product. The reaction mixture was cooled to RT and diluted with three times its volume of water to precipitate a creamy solid. The material was collected by filtration, washed with water, and dried under vacuum (0.58 g). 1 H NMR (DMSO-d6) δ: 10.28 (s, 1H), 9.35 (s, 1H), 8.60 (s, 1H), 8.23-8.49 (m, 1H), 7.58 (t, J = 8.8Hz, 1H), 2.71 (s, 3H). MS m / z 350.1(MH + ).

[0545] Step 3: Nitroaromatic B-2 (0.86 g, 2.45 mmol) and tin(II) chloride dihydrate (2.7 equivalents, 6.6 mmol, 1.49 g) were suspended in anhydrous ethanol (10 mL), and the mixture was stirred at 65 °C for 3 hours. The reaction mixture was partitioned between 1 N NaOH and EtOAc. The organic extract was washed with NaHCO3 and brine and dried (MgSO4). The drying agent was then removed from the extract by filtration through a silica gel pad (40 mL) using EtOAc as the eluent to remove baseline material. The filtrate was concentrated, and the residue was ground with EtOAc / hexane to give aniline B-3 as an orange solid. The solid was collected by filtration, washed with ether, and dried (0.438 g). 1 HNMR(DMSO-d6)δ:9.98(s,1H),9.28(s,1H),8.53(s,1H),6.93(td,J=9.4,1.6Hz,1H),6.77(td,J=9.4,5.5Hz,1H),5.12(br s,2H),2.73(s,3H). MS m / z 321.1(MH + ).

[0546] The mother liquor was purified by rapid chromatography (30 mL) using 10%-70% EtOAc in hexane (Rf = 0.3, in 1:2 hex-EA) to provide an additional 96 mg of aniline B-3.

[0547] Step 4 (Example 12): Aniline B-3 (25 mg, 0.078 mmol) and 4-methoxy-2-methylbenzenesulfonyl chloride (100 mg, 0.23 mmol, 3 equivalents) were dissolved in THF (1 mL), and pyridine (40 μL) was added. The mixture was stirred at 45 °C for 1 hour (LCMS showed a conversion of 50%). Another portion of sulfonyl chloride (100 mg) was added, and stirring was continued at 45 °C for 18 hours (LCMS showed complete conversion). The reaction mixture was acidified with TFA (100 μL) and diluted to 1.8 mL with DMSO. The product was separated by preparative HPLC using a 30%–100% MeOH + 0.1% HCOOH gradient (13 mg) (Example 12).

[0548] Steps 5 and 6 (Example 56): 1.2 equivalents of m-CPBA (160 mg, 0.71 mmol) were added to a suspension of thiomethylpyrimidine (Example 12, 300 mg, 0.59 mmol) in DCM (5 mL) at room temperature. The mixture turned into a yellow solution within 10 minutes. It was stirred at room temperature for a total of 45 minutes, at which point LCMS showed the reaction was complete. The mixture was concentrated to remove most of the DCM and then partitioned between EtOAc and NaHCO3 aqueous solution. The layers were separated, and the organic layer was washed twice with NaHCO3 solution. The combined aqueous layer was back-extracted twice with EtOAc, and the combined organic layer was washed with brine, then dried over MgSO4 and filtered. The filtrate was concentrated to dryness and then dried under vacuum to give 295 mg of a mixture of sulfoxide and sulfone (LCMS showed a ~80:20 ratio). The material was used as is in the next step without further purification.

[0549] 4,5-Dimethyl-1H-imidazolium hydrochloride (19 mg, 0.14 mmol, 3 equivalents) and the sulfoxide-sulfone mixture from above (25 mg, 0.048 mmol, 1 equivalent) were placed in 4 mL vials, followed by the addition of NMP (0.5 mL) and DIEA (42 μL, 0.24 mmol, 5 equivalents). The resulting mixture was stirred at 60 °C for 4 h (LCMS showed complete conversion). The reaction mixture was acidified with AcOH (0.2 mL), and the product was separated by preparative HPLC (MeOH / H₂O / 0.1% formic acid, 30%–100% methanol gradient) (Example 56). The fraction containing the main peak was pooled and partially concentrated to remove methanol. The resulting suspension was dissolved by adding a few mL of acetonitrile, and the solution was then frozen and lyophilized. 14 mg of a yellow powder was obtained, with a purity of only 88% as determined by HPLC. The material was further purified by rapid chromatography using a DCM gradient from 100% DCM to 7% isopropanol on a 3 g silica gel cartridge. The appropriate fractions were collected, concentrated, and co-evaporated once with acetonitrile, then added to a 1:1 MeCN / water mixture. After freeze-drying, 7.5 mg of the desired product (Example 56) was obtained as a yellow solid.

[0550] Other examples of inhibitors of general formula I and general formula II prepared in a similar manner are listed in Tables 2 and 3 along with characterization data.

[0551] Inhibitors III and IV were synthesized using conventional synthetic method C (Examples 29 and 73):

[0552]

[0553] Step 1: Dissolve thioyl chloride (0.051 ml, 0.624 mmol) in DCM (2 ml) and cool the solution to -78°C. Add dropwise a solution of aniline B-3 (50 mg, 0.156 mmol) and triethylamine (0.11 ml, 0.78 mmol) in DCM (5 ml) over 5 minutes. Stir the reaction mixture at -78°C for 90 minutes to obtain a solution of intermediate C-1.

[0554] Step 2 (Example 29): (R)-3-methylpyrrolidine hydrochloride (76 mg, 0.62 mmol) in DCM (3 mL) was added to a cold solution of intermediate C-1, followed by the addition of pyridine (0.5 mL). The reaction mixture was heated to RT and stirred for 2 hours (LCMS showed the mass of the product and the completion of the reaction). The reaction mixture was evaporated to dryness and azeotropically reacted with toluene to remove the pyridine. The residue was purified on ISCO using a RediSep 24 g column (DCM / EtOAc) to provide the inhibitor of Example 29 as a brown solid (25 mg).

[0555] Steps 3 and 4 (Example 73): Thiomethylpyrimidine from Step 1 (Example 29, 20 mg, 0.043 mmol) was dissolved in DCM (5 mL), and m-CPBA (11.5 mg, 0.051 mmol) was added. The mixture was stirred at RT for 30 minutes (LCMS no longer showed the starting material). The reaction mixture was diluted with 25 mL of dichloromethane and washed with a saturated NaHCO3 solution. The organic layer was separated and dried over anhydrous Na2SO4 and filtered. The filtrate was evaporated to dryness to provide a mixture of sulfoxide and sulfone (18 mg) as a brown foamy solid, which was used in the next step without any further purification.

[0556] The crude material (18 mg, 0.037 mmol) was dissolved in NMP (2 mL), and DIEA (0.033 mL, 0.186 mmol) and benzimidazole (13.2 mg, 0.112 mmol) were added. The reaction mixture was heated at 60 °C for 3 h (LCMS showed complete conversion). The reaction mixture was diluted with MeOH (1 mL), acidified with a few drops of acetic acid, and the product was separated by preparative HPLC using a 30%–100% MeOH–water–0.1% TFA gradient. The product fractions were partially evaporated, dissolved in ACN and water, and lyophilized to give Example 73 (12 mg).

[0557] Other examples of inhibitors of general formula I and general formula II prepared in a similar manner, along with characterization data, are listed in Tables 2 and 3.

[0558] Inhibitor V was synthesized using the general synthetic method D (Example 88):

[0559]

[0560] Step 1: Dissolve amino-dichloropyrimidine D-2 (50 mg, 0.23 mmol, 1 equivalent) and aniline hydrochloride A-5 (85 mg, 0.23 mmol, 2 equivalents) in AcOH (1.5 mL) and stir the mixture at 55 °C for 1 hour (LCMS showed conversion to the product, but a small amount of aniline remained). Add 10 mg of dichloro derivative D-2 and continue stirring at 55 °C for 30 minutes. Cool to room temperature, dilute with 3 times the volume of water, collect the cream-colored precipitate, wash with water and dry under vacuum (120 mg). 1 H NMR(DMSO-d6)δ:10.10(s,1H),9.78(s,1H),8.61(br s,1H),8.39(br s,1H),8.37(s,1H),7.66(d,J=9.0Hz,1H),7.17-7.26(m,1H),7.13(t,J=9.4Hz,1 H), 6.94 (d, J = 2.0Hz, 1H), 6.86 (dd, J = 9.0, 2.0Hz, 1H), 3.80 (s, 3H), 2.56 (s, 3H). MS m / z 508.0(MH + ).

[0561] Step 2: Aminochloropyrimidine (25 mg, 0.05 mmol, 1 equivalent) and benzimidazole (10.5 mg, 0.09 mmol, 1.8 equivalent) were suspended in DMSO (0.7 mL), followed by the addition of cesium carbonate (37 mg, 0.11 mmol, 2.3 equivalent), copper powder (0.3 mg, 0.1 equivalent), and racemic BINOL (1.5 mg, 0.1 equivalent). The mixture was stirred at 110 °C for 2 hours (LCMS showed complete conversion to the desired mass). The brown reaction mixture was acidified with TFA (100 μL), and the product was separated by preparative HPLC using a 30%–100% MeOH / 0.1% HCOOH gradient. The inhibitor of Example 88 (10 mg) was separated as a beige solid after lyophilization. Examples 85, 86, and 87 were prepared in a similar manner.

[0562] In Example 84, as described in the last step of Scheme A and Scheme B, a nucleophilic substitution was used to form the product (intermediate D-3, 4 equivalents of DIEA and 2 equivalents of 3-fluoropyrrolidine hydrochloride heated in NMP at 100°C for 1.5 hours).

[0563] Other examples of inhibitors of general formula V prepared in a similar manner are listed in Table 4 along with characterization data.

[0564] Synthesize inhibitor VI using conventional synthetic method E:

[0565]

[0566] Step 1: Oxidize intermediate I (Ar = 3-fluoro-2-methylphenyl, Example 90) prepared from suitable intermediate A-5 into a mixture of sulfoxide and sulfone, as described in steps 1 and 2 of general synthetic method A.

