Inhibitors of MYC and uses thereof

Abstract

Substituted heterocyclic compounds and chimeric molecules (PROTAC) that target proteolysis are disclosed. The substituted heterocycles disclosed herein are demonstrated to be useful for the inhibition of c-MYC. The disclosed PROTACs are demonstrated to induce degradation of the c-MYC protein. The substituted heterocyclic compounds and chimeric molecules targeting proteolysis (PROTAC) disclosed herein are useful as therapeutic agents for the treatment of cancer and cell proliferative disorders.

Description

[0001] Cross-references to related patent applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 369,892, filed on July 29, 2022, the contents of which are incorporated by reference in their entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant Nos. CA180995 and CA257258 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0005] background

[0006] The field of the invention relates to substituted heterocycles as c-MYC targeting agents and proteolytically targeted chimeric molecules (PROTACs) that induce degradation of the c-MYC protein. Specifically, the field of the invention relates to substituted pyrazoles, imidazoles, or triazoles as c-MYC targeting agents, and PROTACs that target c-MYC for degradation, which can be used to treat cell proliferative diseases and disorders such as cancer.

[0007] The c-MYC oncogene is dysregulated and plays a pathogenic role in most human cancers, and c-MYC inhibition significantly affects tumor growth or survival in multiple models. MYC is the most common oncogene associated with human cancer and is overexpressed in up to half of all cancers. Therefore, developing c-MYC inhibitors is one of the most attractive potential anti-cancer strategies. Unfortunately, due to the difficulty of targeting transcription factors with small molecules, c-MYC is currently considered "undruggable." Compounds that selectively target c-MYC-driven cell proliferation and interfere with the binding of c-MYC to DNA can be found, for example, in U.S. Publication No. 2017 / 0253581, published on September 7, 2017, U.S. Publication No. 2019 / 0062281, published on February 28, 2019, U.S. Publication No. 2020 / 0390894, published on December 17, 2020, and U.S. Publication No. 2020 / 0392116, published on December 17, 2020, the contents of which are incorporated herein by reference in their entirety.

[0008] Chimeric molecules targeting proteolysis (PROTACs) are an emerging technology that can be used to target previously "undruggable" targets, such as transcription factors and non-enzymatic proteins. (See, e.g., An et al., "Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs," EBioMedicine. 2018 Oct;36:553-562; and Gu et al., "PROTACs: An Emerging Targeting Technique for Protein Degradation in Drug Discovery," Bioessays. 2018 Apr;40(4):e1700247, the contents of which are incorporated herein by reference in their entirety). PROTACs are chimeric molecules that can be characterized as "heterobifunctional" because PROTACs include a ligand for recruiting an E3 ubiquitin ligase, a linker, and another ligand that binds to the protein targeted for degradation. By design, PROTACs “hijack” E3 ubiquitin ligases to proteins targeted for degradation via ubiquitination, even if the targeted proteins are not physiological substrates for degradation via the ubiquitin-proteasome system.

[0009] Here, we disclose a strategy to selectively target c-MYC-driven cell proliferation and interfere with c-MYC binding to DNA. We also disclose PROTACs that induce c-MYC protein degradation.

[0010] Overview

[0011] Disclosed are substituted heterocycles useful as c-MYC targeting agents. The substituted heterocycles may include substituted pyrazoles, substituted imidazoles, and substituted triazoles. The disclosed heterocycles may be used in pharmaceutical compositions and methods for treating cell proliferative disorders, such as cancer.

[0012] Disclosed substituted heterocycles may include substituted pyrazoles, imidazoles, and triazoles having Formula I:

[0013]

[0014] in

[0015] W is CR 8 or N;

[0016] Y is CH or N;

[0017] Q is CR 2 or N;

[0018] Z is C(Alk 2 ) q (X) p (Alk 1 ) n R 1 or N;

[0019] R 1 is hydrogen, halogen, alkyl, aryl, benzyl, heteroaryl, cycloalkyl, alkoxy or cycloheteroalkyl, wherein R 1 Optionally, at one or more positions, the alkyl, alkoxy, cycloalkyl, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, carboxyl, -(CH2)-NH-R 9 、-(O(CH2)2) m -R 15 OR 11 One or more substitutions in ;

[0020] Alk 1 is a straight-chain or branched alkylene or cycloalkylene group;

[0021] n is 0, 1, or 2;

[0022] p is 0 or 1;

[0023] Alk 2 is a straight-chain or branched-chain alkylene group;

[0024] q is 0 or 1;

[0025] r is 0 or 1;

[0026] m is an integer selected from 1 to 20;

[0027] X is O or NR 13 ;

[0028] R 2 is hydrogen or a halogen;

[0029] R 3 is alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, -OC(O)-alkyl, or halogen;

[0030] R 4 is hydrogen, halogen, amino, alkyl or haloalkyl; or R 4 is aryl or benzyl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, carboxyl, aryloxy, and alkylaryloxy; or R 4 is an alkyl group optionally substituted with a haloaryloxy group; or R 4 With R 1together forming a cycloheteroalkyl group fused to Ring A, wherein the cycloheteroalkyl group fused to Ring A is optionally substituted with an aryl group or an alkylaryl group, which is optionally substituted with a halogen;