[0567] Steps 2 and 3 (X = CH): Cesium carbonate (1.16 g, 3.51 mmol) and methylindole-3-carboxylic acid ester (X = CH; 0.554 g, 3.1 mmol) were suspended in DMSO (62 mL), and the mixture was stirred at RT for 10 min. A mixture of sulfoxide and sulfone from Step 1 (1.55 g, 2.95 mmol) was added, and the mixture was stirred at 80 °C for 18 h. The conversion was then determined to be complete by LCMS analysis.

[0568] NaOH (236 mg, 5.9 mmol) was added to the reaction mixture from step 2, and the mixture was stirred at RT until completely converted to the desired carboxylic acid intermediate E-1 (X = CH) as determined by LCMS analysis. Citric acid solution (1 M) was then added to precipitate the product, which was filtered, washed with water, and dried to give E-1 (X = CH) as a brown solid (1.67 g, 93% yield). 1 HNMR(DMSO-d6)δ12.70(br,1H),10.56(s,1H),10.50(s,1H),9.64(s,1H),9.43(s,1H),8.89(d,J=8.3Hz,1H),8.55(s,1H) ,8.20-8.12(m,1H),7.61(d,J=7.8Hz,1H),7.53-7.45(m,1H),7.45-7.33(m,3H),7.27(t,J=9.2Hz,1H),2.49-2.48(m,3H). MS m / z 620.3(MH + ).

[0569] Steps 2 and 3 (X = N): Sodium tert-butoxyl (341 mg, 3.54 mmol) was added to methyl 1H-indazole-3-carboxylate (573 mg, 3.19 mmol) in anhydrous THF (6.2 mL), and the mixture was stirred at RT for 10 min. A sulfoxide / sulfone mixture (1.55 g, 2.95 mmol) from Step 1 was added, and the mixture was stirred at room temperature for 18 h when LCMS analysis indicated completion of the conversion. The reaction mixture was concentrated to a volume of 20 mL under reduced pressure, and a solution of NaOH (236 mg, 5.9 mmol) in water (20 mL) was added. The mixture was stirred at room temperature for 4 h, at which point LCMS analysis confirmed the conversion to the desired carboxylic acid. The reaction mixture was concentrated under reduced pressure to remove THF, and the aqueous residue was acidified with 1 M citric acid to precipitate the product. The product was collected by filtration, washed with water, and dried to a quantitative yield of brown solid E-1 (X = N): 1 H NMR(DMSO-d6)δ10.53(br,1H),10.03(s,1H),9.69(s,1H),8.89(d,J=8.6Hz, 1H),8.61(s,1H),8.26(d,J=8.1Hz,1H),7.72-7.67(m,1H),7.62(d,J=7.8Hz, 1H),7.55(dt,J=8.0,1.5Hz,1H),7.48(d,J=8.5Hz,1H),7.41(td,J=8.0,5.6H z, 1H), 7.33 (td, J = 8.6, 5.9Hz, 1H), 7.25 (t, J = 9.3Hz, 1H), 2.50-2.49 (m, 3H). MS m / z 607.2 (MH) + ).

[0570] Step 4 (Preparation of Example 103, X = CH): Carboxylic acid E-1 (X = CH; 61 mg, 0.1 mmol) and (S)-3-hydroxypiperidine hydrochloride (17 mg, 0.12 mmol) were dissolved in DMF (0.5 mL), DIPEA (61 μL, 0.35 mmol) was added, followed by HATU (58 mg, 0.15 mmol). The mixture was stirred at ambient temperature for 18 hours. The reaction mixture was then directly purified by preparative reversed-phase HPLC to provide the compound of Example 103 as a yellow powder after lyophilization.

[0571] Other embodiments listed in Table 3 (X = CH or N) were prepared in a similar manner using method E.

[0572] Synthesize inhibitor VII using the general synthesis method F (Example 95):

[0573]

[0574] Step 1: 3-Indolesulfonyl chloride was prepared according to Org. Lett. 2011, 13, 3588. Sulfonyl chloride (200 mg, 0.9 mmol) was placed in a 25 mL flask, and THF (4 mL) was added, followed by dimethylamine hydrochloride (2 equivalents, 150 mg, 1.9 mmol) and DIEA (4 equivalents, 0.65 mL, 3.7 mmol). The solution quickly turned pale yellow, and a yellow, viscous, oily substance deposited at the bottom. After stirring at RT for 20 minutes (LCMS showed complete consumption of sulfonyl chloride), the mixture was partitioned between saturated solutions of EtOAc and NH4Cl. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed again with saturated NH4Cl solution, followed by washing with brine. The mixture was then dried over MgSO4, filtered, and concentrated to dryness to give 65 mg of a beige crystalline solid, which was used as is without further purification. 1 H NMR(DMSO-d6)δ:12.17(br s,1H),7.96(d,J=3.1Hz,1H),7.81(d,J=7.8Hz,1H),7.49-7.57(m,1H),7.22-7.28(m,1H),7.16-7.22(m,1H),2.58(s,6H). MS m / z225.1(MH + ).

[0575] Step 2: As described in general method A, thiomethyl derivative I (Ar = 3-fluoro-2-methylphenyl; Example 90) is oxidized to a mixture of sulfoxide and sulfone.

[0576] Step 3 (Example 95): Prepared using general method A from indolesulfonamide from step 1 and sulfoxide / sulfone mixture from step 2.

[0577] The other embodiments listed in Table 3 were prepared using general method F in a similar manner.

[0578] G was synthesized using the conventional synthesis method (Example 142):

[0579]

[0580] Step 1: Sodium sulfide nonahydrate (414 mg, 1.72 mmol) dissolved in 0.5 mL of water was added to a solution of 1H-indole-3-yl thiocyanate (Phosphorus, Sulfur and Silicon and the Related Elements 2014, 189, 1378) (100 mg, 0.57 mmol) in iPrOH (5 mL). The resulting mixture was then stirred at 50 °C for 2 hours. Subsequently, 4-chlorotetrahydropyran (0.19 mL, 1.72 mmol) was added, and the mixture was stirred overnight at 50 °C. The reaction mixture was diluted with EtOAc (30 mL) and separated. The organic layer was washed with water (15 mL), then with brine (15 mL), dried over MgSO4, and concentrated under vacuum to give a crude sulfide, which was used directly in the next step without further purification.

[0581] Step 2: The sulfide from Step 1 was dissolved in DCM, and 3-chloroperoxybenzoic acid (297 mg, 1.72 mmol) was added and stirred at room temperature for 2 h. After completion, the reaction was quenched by adding 10 mL of a 1:1 solution of saturated NaHCO3 aqueous solution and 10% Na2SO3 aqueous solution. The resulting suspension was stirred at room temperature for 15 min. EtOAc was added and the organic layer was separated. The organic layer was washed with water (15 mL), followed by washing with saturated brine (15 mL). The organic layer was separated, dried (MgSO4), and filtered before being concentrated to dryness to provide the desired sulfone (154 mg, 98%), which was dissolved in DMSO and used directly in the next step without further purification. MS m / z 266.2 (MH) + ).

[0582] Step 3 (Example 142): The indole from step 2 is coupled to intermediate I (Ar = 2,3-dichlorophenyl) using the procedure described in general method A.

[0583] Other examples of inhibitors prepared in a similar manner using a suitable alkylating agent in step 1 are listed in Table 3 under method F.

[0584] The inhibitor IX was synthesized using the conventional synthesis method H (Example 130):

[0585] Iron is used as a reducing agent in step 2 (Example 130):

[0586]

[0587] Step 1: 4-Methylpiperidin-4-ol (0.24 g, 1.84 mmol) and potassium carbonate (0.49 g, 3.52 mmol) were added to a solution of 3-fluoro-2-nitroaniline (0.25 g, 1.60 mmol) in MeCN (2.6 mL). The resulting mixture was stirred at 85 °C for 10 h. MeCN was removed under reduced pressure and EtOAc was added. The suspension was centrifuged and poured into a flask. The concentrated solution, crude 1-(3-amino-2-nitro-phenyl)-4-methylpiperidin-4-ol (0.40 g, 94% yield), was used in the next step without further purification. MS m / z 252.2 (MH) + ).

[0588] Step 2 (using iron as a reducing agent): Iron (0.37 g, 6.70 mmol) and ammonium chloride (0.36 g, 6.7 mmol) were added to a mixture of 1-(3-amino-2-nitro-phenyl)-4-methyl-piperidin-4-ol (0.34 g, 1.34 mmol) and formic acid (1.9 mL, 49.6 mmol) in iPrOH (6.5 mL). The resulting mixture was heated to 90 °C and stirred for 10 hours. The reaction mixture was cooled to room temperature and then... Filter. The concentrated solution was purified by column chromatography (silica gel, 0-15% MeOH in DCM) to give 1-(1H-benzimidazol-4-yl)-4-methyl-piperidin-4-ol (0.17 g, 55% yield) as a red, foamy solid. MS m / z 232.2 (MH) + ). 1 H NMR(400MHz,DMSO-d6)δ:12.21(br s,1H),8.02(s,1H),6.85-7.20(m,2H),6.33-6.67(m,1H),4.24(s,1H),3.15 -3.26(m,2H),2.48(td,J=1.66,3.72Hz,2H),1.41-1.74(m,4H),1.16(s,3H).

[0589] Step 3 (Example 130): The benzimidazole from step 2 is coupled to intermediate I (Ar = 2,3-dichlorophenyl) using the procedure described in general method A.