[0031] R 5 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 5 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy;

[0032] R 6 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 6 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy;

[0033] R 7 is an alkyl group;

[0034] R 8 is hydrogen, cyano, amino, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, benzyl, hydroxy, halogen, amido, and carboxyl;

[0035] R 9 is optionally substituted with one or more -P(O)(OR 10 )2 substituted alkyl;

[0036] R 10 is hydrogen or an alkyl group;

[0037] R 11 is optionally substituted with one or more -P(O)(OR 12 )2 substituted alkyl;

[0038] R 12 is hydrogen or an alkyl group;

[0039] R 13 is hydrogen, alkyl or -C(O)R 14 ;

[0040] R 14 is aryl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl;

[0041] R 15 is OR 16 、-OS(O)2-R 16 or NR17 R 18 ;

[0042] R 16 is an alkyl or aryl group, wherein R 16 is optionally substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl; and

[0043] R 17 and R 18 is independently hydrogen or alkyl.

[0044] The disclosed compounds may exhibit one or more biological activities. The disclosed compounds may inhibit the binding of the Myc / Max complex to DNA (e.g., in a DNA gel shift assay). The disclosed compounds may not produce significant DNA damage (e.g., in an rH2AX staining assay, at a concentration greater than about 0.001 μM, 0.005 μM, 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM or higher). The disclosed compounds may inhibit the growth of cells expressing c-MYC (preferably at a concentration less than about 100 μM, 50 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM, 0.001 μM or lower). The disclosed compounds may not inhibit the growth of cells that do not express c-MYC (preferably at concentrations greater than about 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM or more).

[0045] Also disclosed are chimeric molecules (PROTACs) that target proteolysis and induce c-MYC protein degradation. The disclosed PROTACs comprise a portion that binds to c-MYC, which is covalently linked to a portion that binds to a ubiquitin ligase. The disclosed PROTACs generally include a first targeting moiety (M) that binds to c-MYC. c-MYC ), which may be derived from a substituted heterocycle such as a substituted pyrazole that binds to c-MYC. The first targeting moiety may be covalently linked to a second targeting moiety (M) that binds to an ubiquitin ligase such as an E3 ubiquitin ligase via a bond or linker (L). E3 ). Thus, the disclosed PROTACs can be described as having Formula II or Formula M c-MYC -LM E3 or M E3 -LM c-MYC .

[0046] The disclosed PROTACs target the E3 ubiquitin ligase moiety to c-MYC, which is subsequently ubiquitinated and targeted for degradation. The disclosed PROTACs can be used to treat diseases and disorders associated with c-MYC, such as cell proliferative diseases and disorders, including cancer.

[0047] The disclosed PROTACs generally include a first targeting moiety (M) that binds to c-MYC. c-MYC ), which is derived from a substituted heterocycle that binds to c-MYC. Suitable substituted heterocycles that bind to c-MYC may include, but are not limited to, substituted pyrazoles.

[0048] c-MYC targeting portion of the disclosed PROTAC (M c-MYC ) is typically linked via a bond or linker (L) to a second targeting moiety (M) that binds to an E3 ubiquitin ligase. E3 ).

[0049] Suitable linkers for the disclosed PROTACs may include, but are not limited to, linkers comprising a polyethylene glycol moiety.

[0050] The E3 ubiquitin ligase targeting portion (M) of the disclosed PROTAC E3 ) generally binds to a PROTAC and / or targets a PROTAC to an E3 ubiquitin ligase. Suitable E3 ubiquitin ligases may include, but are not limited to, von Hippel-Lindau (VHL) E3 ubiquitin ligase, cereblon (CRBN) E3 ubiquitin ligase, inhibitor of apoptosis protein (IAP) E3 ubiquitin ligase, and mouse double minute 2 homolog (MDM2) E3 ubiquitin ligase.

[0051] The E3 ubiquitin ligase targeting portion (M) of the disclosed PROTAC E3 ) are generally derived from compounds that bind to E3 ubiquitin ligases, for example, as ligands for E3 ubiquitin ligases. Suitable ligands may include, but are not limited to, thalidomide, pomalidomide, lenalidomide, VHL ligand 1 (VHL-1), VHL ligand 2 (VHL-2), VH032, VL-269, LCL161, hydroxyproline-based ligands, and HIF-1α-derived (R)-hydroxyproline ligands, including residue-bound forms.

[0052] The disclosed PROTACs may exhibit one or more biological activities. The disclosed PROTACs may inhibit the growth of cells expressing c-MYC (preferably at a concentration of less than about 100 μM, 50 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM, 0.001 μM or less). The disclosed PROTACs may not inhibit the growth of cells that do not express c-MYC (preferably at a concentration of greater than about 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM and 100 μM or more).