[0590] Zinc is used as a reducing agent in step 2 (preparation of benzimidazole fragment C328):

[0591] Step 1: Replace 4-methylpiperidine-4-ol with (R)-3-methoxypiperidine hydrochloride. The nitroaromatic hydrocarbon from Step 1 was obtained in 88% yield as a red solid and was used directly in Step 2 without purification: MS m / z 252.1 (MH+). MS m / z 252.1 (MH+) + ). 1 H NMR(400MHz, CDCl3)δ:1.2-1.35(m,1H),1.58-1.73(m,1H),1.74-1.85(m,1H),2.06-2.17(m,1H),2.53-2.61 (m,1H),2.70(td,J=11.5,3.0Hz,1H),3.12(dt,J=12.0,3.8Hz,1H),3.35-3.45(m,2H),3.40(s,3H),4.79(br s, 2H), 6.38 (dd, J = 8.25, 1.13Hz, 1H), 6.41 (dd, J = 8.13, 1.13Hz, 1H), 7.12 (t, J = 8.13Hz, 1H).

[0592] Step 2: In a 250 mL round-bottom flask equipped with a PTFE-coated magnetic stir bar, under nitrogen atmosphere, a mixture of the crude product from Step 1 (1.37 g, 5.45 mmol) and ammonium chloride (4.08 g, 76.3 mmol) in methanol (18 mL) and 2-methyltetrahydrofuran (36 mL) was treated with a single-added addition of zinc powder (2.7 g, 38.16 mmol). An exothermic reaction was observed, and the reaction mixture became colorless after approximately 10 minutes. The reaction mixture was stirred for 1 hour, and LC-MS showed a clean conversion to 1,2-diaminobenzene. The reaction mixture was filtered through a small diatomaceous earth mat and washed with DCM:isopropanol (9:1, 30 mL). The filtrate was diluted with DCM:isopropanol (9:1, 100 mL) and washed with 10% potassium carbonate aqueous solution (30 mL, pH ~10). The organic phase was collected and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure to give 1.2 g: MS m / z 222.2 (MH) + ). 1H NMR(400MHz, CDCl3)δ:1.4-1.6(br s,1H),1.61-1.74(m,1H),1.81-1.94(m,1H),2.0(br s,1H),2.65(br s,1H),2.95(br s,1H),3.17(br s,1H),3.37(br s,2H),3.4-3.5(m,2H),3.41(s,3H),3.79(br s, 2H), 6.53 (dd, J = 7.63, 1.45Hz, 1H), 6.61 (dd, J = 7.94, 1.45Hz, 1H), 6.68 (t, J = 7.8Hz, 1H).

[0593] The crude 1,2-diphenylamine was cyclized to the desired benzimidazole as follows: 2-propanol (20 mL) and the crude product (1.20 g, 5.42 mmol) from above were added to a 100 mL round-bottom flask equipped with a PTFE-coated magnetic stir bar, a reflux condenser, and nitrogen. Formic acid (5 mL, 132 mmol) was then added in a single addition, and the resulting solution was heated at 60 °C for 16 hours. LC-MS indicated the clean formation of the desired benzimidazole (m / z 232). The cooled reaction mixture was diluted with DCM:2-propanol (9:1, 200 mL), washed with 10% potassium carbonate aqueous solution (40 mL, pH 10), brine, and dried over anhydrous magnesium sulfate. Evaporation of the solvent under reduced pressure gave a brown solid. This solid was chromatographically separated on silica gel (3 x 12 cm), eluting ethyl acetate-2-propanol (90:5 to 9:1) to give 1.01 g (81% yield). The mixture was ground with ethyl acetate (10 mL) to obtain 0.88 g of a light orange-brown solid: MS m / z 232.1 (MH). + ). 1 ¹H NMR (400MHz, CDCl₃) δ: 1.47 (br s, 1H) 1.80 (br s, 1H) 1.94 (br s, 1H), 2.13 (br s, 1H), 2.92 (br s, 2H), 3.47 (s, 3H), 3.53–3.63 (m, 1H), 4.1 (br s, 2H), 6.72 (br s, 1H), 7.07 (br s, 1H), 7.19 (t, J = 7.9 Hz, 1H), 7.98 (s, 1H), 9.43 and 9.7 (both br s, ratio 2:1, 1H).

[0594] Other examples of inhibitors prepared in a similar manner using a suitable amine in step 1 and iron or zinc as a reducing agent in step 2 are listed under Method H in Table 3.

[0595] Inhibitor X was synthesized using general synthesis method I (Example 138):

[0596]

[0597] Step 1: 1-Oza-8-azaspiro[4.5]decane hydrochloride (188 mg, 1.06 mmol) and potassium carbonate (2.20 equivalents, 292 mg, 2.11 mmol) were added to a bright red solution of 3-fluoro-2-nitroaniline (150 mg, 0.961 mmol) in ACN (4.5 mL). The resulting mixture was stirred at 90 °C for 16 hours. After completion, the reaction mixture was diluted with ACN and centrifuged. The supernatant was separated and used as is in the next step. MS m / z 278.3 (MH) + ).

[0598] Step 2: Ammonium chloride (1028 mg, 19.2 mmol) and zinc (628 mg, 9.61 mmol) were added to 2-nitro-3-(1-oxa-8-azaspiro[4.5]dec-8-yl)aniline (250 mg, 0.901 mmol) in ACN. The resulting suspension was stirred at 40 °C for 1 hour. After completion, the reaction mixture was diluted with EtOAc and then centrifuged. The supernatant was separated and evaporated under vacuum. The residue 3-(1-oxa-8-azaspiro[4.5]dec-8-yl)phenyl-1,2-diamine (194 mg, 0.784 mmol, 82% yield) was used as is in the next step. MS m / z 248.2 (MH) + ).

[0599] Step 3: 3-(1-oxa-8-azaspiro[4.5]dec-8-yl)phenyl-1,2-diamine (194 mg, 0.78 mmol) was dissolved in AcOH (6 mL), followed by the addition of sodium nitrite (54 mg, 0.78 mmol) and stirring at room temperature for 1 h. After completion, EtOAc and NaHCO3 aqueous solution were added, and the organic layer was separated. The organic layer was washed with NaHCO3 aqueous solution, then with brine, dried over MgSO4, filtered, and concentrated under vacuum to give 8-(1H-benzotriazol-4-yl)-1-oxa-8-azaspiro[4.5]decane (165 mg, 0.64 mmol, 67% yield), which was used without purification. MS m / z 259.2 (MH) + ).

[0600] Step 4 (Example 138): The benzotriazole from step 3 is coupled to intermediate I (Ar = 2,3-dichlorophenyl) using the procedure described in general method A.

[0601] Other examples of inhibitors prepared in a similar manner using a suitable amine in step 1 are listed under Method I in Table 3.

[0602] Preparation of Example 114:

[0603]

[0604] Step 1 – Preparation of 4-(pyridin-3-yl)-1H-benzimidazole: Bromobenzimidazole (70 mg, 0.355 mmol), potassium carbonate (196 mg, 1.42 mmol), and 3-pyridineboronic acid (57 mg, 0.46 mmol) were placed in a 4 mL vial, and dioxane (2 mL) and water (0.7 mL) were added. Argon gas was bubbled through the mixture for 1 minute, and then tetrakis(triphenylphosphine)palladium (0) (16.4 mg, 0.014 mmol) was added. Argon gas was bubbled through the solution again for 3 minutes, the vial was sealed, and heated at 100 °C for 2 hours (LCMS analysis indicated that the conversion to the desired product was complete). The reaction mixture was cooled to RT, diluted with EtOAc, and washed with brine. After drying on MgSO4, the extract was concentrated under reduced pressure, and the residue was purified by rapid chromatography using Et3N-pretreated silica and a DCM-20% iPrOH / DCM gradient to provide the desired benzimidazole intermediate (58 mg, 84% yield): 1 H NMR (DMSO-d6) δ: 12.71 (width s, 1H), 9.24 (s.1H), 8.57 (dd, J = 5.1, 1.6Hz, 1H), 8.43 (width d, J = 5.5Hz, 1H), 8.31 (s, 1H), 7.61 (d, J = 7.8Hz, 1H), 7.52 (ddd, J = 7.8, 4.7, 0.8Hz, 1H), 7.47 (d, J = 7.4Hz, 1H), 7.34 (t, J = 7.8Hz, 1H). MS m / z 196.1(MH + ).

[0605] Step 2: As described in General Method A, thiomethyl derivative I (Ar = 3-fluoro-2-methylphenyl; Example 90) is oxidized to a mixture of sulfoxide and sulfone.

[0606] Step 3 (Example 114): Prepared using general method A from the benzimidazole derivative described in step 1 and the sulfoxide / sulfone mixture from step 2.

[0607] Preparation of (R)-3-(difluoromethoxy)pyrrolidine hydrochloride:

[0608]

[0609] (R)-N-Boc-3-hydroxypyrrolidine was difluoromethylated using 2-fluorosulfonyl-2,2-difluoroacetic acid and copper iodide (I) as catalyzed, as described in J. Org. Chem. 2016, 81, 5803, followed by removal of the N-Boc protecting group with 4N HCl in dioxane.

[0610] Preparation of (S)-3-ethylpyrrolidine hydrochloride:

[0611]

[0612] Step 1: Commercially available (R)-2-(1-(tert-butoxycarbonyl)pyrrolidine-3-yl)acetic acid (2.0 g, 8.72 mmol) was dissolved in anhydrous THF (25 mL), and 1 M BH3·THF (17.45 mL, 17.45 mmol) was added. The reaction mixture was stirred at RT for 3 h. It was then cooled in an ice-water bath and quenched by the slow addition of 1 N HCl. The product was extracted into EtOA and washed with saturated NaHCO3 aqueous solution and brine. The organic layer was dried on anhydrous Na2SO4 and evaporated to dryness to provide an oily (R)-tert-butyl-3-(2-hydroxyethyl)pyrrolidine-1-carboxylic acid ester (1.50 g, 6.98 mmol, 80% yield): 1 H NMR(CDCl3)δ:3.62-3.70(m,2H),3.37-3.60(m,2H),3.15-3.33(m,1H),2.88(dt,J=18.4,9.6Hz,1H),2.13 -2.35(m,1H),1.94-2.08(m,1H),1.47-1.69(m,3H),1.45(s,9H),1.30-1.43(m,1H),0.93(t,J=7.4Hz,1H).

[0613] Step 2: Dissolve (R) tert-butyl 3-(2-hydroxyethyl)pyrrolidine-1-carboxylic acid ester (1.0 g, 4.64 mmol) from Step 1 in DCM (15 mL), and add triethylamine (1.55 mL, 11 mmol). Cool the mixture to 0 °C and add dropwise a solution of methanesulfonyl chloride (0.58 mL, 7.4 mmol) in DCM (2 mL). Stir the reaction mixture at 0 °C for about 1 h. Then dilute the reaction mixture with DCM (15 mL) and wash with saturated NaHCO3. Separate the organic layer, dry with anhydrous Na2SO4 and filter. Evaporate the filtrate to dryness to give an oily crude methanesulfonate ester (1.36 g), which is used in the next step without further purification. 1HNMR(CDCl3)δ:4.26(dt,J=11.3,6.3Hz,2H),3.39-3.66(m,2H),3.21-3.35(m,J=8.2,8.2Hz,1H),3.03(s,3H),2.85-2.99(m,1H), 2.21-2.38(m,1H),1.98-2.15(m,1H),1.80-1.92(m,1H),1.70-1.80(m,1H),1.48-1.70(m,2H),1.46(s,9H),0.97(t,J=7.2Hz,1H).

[0614] Step 3: The crude methanesulfonate from Step 2 (0.60 g, 2.0 mmol) was dissolved in THF (10 mL), and the solution was cooled in an ice-water bath. 1M triethyllithium borohydride (4.70 mL, 4.70 mmol) was slowly added to THF, after which the ice bath was removed, and the mixture was stirred at room temperature for 2 hours. TLC (2:1, ethyl acetate / hexane) showed no starting material. Methanol (5 mL) was slowly added to quench the reaction, and the organic solvent was removed under reduced pressure. The crude reaction mixture was partitioned in EtOAc (30 mL) and water (15 mL), and the organic layer was washed with brine (10 mL). The organic layer was separated, dried over anhydrous Na₂SO₄, filtered, and the filtrate was evaporated to dryness to give the crude product. The material was purified on ISCO using a RediSep 12g column (Hex / EtOAc) to provide an oily (S)-tert-butyl-3-ethylpyrrolidine-1-carboxylic acid ester (300 mg, 73% yield): 1 H NMR(CDCl3)δ:3.48(tt,J=29.7,9.4Hz,2H),3.16-3.34(m,1H),2.85(dt,J=19.8,10 .1Hz,1H),1.90-2.11(m,2H),1.45(s,9H),1.36-1.43(m,3H),0.93(t,J=7.4Hz,3H).

[0615] Step 4: Dissolve the carboxylic acid ester (250 mg, 1.25 mmol) from Step 3 in MeOH (2 mL), and add dioxane (2 mL, 8 mmol) in 4 M HCl. Stir the reaction mixture at RT for 2 hours. Then remove the volatiles under reduced pressure, azeotropically dry the residue with ethyl acetate to dryness, and dry the residue under vacuum to give a viscous oily (S)-3-ethylpyrrolidine hydrochloride (162 mg, 95% yield).

[0616] Preparation of (R)-3-(methoxymethyl)pyrrolidine hydrochloride:

[0617]

[0618] Step 1: Dissolve (R)-tert-butyl-3-(hydroxymethyl)pyrrolidine-1-carboxylic acid ester (1.00 g, 4.97 mmol) in THF (20 mL) and cool the solution in an ice-water bath. Add 60% NaH oil dispersion (0.60 g, 14.91 mmol) in portions and stir the reaction mixture at 0 °C for 15 min. Slowly add iodomethane (1.55 mL, 24 mmol) and stir the reaction mixture at room temperature overnight. Then quench the reaction mixture with saturated NH4Cl solution and extract with EtOAc (2 x 50 mL). Separate the organic layer, dry with anhydrous Na2SO4 and filter. Evaporate the filtrate to dryness and purify it to 100% by silica gel column chromatography (80 g) using EtOAc-hex 0. A colorless oily (R)-tert-butyl-3-(methoxymethyl)pyrrolidine-1-carboxylic acid ester was obtained (700 mg, 3.25 mmol, 65.4% yield): 1 H NMR(CDCl3)δ:3.49(dd,J=11.0,7.6Hz,1H),3.43(br s,1H),3.35(s,3H),3.26-3.35(m,3H),3.06(dd,J=10.7,7.3Hz,1H),2.4 6(spt,J=7.3Hz,1H),1.91-2.02(m,1H),1.58-1.70(m,1H),1.46(s,9H).

[0619] Step 2: The (R)-tert-butyl-3-(methoxymethyl)pyrrolidine-1-carboxylic acid ester (100 mg, 0.46 mmol) from Step 1 was mixed in DCM (2 mL) with 4 M HCl in dioxane (2 mL, 8.0 mmol), and the mixture was stirred at RT for 2 hours. The evaporation product was evaporated under reduced pressure, and the residue was co-evaporated with EtOAc to dryness. The product was dried under vacuum to give (R)-3-(methoxymethyl)pyrrolidine hydrochloride (70 mg), which was an oily substance and could be used without further purification.

[0620] Preparation of Example 89:

[0621]

[0622] Step 1: 10 g of 2-chloro-6-fluoroaniline was placed in a 250 mL flask and dissolved in 40 mL of glacial acetic acid. Acetic anhydride (7.47 mL) was added at room temperature, and the resulting mixture was stirred at 90 °C for 1 hour, at which point LCMS analysis showed the reaction was complete. Volatile substances were removed under reduced pressure, and the residue was dissolved in DCM and slowly neutralized with saturated NaHCO3 solution. The layers were separated, and the aqueous layer was extracted three times with DCM. The combined organic layers were washed once with water, dried over MgSO4, filtered, and concentrated. After vacuum drying, the desired product was obtained as white to pale pink crystals (12.79 g). 1 H NMR(CDCl3)δ:7.16-7.26(m,2H),7.03-7.13(m,1H),6.93(br.s.,1H),2.23(br.s.,3H). MS m / z 188.1(MH + ).

[0623] Step 2: Dissolve 12.75 g of acetanilide from Step 1 in 25 mL of concentrated sulfuric acid and cool to 0 °C in an ice bath. Slowly add 3.31 mL of 90% nitric acid. After 5–10 minutes, the mixture forms solid clumps. Heat to room temperature, producing a thick, deep purple-red muddy sediment. After a total of 4 hours, monitor the reaction by LCMS, showing some remaining starting material. Add another 5 mL of sulfuric acid to improve flowability, followed by 0.3 mL of 90% nitric acid. Stir the mixture at room temperature for another 18 hours. Then cool the mixture to 0 °C and pour it onto crushed ice (~150 mL). Once the ice melts, sonicate the suspension and collect the yellow solid by filtration, wash with water, and dry (15.1 g crude product). Dissolve the crude solid in 50 mL of acetonitrile and reflux to give a clear, deep red solution. Stop heating and cool the mixture to room temperature for 1 hour, then stir at room temperature for 2 hours. The mixture had now solidified into clumps, which were broken up with a spatula and sonicated. The solids were then collected by filtration and washed with a small amount of cold acetonitrile. As indicated by NMR, the desired off-white nitro compound (6.63 g) in a single regioisomer was obtained (the mother liquor yielded a second batch of 2.16 g containing 7% 6-chloro-2-fluoro-3-nitroacetanilide). 1 H NMR (CDCl3) δ: 7.90 (dd, J = 9.2, 4.9 Hz, 1H), 7.24 (dd, J = 9.2, 8.4 Hz, 1H), 6.98 (br.s., 1H), 2.28 (s, 3H). MS m / z 233.0(MH + ).

[0624] Step 3: Add a solution of NH4Cl (60 mg, 1.12 mmol) in 1.35 mL of water to the solution of nitroacetanilide (500 mg, 2.15 mmol) in 15 mL of ethanol from Step 2. Heat the mixture to 70°C, then add iron powder (600 mg, 10.75 mmol) in three batches at 10-minute intervals. Stir the resulting deep red to deep purple-red mixture at 70°C for 20 hours. At this point, LCMS analysis of filtered aliquots of the reaction mixture showed that the reaction was complete. Pass the mixture through… Filter the solution. Concentrate the dark brown filtrate to dryness, then dissolve it in EtOAc, adding MgSO4. Stir the suspension, then filter to obtain a clear, pale yellow solution. Concentrate the solution to dryness to give the desired product (440 mg) as a pale yellow solid, which can be used directly without further purification. 1 H NMR (CDCl3) δ: 6.92 (t, J = 9.0 Hz, 1H), 6.76 (br.s., 1H), 6.68 (dd, J = 8.6, 4.7Hz, 1H), 3.98 (br.s., 2H), 2.24 (br.s., 3H). MS m / z 203.1(MH + ).

[0625] Step 4: Sulfonate the aniline from Step 3 using 4-methoxybenzenesulfonyl chloride in the general manner described in A-9 of Scheme A: 1 H NMR(DMSO-d6)δ:9.89(s,1H),9.67(s,1H),7.57-7.74(m,2H),7.23(t,J=9.2Hz ,1H),7.13(dd,J=8.8,5.3Hz,1H),7.02-7.10(m,2H),3.82(s,3H),2.00(s,3H). MSm / z 373.0(MH + ).

[0626] Step 5: Dissolve acetanilide (200 mg, 0.54 mmol) from Step 4 in 1.5 mL of ethanol, then slowly add a 1:1 mixture of concentrated HCl and water (2 mL). Heat the yellow slurry to 80 °C and stir for 1 hour. At this point, add 1 mL of ethanol to improve solubility. Stir the mixture at the same temperature for another 5 hours (until the mixture becomes a clear yellow solution). LCMS analysis at this point shows <3% remaining starting material. Concentrate the mixture to remove most of the ethanol, then cool on ice. Alkalize it to pH 5-6 with 4N NaOH. Sonicate the resulting suspension and collect the solid by filtration and washing with water. After drying under reduced pressure, 156 mg of the desired product is obtained as a beige solid. 1H NMR(DMSO-d6)δ:9.53(s,1H),7.53-7.72(m,2H),7.00-7.14(m,2H),6.95(dd ,J=10.8,8.8Hz,1H),6.36(dd,J=8.6,5.1Hz,1H),5.39(s,2H),3.81(s,3H). MS m / z 329.0(MH).