[0053] In certain embodiments, the PROTAC has the formula: c-MYC -LM E3 , where M c-MYC It is the part that binds to c-MYC, and L is covalently linked to M MYC and M E3 The key or joint, and M E3 is a moiety that binds to an E3 ubiquitin ligase. In certain embodiments, the PROTAC has Formula II:

[0054]

[0055] in

[0056] R 1 is hydrogen, cyano, amino, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, benzyl, hydroxy, halogen, amido, and carboxyl;

[0057] R 2 is an alkyl group;

[0058] R 3 is alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, -OC(O)-alkyl, or halogen;

[0059] R 4 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 4 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy;

[0060] X is O or NR 5 ;

[0061] R 5 is hydrogen, alkyl or -C(O)R 6 ;

[0062] R 6is aryl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl;

[0063] Y is alkylene, arylene, benzylene, heteroarylene, cycloalkylene, or cycloheteroalkylene;

[0064] L is a bond or a linker selected from: -(O(CH2)2) a -、-(O(CH2)2) a -NH-C(O)-Alk 3 -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -、-(O(CH2)2) a -C(O)-NH-Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-Alk 3 -NH-C(O)-CH2- and -(O(CH2)2) a -C(O)-NH-Alk 3 -;

[0065] a is an integer selected from 1-20;

[0066] b is an integer selected from 1 to 20;

[0067] Alk 3 is a straight-chain or branched-chain alkylene group;

[0068] M E3 Selected from

[0069]

[0070] R 7 is optionally amino or -S(O)2R 8 One or more substituted alkyl groups;

[0071] R 8 is an alkyl or aryl group, wherein R 8is optionally substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl; and

[0072] R 9 、R 10 and R 11 is independently hydrogen or alkyl.

[0073] Also disclosed are pharmaceutical compositions comprising the disclosed compounds and / or disclosed PROTACs and a suitable pharmaceutical carrier, excipient, or diluent. The disclosed pharmaceutical compositions may comprise an effective amount of the disclosed compounds and / or PROTACs for inhibiting the growth of cancer cells when administered to a subject in need thereof.

[0074] Also disclosed are methods for treating cell proliferative diseases and disorders (such as cancer). The methods may include administering the disclosed compounds and / or disclosed PROTACs or pharmaceutical compositions comprising the disclosed compounds to a subject in need thereof (e.g., a subject suffering from cancer). The disclosed compounds and / or PROTACs or pharmaceutical compositions comprising the disclosed compounds and / or PROTACs may be administered together with another therapeutic agent, optionally in combination, to treat cell proliferative diseases and disorders. Cell proliferative diseases and disorders treated by the disclosed methods may include, but are not limited to, cancers selected from multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Describes the compound A4BC1R2 (NUCC-0226279) 19 F NMR.

[0077] Figure 2 Shown is a PK study of NUCC-0226545 administered orally ("545" represents compound NUCC-0226545; "975" represents compound NUCC-0200975).

[0078] Figure 3 Shown is a PK study of NUCC-0226605 via oral administration ("605" represents compound NUCC-0226605; "606" represents compound NUCC-0226606; "975" represents compound NUCC-0200975).

[0079] Figure 4PK studies of NUCC-0226606 via oral administration are shown ("7037" represents compound NUCC-0227037; "416" represents compound NUCC-0202416; "975" represents compound NUCC-0200975).

[0080] Figure 5 Shown is a PK study of NUCC-0227037 ("7037" represents compound NUCC-0227037; "975" represents compound NUCC-0200975) administered orally.

[0081] Figure 6 A PK study of NUCC-0202416 via oral administration is shown ("416" represents compound NUCC-0202416; "975" represents compound NUCC-0200975).

[0082] Figure 7A Depicted are changes in tumor volume in test mice following once-daily oral administration of 100 mg / kg MYCi 975 to mice ("MYCi 975" stands for compound NUCC-0200975).

[0083] Figure 7B Depicted are changes in tumor volume in test mice following once-daily oral administration of 100 mg / kg MYCi 605 to mice ("MYCi 605" stands for compound NUCC-0226605).

[0084] Figure 7C Depicted are changes in tumor volume in test mice following once-daily oral administration of 30 mg / kg and 10 mg / kg MYCi 605 to mice ("MYCi 605" stands for compound NUCC-0226605).

[0085] Figure 7D Depicted are changes in tumor volume in test mice following once-daily oral administration of 100 mg / kg, 30 mg / kg, and 10 mg / kg of MYCi 606 to mice ("MYCi 606" stands for compound NUCC-0226606).

[0086] Figure 7E Depicted are changes in tumor volume in test mice following once-daily oral administration of 100 mg / kg, 30 mg / kg, and 10 mg / kg of MYCi 7037 to mice ("MYCi 7037" stands for compound NUCC-0227037).

[0087] Figure 8ADepicted are changes in body weight in test mice following once-daily oral administration of 100 mg / kg MYCi 975 to mice ("MYCi 975" stands for compound NUCC-0200975).

[0088] Figure 8B Depicted are changes in body weight in test mice following oral administration of 100 mg / kg MYCi 605 to mice once daily ("MYCi 605" stands for compound NUCC-0226605).