[0627] Step 6: Using the method described in Scheme A for preparing an inhibitor of Formula I from synthetic intermediate A-5 (Step 1, Example 1), aniline from Step 5 is reacted with intermediate A-10 to provide Example 89.

[0628] Preparation of Example 135:

[0629]

[0630] Step 1: Add 1.3 mL of 2,6-difluoronitrobenzene (12.6 mmol) and 1.6 mL of ethyl cyanoacetate (15.1 mmol) to a 100 mL round-bottom flask containing 15 mL of DMF. Next, slowly add sodium hydride (754 mg, 18.9 mmol) at room temperature. Stir the reaction at room temperature for 15 minutes. Quench the reaction with 1 M HCl until the deep red solution turns yellow, then dilute in EtOAc. Separate the organic layer, wash with aqueous NH4Cl solution, then wash with brine. Dry the organic layer with MgSO4, filter, and then concentrate under reduced pressure. Dissolve the crude material in DMSO (9 mL) and water (1 mL) and transfer to a 20 mL microwave-safe vial. Heat the reaction mixture to 120 °C and stir for 16 hours. Cool the reaction mixture to room temperature and dilute in EtOAc, then wash with aqueous NH4Cl solution, then wash with brine. Dry the organic layer with MgSO4, filter, and then concentrate under reduced pressure. The crude material was purified by normal-phase rapid column chromatography using hexane:EtOAc to obtain the desired benzyl nitrile (2.12 g, 93%) as an orange solid. 1 H NMR (400MHz, DMSO-d6) δ: 7.80 (td, J = 7.8, 5.5Hz, 1H), 7.65 (t, J = 9.6Hz, 1H), 7.54 (d, J = 7.4Hz, 1H), 4.28 (s, 2H). MS m / z 725.4(MH + MS m / z 181.2 (MH) + ).

[0631] Step 2: The phenylacetonitrile derivative from Step 1 (200 mg, 1.11 mmol) and DMSO (4 mL) were placed in a 25 mL flask. Diphenyl(vinyl)sulfonate trifluoromethanesulfonate (479 mg, 1.32 mmol) was then added at room temperature, followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (0.37 mL, 2.48 mmol). The mixture was stirred at this temperature for 16 hours. After completion, an aqueous solution of NH4Cl was added, and the aqueous layer was extracted with EtOAc. The organic layer was washed with water and then once with brine. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The residue was purified by rapid chromatography using EtOAc in hexane via silica gel column chromatography to give the desired cyclopropane derivative (138 mg, 60% yield). 1 H NMR (400MHz, CDCl3) δ: 7.47-7.59(m,1H), 7.29-7.41(m,2H), 1.69-1.85(m,2H), 1.29-1.39(m,2H). MS m / z207.2(MH + ).

[0632] Step 3: Add the cyclopropane derivative from Step 2 (138 mg, 0.67 mmol) to a 10 mL microwave-safe vial. Next, add 3 mL of concentrated ammonium hydroxide to the reaction mixture. Heat the reaction mixture in a microwave at 130 °C for 1 h. After completion, dilute the reaction mixture with water and then extract with EtOAc. Wash the organic layer with brine, dry with MgSO4, filter, and then concentrate under reduced pressure to give 1-(3-amino-2-nitro-phenyl)cyclopropane nitrile (120 mg, 88% yield) as an orange solid. 1 H NMR (400MHz, CDCl3) δ: 7.27 (d, J = 15.26Hz, 1H), 6.83 (d, J = 8.38Hz, 1H), 6.86 (d, J = 7.50Hz, 1H), 5.36 (br s, 2H), 1.71 (br s, 2H), 1.24 (br s, 2H).

[0633] Step 4: The reduction and ring closure of intermediate 1,2-phenylenediamine to a benzotriazole ring are performed using the procedures described in steps 2 and 3 of the general method I for Example 138.

[0634] Step 5 (Example 135): The benzotriazole from step 4 is coupled to intermediate I (Ar = 2,3-dichlorophenyl) using the procedure described in general method A.

[0635] Preparation of Example 137:

[0636]

[0637] Step 1: Potassium carbonate (0.35 g, 2.56 mmol) and 2-methoxyethanol (0.40 mL, 5.1 mmol) were added to a DMF (3.2 mL) solution of 3-fluoro-2-nitroaniline (0.10 g, 0.641 mmol). The resulting mixture was stirred at 80 °C for 10 hours. Water was added and the aqueous mixture was extracted with EtOAc. The organic layers were combined, washed with brine, dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (silica gel, 0-100% EtOAc in hexane) to give 3-(2-methoxyethoxy)-2-nitroaniline (52 mg, 38% yield). MS m / z 213.1 (MH) + ).

[0638] Step 2: The reduction and ring closure of intermediate 1,2-phenylenediamine to a benzotriazole ring is performed using the procedures described in steps 2 and 3 of the general method I for Example 138.

[0639] Step 3 (Example 137): The benzotriazole from step 2 is coupled with intermediate I (Ar = 2,3-dichlorophenyl) using the procedure described in general method A.

[0640] Preparation of Examples 160 to 163 (Table 5):

[0641]

[0642] Step 1: The carbon methoxylation of 2,4,5-trifluoroaniline is carried out as described in patent WO2020 / 261156.

[0643] Step 2: At room temperature, 2,3-dichlorobenzenesulfonyl chloride (3.91 g, 15.9 mmol) was added fractionally to a solution of methyl 3-amino-2,5,6-trifluorobenzoate (2.72 g, 13.26 mmol) in DCE / pyridine (1:1, 16 mL). The reaction was heated to 70 °C and continued for 16 hours. The reaction was monitored by LC-MS. When the reaction was complete, it was quenched with 1 M HCl. The aqueous layer was extracted three times with EtOAc (15 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated to dryness. The residue was purified by chromatography on a silica gel column using 0–30% EtOAc in hexane. The pure fraction was collected and evaporated to give a light brown solid of methyl 3-((2,3-dichlorophenyl)sulfonamide)-2,5,6-trifluorobenzoate (5.14 g, 91% yield): m / z = 412.0.

[0644] Step 3: 2M KOH (37mL, 74.5mmol) was added to a solution of methyl 3-((2,3-dichlorophenyl)sulfonamide)-2,5,6-trifluorobenzoate (5.14g, 12.4mmol) from Step 2 in 30mL of THF:MeOH (5:1) at room temperature. The reaction mixture was stirred overnight at room temperature. When the reaction was complete, it was evaporated to dryness, and water (30mL) and diethyl ether (30mL) were added to the residue. The aqueous layer was washed twice with ether (20mL). The aqueous layer was acidified to pH 2 with 1M HCl. The aqueous layer was extracted three times with EtOAc (30mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated to give a light orange oil of 3-((2,3-dichlorophenyl)sulfonamide)-2,5,6-trifluorobenzoic acid (4.5g, 91% yield). In the next step, use the compound as is: m / z = 398.0.

[0645] Step 4: At room temperature, triethylamine (1.71 mL, 12.37 mmol) and diphenylphosphoazide (2.91 mL, 13.50 mmol) were added to a solution of 3-((2,3-dichlorophenyl)sulfonamide)-2,5,6-trifluorobenzoic acid (4.50 g, 11.2 mmol) from Step 3 in acetonitrile (30 mL). The reaction was heated to 80 °C overnight. The reaction was cooled to rt and water (30 mL) was added. The aqueous layer was extracted three times with EtOAc (30 mL). The combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated to a deep yellow residue. The crude material was purified by chromatography on a silica gel column using 0–50% hexane of EtOAc. The pure fraction was collected and evaporated to give 2,3-dichloro-N-(2,4,5-trifluoro-3-isocyanophenyl)benzenesulfonamide (2.29 g, 51% yield) as a brown solid: m / z = 391.0.

[0646] Step 5: To a solution of 2,3-dichloro-N-(2,4,5-trifluoro-3-isocyanophenyl)benzenesulfonamide (1.35 g, 3.39 mmol) from Step 4 in THF (17 mL), add 17 mL of 4M LiOH aqueous solution. Tighten the pressure vessel and heat in an oil bath to 100 °C for 1 hour. When the reaction is complete, quench with saturated NH4Cl. Add EtOAc and separate the layers. Extract the aqueous layer twice more with EtOAc (20 mL). Wash the combined organic layers with brine, dry with MgSO4, filter and evaporate to give N-(3-amino-2,4,5-trifluorophenyl)-2,3-dichlorobenzenesulfonamide (1.09 g, 86% yield) as a brown solid. This compound was proceeded to the next step without further purification. 1H NMR(400MHz,DMSO-d6)δ:10.59(br s,1H),7.94(dd,J=8.2,1.6Hz,1H),7.89(dd,J=8.2,1.6Hz,1H),7.51(dd,J=8.0Hz,1H),6.34-6.43(m,1H),5.72(s,2H). m / z=369.0.

[0647] Step 6: As described in General Method A (Example 36), the aniline from Step 5 is converted to pyrimidine and pyrimidine, wherein Ar = 2,6-dichlorophenyl.

[0648] Step 7 (Examples 160 to 163, Table 5): The thiomethyl intermediate from step 6 is converted into an inhibitor using a suitably substituted benzimidazole (Examples 160, 162, 163) or benzotriazole (Example 161), according to the scheme in General Method A.

[0649] Bioactivity

[0650] In vitro bioactivity

[0651] (a) Kinase activity assays of BRAF, CRAF and ARAF

[0652] Compound preparation: Suspend the solid sample of each substance in one vial in DMSO (Fisher Scientific) at a stock concentration of 20 mM. Store the stock solution at -20°C and protected from light. If the solubility of the compound at 20 mM is problematic, change the initial concentration of the DMSO stock solution to 10 mM or 5 mM.