[0089] Figure 8C Depicted are changes in body weight in test mice following once-daily oral administration of 30 mg / kg and 10 mg / kg MYCi 605 to mice ("MYCi 605" stands for compound NUCC-0226605).

[0090] Figure 8D Depicted are changes in body weight in test mice following once daily oral administration of 100 mg / kg, 30 mg / kg, and 10 mg / kg MYCi 606 to mice ("MYCi 606" stands for compound NUCC-0226606).

[0091] Figure 8E Depicted are changes in tumor volume in test mice following once-daily oral administration of 100 mg / kg, 30 mg / kg, and 10 mg / kg of MYCi 7037 to mice ("MYCi 7037" stands for compound NUCC-0227037).

[0092] Figure 9 NUCC-0227247 (ie, P2-2203-1) is shown 1 H NMR.

[0093] Figure 10 NUCC-0227245 (ie, P2-2203-2) is shown 1 H NMR.

[0094] Figure 11 NUCC-0227246 (i.e., P2-2203-2A) is shown 1 H NMR.

[0095] Figure 12 NUCC-0227244 (ie, P2-2112-3) is shown 1 H NMR.

[0096] Detailed description

[0097] The invention is described herein using several definitions, as set forth below and throughout this application.

[0098] Unless the context indicates otherwise, the terms "a", "an" and "the" mean "one or more". For example, "a compound" should be interpreted as meaning "one or more compounds".

[0099] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If the context in which they are used gives use of these terms that is unclear to one of ordinary skill in the art, "about" and "approximately" will mean plus or minus ≤10% of the particular term, and "substantially" and "significantly" will mean plus or minus >10% of the particular term.

[0100] As used herein, the term "comprising" has the same meaning as the term "including," in that these latter terms are "open" transitional terms that do not limit the claim to only the listed elements that follow the transitional term. Although encompassed by the term "comprising," the term "consisting of" should be interpreted as a "closed" transitional term that limits the claim to only the listed elements that follow the transitional term. Although encompassed by the term "comprising," the term "consisting essentially of" should be interpreted as a "partially closed" transitional term that allows for additional elements that follow the transitional term, provided that those additional elements do not materially affect the basic and novel characteristics of the claim.

[0101] As used herein, "subject" is interchangeable with "patient" or "individual" and refers to an animal, which may be a human or non-human animal, in need of treatment.

[0102] A "subject in need of treatment" can include a subject having a disease, disorder, or condition that is responsive to treatment with a substituted heterocycle (such as the presently disclosed substituted pyrazoles, substituted imidazoles, and substituted triazoles, or a proteolysis-targeting chimeric molecule (PROTAC) that is targeted to c-MYC to achieve degradation of c-MYC). For example, a "subject in need of treatment" can include a subject having a cell proliferative disease, disorder, or condition such as cancer (e.g., cancers such as multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer).

[0103] As used herein, the phrase "effective amount" shall mean a dosage of a drug that provides a specific pharmacological response when administered to a large number of subjects in need of such treatment. An effective amount of a drug administered to a particular subject under a particular circumstances is not always effective to treat the conditions / diseases described herein, even if such a dosage is considered a therapeutically effective amount by one skilled in the art.

[0104] Chemical Entity

[0105] Disclosed herein are novel chemical entities and uses of chemical entities. Chemical entities can be described using terms known in the art and are discussed further below.

[0106] The wavy lines used in this article Can be used to indicate the point of attachment of any residue group or substituent.

[0107] The term "alkyl" as contemplated herein includes straight or branched chain alkyl residues in all isomeric forms thereof, such as straight or branched chain groups of 1-12, 1-10 or 1-6 carbon atoms, referred to herein as C1-C2, C3-C4, C5-C6, C6-C7, C7-C8, C8-C9, C9-C10, C11-C12, C12-C10, C11-C12, 12 -alkyl, C1-C 10 -alkyl and C1-C6-alkyl. The number of carbon atoms in an alkyl group can be specified using the Cx-Cy nomenclature, where x and y are integers representing the number of carbon atoms.

[0108] The term "alkylene" refers to the di-residue of a straight-chain or branched alkyl group (i.e., the di-residue of a straight-chain or branched C1-C6 alkyl group). Exemplary alkylene groups include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)CH2-, -CH(CH2CH3)CH2-, and the like.

[0109] The term "halo" or "halogen" refers to the residue of -F, -Cl, -Br or -I.

[0110] The term "haloalkyl" refers to an alkyl group substituted with at least one halogen, for example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, etc.

[0111] The term "alkoxy" or "alkoxy" is art-recognized and refers to an alkyl group as defined above having an oxy group attached thereto. Representative alkoxy groups include methoxy, ethoxy, tert-butoxy, and the like.

[0112] The term "haloalkoxy" denotes an alkoxy group as defined above substituted with at least one halogen.

[0113] The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8 or 4-6 carbon atoms, for example, a "C" derived from a cycloalkane. 4-8-cycloalkyl". The number of carbon atoms in a cycloalkyl group can be designated using the Cx-Cy nomenclature, where x and y are integers representing the number of carbon atoms. Unless otherwise indicated, a cycloalkyl group is optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxamido (or amidocarboxy), amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl, or thiocarbonyl. In certain embodiments, the cycloalkyl group is unsubstituted, i.e., it is unsubstituted.

[0114] The term "cycloheteroalkyl" refers to a monovalent saturated cyclic, bicyclic or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8 or 4-6 carbons, wherein at least one carbon of the cycloalkane is replaced by a heteroatom such as, for example, N, O and / or S.

[0115] As used herein, the term "cycloheteroalkylene" denotes a diradical of a cycloheteroalkyl group as defined above.

[0116] The term "cycloalkylene" refers to a diradical of a cycloalkyl group as defined above. Exemplary cycloalkylene groups include, but are not limited to wait.

[0117] The term "aryl" is generally recognized in the art and represents a carbocyclic aromatic group containing, for example, 5-12, 6-10 or 5-8 carbon. The number of carbon atoms in the aryl group can be specified using the Cx-Cy nomenclature, where x and y are integers representing the number of carbon atoms. Representative aryl groups include phenyl, naphthyl, anthracenyl, etc. The term "aryl" includes a polycyclic ring system with two or more carbocyclic rings, wherein two or more carbons are shared by two adjacent rings (the ring is a "fused ring"), wherein at least one of the rings is aromatic, and for example, other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl and / or aryl. Unless otherwise indicated, the aromatic ring can be substituted by, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, sulfydryl, imino, amido or carboxyamido (or amidocarboxyl), carboxylic acid,-C (O) alkyl,-CO alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclic radical, aryl or heteroaryl moiety,-CF ,-CN etc. at one or more ring positions. In certain embodiments, the aromatic ring is substituted by halogen, alkyl, hydroxyl or alkoxy at one or more ring positions. In certain other embodiments, the aromatic ring is unsubstituted, that is, it is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure.

[0118] The term "arylene" as used herein denotes a di-residue of an aryl group as defined above.

[0119] The term "alkylaryl" refers to a single radical having an alkylene group attached to an aryl group.

[0120] The term "aryloxy" is art-recognized and refers to an aryl group as defined above having an oxygen radical attached thereto. Representative alkoxy groups include phenoxy and the like.

[0121] The term "alkylaryloxy" refers to a monoradical having an alkylene group attached to an aryloxy group.

[0122] As used herein, the term "heteroaryl" refers to monocyclic and bicyclic heteroaryl groups. A monocyclic heteroaryl group is a 5- or 6-membered ring. The 5-membered ring contains two double bonds. The 5-membered ring may contain at least one heteroatom selected from the group consisting of O, S, and / or nitrogen; or 1, 2, 3, or 4 nitrogen atoms and optionally 1 oxygen or 1 sulfur atom. The 6-membered ring may contain 3 double bonds and 1, 2, 3, or 4 nitrogen, oxygen, and / or sulfur atoms. Representative examples of monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1,3-oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, 1,3-thiazolyl, thienyl, triazolyl, and triazinyl. Bicyclic heteroaryl consists of a monocyclic heteroaryl fused to a phenyl group, a monocyclic heteroaryl fused to a monocyclic cycloalkyl group, a monocyclic heteroaryl fused to a monocyclic cycloalkenyl group, a monocyclic heteroaryl fused to a monocyclic heteroaryl group, or a monocyclic heteroaryl fused to a monocyclic heterocycle. Representative examples of bicyclic heteroaryl include, but are not limited to, benzofuranyl, benzothiophenyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, phthalazinyl, 2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl, 6,7-dihydro-pyrazolo[1,5-a]pyrazin-5(4H)-yl, 6,7-dihydro-1,3-benzothiazolyl, imidazole [1,2-a]pyridinyl, indazolyl, indolyl, isoindolyl, isoquinolinyl, naphthyridinyl, pyridoimidazolyl, quinolinyl, 2,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl, thiazolo[5,4-b]pyridin-2-yl, thiazolo[5,4-d]pyrimidin-2-yl and 5,6,7,8-tetrahydroquinolin-5-yl. Unless otherwise indicated, the monocyclic and bicyclic heteroaryl groups (including the exemplary rings) are optionally substituted. The monocyclic and bicyclic heteroaryl groups are attached to the parent molecular moiety through any substitutable carbon atom or any substitutable nitrogen atom contained within the ring system. The nitrogen atom in the heteroaryl ring can be optionally oxidized and can be optionally quaternized.

[0123] The term "heteroaryloxy" denotes a heteroaryl group as defined above having an oxygen residue attached thereto.

[0124] The term "heteroarylene" as used herein denotes a diradical of a heteroaryl group as defined above.