[0653] In vitro enzymatic reactions were used to evaluate the intrinsic activity of compounds against BRAF, CRAF, and ARAF. For BRAF and CRAF, 0.375 nM purified GST-labeled kinase (Millipore Sigma catalogue numbers B4062-10UG and R1656-10UG, respectively) was incubated with 75 nM kinase-death MEK1 substrate (catalog number 40075; BPS Bioscience) in the presence of 10 μM Ultrasound ATP (catalog number V9102; Promega; part V915A), with and without the test compound, in a buffer containing 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-35, and 2 mM DTT. Separate reactions were performed using MEK1 substrate and ATP as blank controls. ARAF and kinase responses are strictly identical, except that the kinase concentration increases to 3.75 nM (catalog number 1768-0000-1; Reaction Biology).

[0654] For compound preparation, place 5 μL / well of the test substance solution in a 384-well substitute plate (Perkin Elmer) and mix with 2x concentrated kinase reactant. Select a dilution series to cover nine concentrations from 100 nM to 0.01 nM. If necessary (if the compound exhibits low intrinsic potency), change the initial concentration of 100 nM to 1 μM or 0.5 μM and perform further dilutions accordingly. The final concentration of DMSO in the assay is set to 0.05%.

[0655] BRAF and CRAF kinase reactions were carried out at 30°C for a total of 2 hours, then stopped by a 1 / 2 dilution in ADP-Glo ​​reagent (catalog number V9102; Promega; part V912C). The reactants were then incubated at room temperature for 1 hour, followed by the addition of one volume of kinase assay reagent (catalog number V9102; Promega; part V917A). The plates were then equilibrated at room temperature for 30 minutes, and luminescence was detected on a Synergy Neo2 plate reader (Biotek). The effect of each compound dilution on BRAF and CRAF kinase activity is expressed as a percentage of inhibition and calculated as follows. First, the mean of the internal 100% inhibition control (containing only the luminescence mean of the kinase reaction containing the kinase-dead MEK1 substrate) was subtracted from each data point. The mean of the DMSO (mediator) control (set to 0% inhibition) was determined and used to calculate the percentage of inhibition:

[0656] Suppression % = 100 * (1 - ((luminous signal)) 化合物 ) / (Light emission signal) DMSO )))

[0657] The ARAF kinase reaction was carried out at 30°C for a total of 2 hours, and then terminated by adding EDTA to a final concentration of 40 mM. Then, [the reaction was] used... Ultra TM The reaction was detected using the p-MEK 1 / 2 (Ser218 / 222) (PerkinElmer) kit. Following the manufacturer's instructions, 5 μL of the kinase reactant was used in a 384-well Proxyplate (Perkin Elmer), followed by overnight incubation at room temperature in a humidified chamber. After the reaction was complete, the reaction was detected using a kit equipped with... Signals were recorded on a Synergy Neo2 plate reader (Biotek) using the filter. The effect of each compound dilution on the pMEK signal generated by the ARAF reaction was expressed as a percentage of inhibition and calculated as follows. Each plate included an internal 100% inhibition control (containing only the average luminescence in the kinase reaction of the kinase-dead MEK1 substrate) to measure pMEK background and subtract it from each data point.

[0658] The mean of the DMSO (mediator) control (set to 0% inhibition) was also determined and used to calculate the inhibition %:

[0659] Suppression % = 100 * (1 - ((pMEK signal)) 化合物 ) / (pMEK signal DMSO )))

[0660] IC 50 The values ​​were obtained by plotting kinase inhibition values ​​and fitting a dose-activity curve using a log(agonist) versus response-variable slope (four parameters) function (using GraphPadPrism (V7.0) or the Dotmatics Screening Ultra platform). The standards included in the ARAF kinase assay were bevacizumab (Belvarafenib) (MedChem Express catalog number HY-109080; CAS#1446113-23-0), LXH254 (MedChem Express catalog number HY-112089; CAS#1800398-38-2), and BGB283 (catalog number HY-18957; CAS#1446090-79-4).

[0661] Therefore, all substances reported herein are BRAF, CRAF, and ARAF ATP-competitive kinase inhibitors, as demonstrated by direct inhibition of their enzymatic activity in vitro. The BRAF and CRAF inhibitory efficiencies of the compounds are listed in Tables 2 through 5, while the ARAF kinase inhibitory efficiencies of representative analogs are listed in Table A. Preferred embodiments defined in the embodiments show BRAF IC50. 50Value <10nM, and even more preferred embodiments have BRAF IC. 50 Value < 1nM. The preferred embodiment as defined in the implementation shows the CRAF IC. 50 Value <50nM, and even more preferred embodiments have CRAF IC. 50 Value < 10nM.

[0662] Table A. Results of ARAF kinase inhibition

[0663]

[0664] For ARAF biochemical kinase assays, * indicates IC50. 50 >20nM,

[0665] **Indicates ICs ranging from 10nm to 20nm. 50 Range, *** indicates IC 50 <10nM.

[0666] (b) General cell culture methods

[0667] All cancer cell lines (A375, A101D, A2058, RKO, HT29SK-MEL 30, IPC298, HepG2, HCT-116, Lovo, SW620, SW480, NCI-H358, NCI-H2122, Calu-6, NCIH2087, NCIH1755, NCIH1666, and Mewo) were obtained from ATCC and cultured in RPMI-1640 medium (Gibco) supplemented with 5% heat-inactivated fetal bovine serum (FBS, Wisent) at 37°C and 5% CO2. Cells were maintained in T175 flasks (Greiner). They were passaged by removing the medium, washing once in 10 mL of room temperature phosphate-buffered saline (PBS; Wisent), and incubating with 2 mL of 0.05% trypsin (Thermo-Fisher) at 37°C. Trypsin was then inactivated by adding complete growth medium, and the cells were re-plated into T175 culture dishes at appropriate dilutions. All cell lines were periodically tested for mycoplasma contamination. Histiotypes and mutation statuses of each cell line are shown in Table B.

[0668] Table B. Tumor type and RAS-ERK pathway mutation status of cancer cell lines (CCLs) used for pERK and antiproliferative profile analysis of the substances described in this application.

[0669] cell lines Organization type Mutation state A375 skin BRAF V600E A101D skin BRAF V600E A2058 skin BRAF V600E RKO colon BRAF V600E HT29 colon BRAF V600E NCIH2087 lung BRAF L597V; KRAS Q61K NCIH1755 lung BRAF G469A NCIH1666 lung BRAF G466V SK-MEL30 skin NRAS-Q61K IPC298 skin NRAS-Q61L HepG2 liver NRAS-Q61L HCT-116 colon KRAS G13D Lovo colon KRAS-G13D SW620 colon KRAS-G12V SW480 colon KRAS G12D NCI-H358 lung KRAS-G12C NCI-H2122 lung KRAS-G12C Calu-6 lung KRAS Q61K Mewo skin NF1LOF

[0670] (c) pass Ultra TM p-ERK 1 / 2 (Thr202 / Tyr204) measurement cultured human Phospho-ERK inhibition in cancer cell lines

[0671] Ultra TM p-ERK 1 / 2 (Thr202 / Tyr204) analysis was performed on cells plated at the density specified in Table C in 100 μL of complete RPMI-1640 growth medium in 96-well flat-bottomed transparent culture dishes (Costar). Cells were incubated overnight at 37°C and 5% CO2 one hour before being serially treated with the compound dilutions. Results were expressed as cells / cm². 2 Cell density in units is equivalent to the number of cells divided by the area of ​​one well in a 96-well plate (0.143 cm²). 2 ).

[0672] Table C. Number of cells coated per well for each cancer cell line.

[0673]

[0674]

[0675] In the dilution series, 100 μL / well of the test substance dilution prepared in complete RPMI-1640 growth medium is added to the cells. Select a dilution series to cover ten concentrations from 30 μM or 10 μM to 0.33 nM. If necessary, increase the initial concentration of 10 μM to 100 μM or decrease it to 1 μM (as in the case of A375 and NCIH1666 cells, which are generally more sensitive to the compound) and perform further dilutions accordingly. The final concentration of DMSO in the assay is set to 0.5%.

[0676] After treatment, the culture medium was removed, and the cells were lysed in 50 μL of 1X AlphaScreen Ultra Lysis Buffer (PerkinElmer). Follow the manufacturer's instructions. Ultra TM The p-ERK 1 / 2 (Thr202 / Tyr204) (PerkinElmer) reaction was performed using 5 μL of cell lysate in a 384-well Proxyplate (PerkinElmer), followed by overnight incubation at room temperature in a humidified chamber. After the reaction, the reaction mixture was transferred to a pre-filled container. The signal was recorded on the EnVision reader (Perkin Elmer).

[0677] The effect of each compound dilution on the pERK signal was expressed as inhibition % and calculated as follows. Each plate contained an internal 100% inhibition control (1 μM trametinib, catalog number HY-10999; MedChem Express; catalog number 871700-17-3) and used as a measure of pERK background. First, the value obtained for trametinib was subtracted from each data point. The mean of the DMSO (mediator) control (set to 0% inhibition) was determined and used to calculate the inhibition %:

[0678] Suppression % = 100 * (1 - ((pERK signal)) 化合物 ) / (pERK signal DMSO )))

[0679] The ability of each compound to suppress the pERK signal is expressed as IC. 50 The values ​​were obtained by plotting the inhibition value for each data point in the diluted series and fitting the obtained curve using the log(agonist) contrast-response-variable slope (four parameters) function (using GraphPadPrism (V7.0)).

[0680] When present, the paradoxical pERK induction originates from the pERK IC of the compound. 50 The negative % inhibition value observed in the curve is inferred. To classify the compound as a pERK paradoxical inducer, the minimum data point of inhibition (%Y) on the dose-activity curve is used. MIN The value was set below -20%, which was considered to be within the expected measurement range (e.g., %Y). MIN Compounds with concentrations of -30%, -50%, or -150% are considered to produce paradoxical induction of this pathway. This is illustrated by the presence of Y. MIN = -10% of IC 50 Compounds whose curves are drawn are considered not to produce paradoxical activation of this pathway. Therefore, compounds are said to inhibit the pathway in a given cell line without paradoxical induction when the following criteria are met:

[0681] 1. Inhibition % exceeds 50% at the highest tested dose (30μM, 10μM, or 1μM).