[0125] The terms "heterocyclyl" and "heterocyclic group" are well known in the art and represent saturated, partially unsaturated or aromatic 3-10 ring structures, alternatively represent 3-7 rings, whose ring structures include 1 to 4 heteroatoms, such as nitrogen, oxygen and sulfur. The number of ring atoms in the heterocyclyl group can be specified using the "x yuan to y yuan" nomenclature, where x and y are integers representing the number of ring atoms. For example, a 3 yuan to 7 yuan heterocyclyl group represents a saturated or partially unsaturated 3-7 ring structure containing 1 to 4 heteroatoms (such as nitrogen, oxygen and sulfur). The name indicates that the heterocycle contains a total of 3-7 ring atoms, including any heteroatoms occupying the ring atom positions.

[0126] The terms "amine" and "amino" are art-recognized and refer to unsubstituted and substituted amines (e.g., monosubstituted amines or disubstituted amines), where substituents can include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl groups.

[0127] An "ether" is two hydrocarbons covalently linked by an oxygen. Thus, an alkyl substituent that makes an alkyl group an ether is or resembles an alkoxy group, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, and the like.

[0128] As used herein, the term "carbonyl" refers to the residue -C(O)-.

[0129] The term "oxo" refers to a divalent oxygen atom

[0130] As used herein, the term "carboxamido" refers to the residue -C(O)NRR', wherein R and R' can be the same or different. For example, R and R' can independently be hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, heteroaryl, or heterocyclyl.

[0131] As used herein, the term "carboxy" or "carboxyl" refers to the residue -COOH or its corresponding salts, such as -COONa and the like.

[0132] As used herein, the term "amide" or "amido" or "amido" refers to a group of the form -R 1 C(O)N(R 2 )-、-R 1 C(O)N(R 2 )R 3 -、-C(O)NR 2 R 3 or -C(O)NH2 residue, where R 1 、R 2 and R 3For example, each is independently hydrogen, alkyl, alkoxy, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxy, ketone or nitro.

[0133] The term "alkenyl" as used herein refers to an unsaturated straight or branched chain hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched chain group of 2-12, 2-10, or 2-6 carbon atoms, respectively referred to herein as C2-C 12 -alkenyl, C2-C 10 -alkenyl and C2-C6-alkenyl.

[0134] The term "alkynyl" as used herein refers to an unsaturated straight or branched chain hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched chain group of 2-12, 2-10, or 2-6 carbon atoms, respectively referred to herein as C2-C 12 -alkynyl, C2-C 10 -alkynyl and C2-C6-alkynyl.

[0135] The term "benzyl" as used herein refers to a group consisting of -C6H4-CH2- ).

[0136] The term "benzylidene" as used herein denotes the di-residue of a benzyl group as defined above.

[0137] The term "cyano" refers to a substituent of "-CN".

[0138] The term "hydroxy" refers to a substituent of "-OH".

[0139] The compounds and molecules of the present disclosure (e.g., PROTACs) may contain one or more chiral centers and / or double bonds and therefore exist as stereoisomers (such as geometric isomers, enantiomers, or diastereomers). When used herein, the term "stereoisomer" consists of all geometric isomers, enantiomers, or diastereomers. These compounds and molecules can be represented by the symbols "R" or "S", or "+" or "-", depending on the configuration of substituents around the stereogenic carbon atom and / or the observed optical rotation. The present invention encompasses various stereoisomers of these compounds and molecules and mixtures thereof. Stereoisomers include enantiomers and diastereomers. A mixture of enantiomers or diastereomers can be represented by (±) in nomenclature, but a skilled artisan will recognize that a structure can implicitly represent a chiral center. It should be understood that, unless otherwise indicated, a diagrammatic depiction of a chemical structure (e.g., a generic chemical structure) encompasses all stereoisomeric forms of a given compound and molecule. Compositions comprising, consisting essentially of, or consisting of enantiomerically pure compounds are also contemplated herein, which compositions may comprise, consist essentially of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 99% of the R enantiomer of a given compound).

[0140] The formulas of the compounds and molecules disclosed herein should be interpreted as encompassing all possible stereoisomers, enantiomers, or epimers of the compounds and molecules, unless the formula indicates a specific stereoisomer, enantiomer, or epimer. The formulas of the compounds and molecules disclosed herein should be interpreted as encompassing salts, esters, amides, or solvates thereof of the compounds and molecules.

[0141] Substituted heterocycles as MYC inhibitors

[0142] Disclosed herein are substituted heterocycles. The disclosed heterocycles have been shown to inhibit the biological activity of c-MYC. The disclosed substituted heterocycles may include substituted pyrazoles, substituted imidazoles, and substituted triazoles.

[0143] In certain embodiments, the disclosed substituted heterocycles may have Formula I:

[0144]

[0145] in

[0146] W is CR 8 or N;

[0147] Y is CH or N;

[0148] Q is CR2 or N;

[0149] Z is C(Alk 2 ) q (X) p (Alk 1 ) n R 1 or N;

[0150] R 1 is hydrogen, halogen, alkyl, aryl, benzyl, heteroaryl, cycloalkyl, alkoxy or cycloheteroalkyl, wherein R 1 Optionally, at one or more positions, the alkyl, alkoxy, cycloalkyl, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, carboxyl, -(CH2)-NH-R 9 、-(O(CH2)2) m -R 15 OR 11 One or more substitutions in ;

[0151] Alk 1 is a straight-chain or branched alkylene or cycloalkylene group;

[0152] n is 0, 1, or 2;

[0153] p is 0 or 1;

[0154] Alk 2 is a straight-chain or branched-chain alkylene group;

[0155] q is 0 or 1;

[0156] r is 0 or 1;

[0157] m is an integer selected from 1 to 20;

[0158] X is O or NR 13 ;

[0159] R 2 is hydrogen or a halogen;

[0160] R 3 is alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, -OC(O)-alkyl, or halogen;

[0161] R 4 is hydrogen, halogen, amino, alkyl or haloalkyl; or R 4 is aryl or benzyl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, carboxyl, aryloxy, and alkylaryloxy; or R 4 is an alkyl group optionally substituted with a haloaryloxy group; or R4 With R 1 together forming a cycloheteroalkyl group fused to Ring A, wherein the cycloheteroalkyl group fused to Ring A is optionally substituted with an aryl group or an alkylaryl group, which is optionally substituted with a halogen;

[0162] R 5 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 5 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy;

[0163] R 6 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 6 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy;

[0164] R 7 is an alkyl group;

[0165] R 8 is hydrogen, cyano, amino, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, benzyl, hydroxy, halogen, amido, and carboxyl;

[0166] R 9 is optionally substituted with one or more -P(O)(OR 10 )2 substituted alkyl;

[0167] R 10 is hydrogen or an alkyl group;

[0168] R 11 is optionally substituted with one or more -P(O)(OR 12 )2 substituted alkyl;

[0169] R 12 is hydrogen or an alkyl group;

[0170] R 13 is hydrogen, alkyl or -C(O)R 14 ;and

[0171] R 14 is aryl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl;

[0172] R 15 is OR 16、-OS(O)2-R 16 or NR 17 R 18 ;

[0173] R 16 is an alkyl or aryl group, wherein R 16 is optionally substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl; and

[0174] R 17 and R 18 is independently hydrogen or alkyl.

[0175] In certain embodiments, the disclosed substituted heterocycles can have the structure of Formula I, wherein:

[0176] R 1 is hydrogen, alkyl, aryl, benzyl, where R 1 Optionally, at one or more positions, alkyl, alkoxy, aryl, halogen, -(CH2)-NH-R 9 OR 11 One or more substitutions in ;

[0177] R 2 is hydrogen or chlorine;

[0178] R 3 is hydroxy or -OC(O)Me;

[0179] R 4 is hydrogen or a halogen;

[0180] R 5 is hydrogen or a halogen;

[0181] R 6 is hydrogen, aryl, benzyl, optionally R 6 substituted at one or more positions with one or more of haloalkyl, halogen, and cyano;

[0182] R 8 is cyano, amino, haloalkyl or acylamino;

[0183] R 14 is aryl optionally substituted at one or more ring positions with one or more of haloalkyl or halogen; and

[0184] The remaining substituents of the compound of Formula I are as defined herein.

[0185] In certain embodiments, the compound is not 4'-chloro-6-((4-chlorobenzyl)oxy)-3-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-3'-(trifluoromethyl)-[1,1'-biphenyl]-2-ol or a compound disclosed in U.S. Publication No. 2017 / 0253581, published on September 7, 2017, U.S. Publication No. 2019 / 0062281, published on February 28, 2019, U.S. Publication No. 2020 / 0390894, published on December 17, 2020, or U.S. Publication No. 2020 / 0392116, published on December 17, 2020.

[0186] In certain embodiments, the disclosed substituted heterocycles may have Formula I(a), wherein the substituents are as defined in Formula I:

[0187]

[0188] In certain embodiments, the disclosed substituted heterocycles may include substituted pyrazoles wherein Y is N and W is CCF3.

[0189] In certain embodiments, the disclosed substituted heterocycles may have R 3 .

[0190] In certain embodiments, the disclosed substituted heterocycles may have r = 0 and R = phenyl substituted with chloro and trifluoromethyl. 6 .

[0191] In certain embodiments, the disclosed substituted heterocycles may have R being hydrogen. 2 .

[0192] In certain embodiments, the disclosed substituted heterocycles can be selected from:

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] Proteolytically targeted chimeric molecules (PROTACs) that induce c-MYC protein degradation

[0204] Also disclosed herein are chimeric molecules targeting proteolysis (PROTACs) that induce degradation of c-MYC protein. In certain embodiments, the disclosed molecules can be described as having the formula: c-MYC -LM E3 , or alternatively described as having the formula: M E3 -LM c-MYC , where M c-MYC It is the part that binds to c-MYC, and L is covalently linked to M MYC and M E3 The key or joint, and M E3 It is a portion that binds to E3 ubiquitin ligase. Compounds that bind to c-MYC are disclosed in the prior art and may include, but are not limited to, compounds disclosed in U.S. Publication No. 2019 / 0062281, published on February 28, 2019 (the contents of which are incorporated herein by reference in their entirety).