[0682] 2.IC 50 %Y of the curve MIN Greater than -20%; where Y MIN IC corresponding to the compound 50 The data point with the lowest value in the curve.

[0683] As is well known to those skilled in the art, some variation in inhibition values ​​is expected in such experiments. ±20% Y MINThe values ​​were considered to be within the experimental error range and not significant. Therefore, only compounds with negative values ​​exceeding the measured change (approximately >20%) were considered to induce paradoxical activation of the signal transduction cascade and were not included in the scope of this disclosure. Figure 1 The IC50 of compounds that induce paradoxical pathway activation (PLX4720, commercially available from Selleck Chemicals; catalog number 918505-84-7) and representative compounds as described herein that exhibit unexpected and unique non-induction curves are provided. 50 Visualization of curves.

[0684] Figure 1 This demonstrates paradoxical induction of pERK signaling in RAS mutant HCT116 cells without inducing Y-cell signaling. MIN >-20%) of the compounds described herein (Examples 80 and 81) and those that caused strong induction of this pathway in the same cell lines (Y MIN Representative IC50 of the compound (PLX4720) with a concentration of ~-600% 50 Inhibitor response curve.

[0685] Significantly, according to the above criteria, compounds as defined herein do not induce paradoxical activation of this pathway. Further illustration of this highly desirable property can be provided using the immunoblotting analysis described below, and... Figure 2 The non-inducing compound (Example 80) and the inducer PLX4720 are described.

[0686] For immunoblotting analysis, 500,000 HCT-116 cells were plated in 1 mL of intact RPMI-1640 growth medium in 24-well flat-bottomed clear culture dishes (Costar). Cells were incubated overnight at 37°C with 5% CO2, followed by serial treatment with dilutions of the compound for one hour. Cells were then washed once in PBS and lysed at 4°C with gentle agitation in 250 μL of Igepal Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Igepal-CA630, 1 mM EDTA, 10% glycerol) supplemented with leucopeptidase, aprotinin, PMSF, a mixture of phosphatase inhibitors (Sigma), and Na3VO4 for 15 minutes. The cell extract was then clarified by centrifugation at 4°C, 20,000 g for 10 minutes. The clarified lysate was then transferred to ice in a new tube and boiled for 5 minutes in sample loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM β-mercaptoethanol). It was then fractionated by SDS-PAGE and transferred to a nitrocellulose membrane (PALL). The membrane was blocked for 1 hour in Tris buffered saline containing 2% BSA (Sigma) and 0.2% Tween-20 (TBST; 10 mM Tris-HCl pH 8.0, 0.2% Tween-20, 150 mM NaCl), and then incubated overnight at 4°C with the following primary antibody dilutions prepared in TBST: anti-pERK (1:2000 dilution; Sigma-Aldrich; catalog number M9692), anti-total ERK (1:1000 dilution; Cell Signaling Technology; catalog number 4695), anti-pMEK (1:1000 dilution; Cell Signaling Technology; catalog number 9121) and anti-total MEK (1:1000 dilution; Cell Signaling Technology; catalog number 9122). Secondary anti-mouse HRP and anti-rabbit HRP (Jackson Immunoresearch Labs; catalog numbers 115-035-146 and 111-035-144, respectively) were prepared in TBST at dilutions of 1:5000 and 1:10000, respectively. Immunoblotting was performed by exposure to X-ray film after one minute of incubation in ECL reagent.

[0687] Figure 2The diagram shows representative compounds that induce paradoxical signaling without inducing pERK or pMEK (Example 80; top panel). As a comparison, immunoblotting analysis results of RAS-mutant HCT-116 cells treated with a compound that induces this pathway (PLX4720; bottom panel) in the same cell line are also shown. Total MEK and total ERK signals were also detected by immunoblotting to ensure that the protein sample loading was equal under different conditions. Compound concentrations in micromoles are shown above the immunoblotting plots. The concentration range used for treatment was the same as in Example 80 and with PLX4720.

[0688] Compounds 1 through 163 of Examples showed pERK inhibitory activity in the HCT-116 cell line with a G13D Ras mutation in the colon, as shown in Tables 2 through 5. Furthermore, some examples also showed paradoxically induced inhibition of pERK signaling in the SW480 colon cell line carrying the G12D allele of KRAS (Tables 2 through 5). Additionally, some examples from Tables 2 through 5 were tested in BRAF-containing... V600E The inhibitory effect of the driver mutant on pERK was observed in A375 cells, and they were also found to be active (Table D). In Tables 2 through 5, the pERK IC50 of all compounds in Examples 1 through 163 in the HCT116 cell line is shown. 50 Value <30 μM. Preferred compound IC50 value. 50 The IC50 value is 1 μM to 10 μM, and the IC50 value of the more preferred compound is... 50 IC50 values ​​for compounds ranging from 0.5 μM to -1 μM, or even more preferred, are... 50 Value <0.5μM.

[0689] The pERK inhibitory activity of the representative compounds as defined herein was also tested on other tumor cell lines, and showed good to very good pERK inhibitory activity in cancer cell lines carrying various NRAS, KRAS, and NF1 alleles and representing multiple tissue types (i.e., SK-MEL 30, IPC298, HepG2, HCT-116, Lovo, SW620, SW480, NCI-H358, NCI-H2122, Calu-6, and Mewo; Table D, and refer to Table B for genotypes). The pERK inhibitory activity of the compounds was stronger in cancer cell lines carrying BRAF alleles (A375, A101D, A2058, RKO, HT29, NCIH2087, NCIH1755, and NCIH1666) (Table D).

[0690] Table D (D-1 and D-2). A group of RAS-mutated cancer cell lines (genotypes shown in Table B) and BRAF V600E Non-inducible pERK IC of selected compounds in mutant A37550 Value and anti-proliferative EC 50 value.

[0691] D-1.

[0692]

[0693]

[0694] In pERK: + indicates IC 50 >300nM, ++ indicates ICs with a range of 30nM to 300nM. 50 Range, +++ indicates IC 50 <30nM. For proliferation: * indicates EC 50 >3000nM, ** indicates EC 300nM-3000nM. 50 Range, *** indicates EC 50 <300 nM. Belv.: Bevarafenib. Values ​​in parentheses represent inhibition percentages. Blank indicates that the value was not measured.

[0695] D-2.

[0696]

[0697]

[0698] In pERK: + indicates IC 50 >300nM, ++ indicates ICs with a range of 30nM to 300nM. 50 Range, +++ indicates IC 50 <30nM. For proliferation: * indicates EC 50 >3000nM, ** indicates EC 300nM-3000nM. 50 Range, *** indicates EC 50 <300 nM. Belv.: Bevarafenib. Values ​​in parentheses represent inhibition percentages. Blank indicates that the value was not measured.

[0699] For RAS-mutated cancer cell lines, pERK IC 50 %Y of the curve min The values ​​were all above -20% and were considered to show minimal or no induction, therefore the compound did not induce detectable paradoxical activation of the pathway in this group of cancer cell lines. In contrast, comparisons with the molecule bevacirafenib (obtained from MedChem Express catalog HY-109080; catalog 1446113-23-0) showed mild to strong induction of this pathway in the same cell lines (in 11 of the 13 RAS-mutant cell lines tested, Y...). MIN <-30%).

[0700] (d) Using CellTiter- Reagents measure the inhibition of proliferation in cultured human cancer cell lines (CCLs)

[0701] CellTiter- Viability analysis was performed on cells plated in 40 μL of complete RPMI-1640 growth medium in 96-well flat-bottomed white opaque plates (Greiner or Corning) at the density indicated in Table E (number of cells plated per well for each CCL, to be determined for CellTiter analysis). Cell viability assay). (cells / cm²) 2 Cell density in units is equivalent to the number of cells divided by the area of ​​one well in a 96-well plate (0.32 cm²). 2 Cells were kept overnight at 37°C and 5% CO2, and then treated with a series of diluted compounds for 3 days.

[0702] Table E. Number of cells coated per well in a 96-well plate for CellTiter- Cell viability assay

[0703]

[0704]

[0705] In the dilution series, 100 μL / well of the test substance dilution prepared in complete RPMI-1640 growth medium is added to the cells initially plated in 100 μL of growth medium. A dilution series is selected to cover ten concentrations from 30 μM or 10 μM to 0.33 μM. If necessary (e.g., in the case of A375 cells, which are more sensitive to the compound), the initial concentration of 10 μM is reduced to 1 μM and further diluted accordingly. The final concentration of DMSO in the assay is set to 0.5%.

[0706] After 3 days of incubation, the growth medium was removed by aspiration, and 60 μL of diluted CellTiter was added to each well. Reagent (10 μL CellTiter- Reagent + 50 μL of PBS). Cells were incubated in CellTiter- by incubating for 5 minutes on a plate shaker, followed by 10 minutes at room temperature. The reagents were cleaved and equilibrated. The luminescence signal was then acquired using a Synergy Neo2 plate reader (Biotek).

[0707] The effect of each compound dilution on cancer cell line proliferation was expressed as a percentage of inhibition and calculated as follows. Each plate contained an internal 100% inhibition control (1 μM trametinib, catalog number HY-10999; MedChem Express; catalog number 871700-17-3) and was used as a CellTiter control. A measure of background signal. The value obtained by subtracting trametinib from each data point. The mean of the DMSO (mediator) control (set to 0% inhibition) was determined and used to calculate the inhibition percentage:

[0708] Inhibition % = 100 * (1 - ((CellTiter- Signal 化合物 ) / (CellTiter- Signal DMSO )))

[0709] The ability of each compound to inhibit proliferation is expressed as EC. 50 The values ​​were obtained by plotting the effect value for each data point in the diluted series and fitting the obtained curves using a log(agonist) versus response-variable slope (four parameters) function (using GraphPadPrism (V7.0) or the Dotmatics Screening Ultra platform).

[0710] As shown in Table D, the active substance exhibited antiproliferative activity in various NRAS-, KRAS-, and NF1- mutant cancer cell lines representing various tissue types (i.e., SK-MEL 30, IPC298, HepG2, HCT-116, Lovo, SW620, SW480, NCI-H358, NCI-H2122, Calu-6, and Mewo; Table D and refer to Table B for genotypes). The antiproliferative activity was generally even stronger in cell lines carrying BRAF driver mutations (Table D). Notably, pERK-reduced IC50... 50 EC values ​​and the antiproliferative activity of substances in KRAS- and BRAF- mutant cell lines 50 The values ​​correlated quite well with each other (Table D). Therefore, the compounds of the present invention are effective against a variety of tumor types and can be used for these and other indications. This demonstrates the usefulness of compounds as defined herein for treating different types of tumors.

[0711] (e) result

[0712] Tables 2 through 5 below summarize the structures, synthetic methods, and biological results of exemplary compounds. Each of these tables is followed by its own table summarizing the chemical characteristics of the compound.

[0713] Table 2

[0714]

[0715]

[0716]

[0717] For pERK assays, + indicates an IC50 concentration of 10 μM-30 μM. 50 Range, ++ indicates IC size from 1μM to 10μM 50 Range, +++ indicates IC size of 0.5μM-1μM. 50 Range, ++++ indicates IC 50 <0.5μM. Y min The % value represents each IC 50 The lowest value of the curve. This represents the Y-axis. min IC values ​​higher than -20% 50 Compounds in the curves are considered to exhibit minimal or no induction, not causing detectable paradoxical activation of the pathway. For BRAF biochemical kinase assays, * indicates IC50. 50 >10nM, ** indicates IC with a range of 1nM to 10nM. 50 Range, *** indicates IC 50 <1 nM. For CRAF biochemical kinase assays, § indicates IC50. 50 >50nM, §§ indicates IC 10nM-50nM. 50 Range, §§§ indicates IC 50 <10nM.

[0718] Characterization of compounds in Table 2

[0719]

[0720]

[0721]

[0722]

[0723] Table 3

[0724]

[0725]

[0726]

[0727]

[0728]

[0729] For pERK assays, + indicates an IC50 concentration of 10 μM-30 μM. 50 Range, ++ indicates IC size from 1μM to 10μM 50 Range, +++ indicates IC size of 0.5μM-1μM. 50 Range, ++++ indicates IC 50 <0.5μM. Y min The % value represents each IC 50 The lowest value of the curve. This represents the Y-axis. min IC values ​​higher than -20% 50 Compounds in the curves are considered to exhibit minimal or no induction, not causing detectable paradoxical activation of the pathway. For BRAF biochemical kinase assays, * indicates IC50. 50 >10nM, ** indicates IC with a range of 1nM to 10nM. 50 Range, *** indicates IC 50 <1 nM. For CRAF biochemical kinase assays, § indicates IC50. 50 >50nM, §§ indicates IC 10nM-50nM. 50 Range, §§§ indicates IC 50 <10nM.

[0730] Characterization of compounds in Table 3

[0731]

[0732]

[0733]

[0734]

[0735]

[0736]

[0737]

[0738]

[0739]

[0740]

[0741]

[0742]

[0743]

[0744]

[0745] Table 4

[0746]

[0747]

[0748] For pERK assays, + indicates an IC50 concentration of 10 μM-30 μM. 50 Range, ++ indicates IC size from 1μM to 10μM 50 Range, +++ indicates IC size of 0.5μM-1μM. 50 Range, ++++ indicates IC 50 <0.5μM. Y min The % value represents each IC 50 The lowest value of the curve. This represents the Y-axis. min IC values ​​higher than -20% 50 Compounds in the curves are considered to exhibit minimal or no induction, not causing detectable paradoxical activation of the pathway. For BRAF biochemical kinase assays, * indicates IC50. 50 >10nM, ** indicates IC with a range of 1nM to 10nM. 50 Range, *** indicates IC 50 <1 nM. For CRAF biochemical kinase assays, § indicates IC50. 50 >50nM, §§ indicates IC 10nM-50nM. 50 Range, §§§ indicates IC 50 <10nM.

[0749] Characterization of compounds in Table 4

[0750]

[0751] Table 5

[0752]

[0753]

[0754] For pERK assays, + indicates an IC50 concentration of 10 μM-30 μM. 50 Range, ++ indicates IC size from 1μM to 10μM 50 Range, +++ indicates IC size of 0.5μM-1μM. 50 Range, ++++ indicates IC 50 <0.5μM. Y min The % value represents each IC 50The lowest value of the curve. This represents the Y-axis. min IC values ​​higher than -20% 50 Compounds in the curves are considered to exhibit minimal or no induction, not causing detectable paradoxical activation of the pathway. For BRAF biochemical kinase assays, * indicates IC50. 50 >10nM, ** indicates IC with a range of 1nM to 10nM. 50 Range, *** indicates IC 50 <1 nM. For CRAF biochemical kinase assays, § indicates IC50. 50 >50nM, §§ indicates IC 10nM-50nM. 50 Range, §§§ indicates IC 50 <10nM.

[0755] Characterization of compounds in Table 5

[0756]

[0757] Various modifications may be made to any of the above embodiments without departing from the scope of the invention. Any references, patents or scientific literature mentioned in this document are incorporated herein by reference in their entirety for all purposes.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: Formula I in: R 1 It is a group of the following formula: ， R 17 Selected from H, OH, halogens, CN, NO2, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 acetylinyl, OC 1-6 Alkyl, C 5-10 heteroaryl, C 3-10 cycloalkyl, C 4-10 Heterocyclic alkyl, C(O)R 15 C(O)N(R) 14 2. SO2R 15 SO2N(R) 14 )2、N(R 16 )C(O)R 15 、N(R 16 SO2R 15 、N(R 16 )C(O)N(R 14 )2、N(R 16 SO2N(R) 14 )2、N(R 14 )2、P(O)(R 15 )2、CH2C(O)R 15 CH2C(O)N(R) 14 2. CH2SO2R 15 CH2SO2N(R) 14 )2、CH2N(R 16 )C(O)R 15 CH2N(R) 16 SO2R 15 CH2N(R) 16 )C(O)N(R 14 )2、CH2N(R 16 SO2N(R) 14 )2, and CH2N(R 14 )2; X 6 It is N or CH; and X 7 It is N and R 18 It does not exist; or, X 7 It is C and R 18 Selected from C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 acetylinyl, OC 1-6 Alkyl, C 5-10 heteroaryl, C 3-10 cycloalkyl, C 4-10 Heterocyclic alkyl, C(O)R 15 C(O)N(R) 14 2. SO2R 15 SO2N(R) 14 )2、N(R 16 )C(O)R 15 、N(R 16 SO2R 15 、N(R 16 )C(O)N(R 14 )2、N(R 16 SO2N(R) 14 )2、N(R 14 )2、P(O)(R 15 )2、CH2C(O)R 15 CH2C(O)N(R) 14 2. CH2SO2R 15 CH2SO2N(R) 14 )2、CH2N(R 16 )C(O)R 15 CH2N(R) 16 SO2R 15 CH2N(R) 16 )C(O)N(R 14 )2、CH2N(R 16 SO2N(R) 14 )2, and CH2N(R 14 )2; R 14 Each time it appears, it is independently selected from H and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 4-10 Heterocyclic alkyl, C6 aryl, and C 5-10 heteroaryl, or two R 14 Together with the nitrogen atoms adjacent to them, they form C 4-10 Heterocyclic alkyl groups; R 15 Each time it appears, it is independently selected from C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C6 aryl, and C 5-10 heteroaryl; and R 16 Each time it appears, it is independently selected from H and C. 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C6 aryl, and C 5-10 Mixed aromatics; The alkyl, alkenyl, ynyl, cycloalkyl, heterocycloalkyl, or heteroaryl groups are optionally further substituted; and Where (---) represents a key; R 2 The groups selected are B21, B36, B41, B42, B52, B53, B54, B59, B65, or B73 as defined below: X 1 It is a halogen or an electron-withdrawing group, wherein the electron-withdrawing group is selected from perhaloalkyl, CN, NO2, sulfonate, alkylsulfonyl, alkyl carbonyl, carboxylic acid ester, alkoxy carbonyl, and amino carbonyl; X 2 The group is selected from H, halogens, and electron-withdrawing groups, wherein the electron-withdrawing groups are selected from perhaloalkyl, CN, NO2, sulfonates, alkylsulfonyl, alkyl carbonyl, carboxylic acid esters, alkoxy carbonyl, and amino carbonyl. X 3 and X 4 Each is selected from H, halogen, electron-withdrawing group, C 1-3 Alkyl, C 3-4 cycloalkyl and OC 1-3 Alkyl group, wherein the electron-withdrawing group is selected from perhaloalkyl, CN, NO2, sulfonate, alkylsulfonyl, alkyl carbonyl, carboxylic acid ester, alkoxy carbonyl, and amino carbonyl; Y is selected from H, halogen, CN, OH, OC. 1-8 Alkyl, NH2, NHC 1-8 Alkyl, N(C) 1-8 Alkyl groups 2 and substituted or unsubstituted C 1-8 alkyl.

2. The compound of claim 1, wherein R 1 Selected from: Where R 14 and R 15 As defined in claim 1, and (---) represents a key.

3. The compound of claim 1, wherein the compound is selected from compounds of the following formula or salts thereof: Where R 1 and R 2 As defined in the table below:

4. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 3 and a pharmaceutically acceptable carrier, diluent, or excipient.

5. Use of the compound as defined in any one of claims 1 to 3 for the preparation of a medicament for treating a disease or condition selected from proliferative disorders or conditions, developmental abnormalities caused by RAS-ERK signaling cascade dysregulation, or inflammatory diseases or immune system disorders.

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