[0205] In certain embodiments of the disclosed PROTACs, the PROTAC has the formula: c-MYC -LM E3 , where M c-MYC It is the part that binds to c-MYC, and L is covalently linked to M MYC and M E3 The key or joint, and M E3 It is the part that binds to the E3 ubiquitin ligase.

[0206] In certain embodiments of the disclosed PROTACs, the PROTAC has Formula II:

[0207]

[0208] in

[0209] R 1 is hydrogen, cyano, amino, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, benzyl, hydroxy, halogen, amido, and carboxyl;

[0210] R 2 is an alkyl group;

[0211] R 3is alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, -OC(O)-alkyl, or halogen;

[0212] R 4 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 4 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy;

[0213] X is O or NR 5 ;

[0214] R 5 is hydrogen, alkyl or -C(O)R 6 ;

[0215] R 6 is aryl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl;

[0216] Y is alkylene, arylene, benzylene, heteroarylene, cycloalkylene, or cycloheteroalkylene;

[0217] L is a bond or a linker selected from: -(O(CH2)2) a -、-(O(CH2)2) a -NH-C(O)-Alk 3 -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -、-(O(CH2)2) a -C(O)-NH-Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-Alk 3 -NH-C(O)-CH2- and -(O(CH2)2) a -C(O)-NH-Alk 3 -;

[0218] a is an integer selected from 1-20;

[0219] b is an integer selected from 1 to 20;

[0220] Alk 3 is a straight-chain or branched-chain alkylene group;

[0221] M E3 Selected from

[0222] R 7 is optionally amino or -S(O)2R 8 One or more substituted alkyl groups;

[0223] R 8 is an alkyl or aryl group, wherein R 8 is optionally substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl; and

[0224] R 9 、R 10 and R 11 is independently hydrogen or alkyl.

[0225] In certain embodiments, the PROTAC is a trifluoromethyl-substituted 1-methyl-1H-pyrazole having Formula II(a):

[0226]

[0227] in

[0228] R 3 is hydroxy or -OC(O)-alkyl;

[0229] R 4 is hydrogen or phenyl substituted at one or more positions with one or more of haloalkyl or halogen;

[0230] X is O or -NC(O)R 6 ;

[0231] R 6 is an aryl group substituted at one or more ring positions with one or more of a haloalkyl group or a halogen;

[0232] Y is an alkylene group or a benzylene group;

[0233] L is a bond or a linker selected from: -(O(CH2)2) a -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -、-(O(CH2)2)a -C(O)-NH-Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-Alk 3 -NH-C(O)-CH2- and -(O(CH2)2) a -C(O)-NH-Alk 3 -;and

[0234] R 8 is an alkyl or aryl group, wherein R 8 Optionally substituted at one or more positions with one or more alkyl groups.

[0235] In certain embodiments of the disclosed PROTACs, the PROTAC has the structure of Formula II(a), wherein R 3 is a hydroxyl group, and R 4 It is a phenyl group substituted by trifluoromethyl and chlorine.

[0236] In certain embodiments of the disclosed PROTACs, the PROTAC has a structure of Formula II(a), wherein X is -NC(O)R 6 , and R 6 It is a phenyl group substituted by trifluoromethyl and chlorine.

[0237] In certain embodiments of the disclosed PROTACs, the PROTAC has the structure of Formula II(a), wherein X is O, and Y is propylene or benzylene.

[0238] In certain embodiments of the disclosed PROTACs, the PROTAC has an M selected from the group consisting of E3 Group:

[0239]

[0240]

[0241]

[0242] where R 19 is hydrogen or methyl.

[0243] In certain embodiments of the disclosed PROTACs, the PROTAC is selected from:

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251] The formulas of the compounds disclosed herein should be interpreted as encompassing all possible stereoisomers, enantiomers, or epimers of the compounds, unless the formula indicates a specific stereoisomer, enantiomer, or epimer. The formulas of the compounds disclosed herein should be interpreted as encompassing salts, esters, amides, or solvates thereof of the compounds.

[0252] The disclosed compounds may exhibit one or more biological activities. The disclosed compounds may inhibit the binding of the Myc / Max complex to DNA (e.g., in a DNA gel shift assay). In certain embodiments, the disclosed compounds inhibit the binding of the Myc / Max complex to DNA by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% at a concentration of less than about 100 μM, 50 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM, 0.001 μM or less. The disclosed compounds may not produce significant DNA damage (e.g., in an rH2AX staining assay, at a concentration greater than about 0.001 μM, 0.005 μM, 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM or more). The disclosed compounds may inhibit the growth of cells expressing c-MYC (preferably by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% at concentrations less than about 100 μM, 50 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM, 0.001 μM or less). The disclosed compounds may not inhibit the growth of cells that do not express c-Myc (preferably by no more than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or less at concentrations greater than about 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM and 100 μM or more). Concentration ranges are also contemplated herein, for example, those bounded by endpoint concentrations selected from 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM.

[0253] The disclosed compounds are effective in inhibiting cell proliferation of cancer cells, including cancer cells that express c-MYC and whose proliferation is inhibited by inhibiting the biological activity of c-MYC. The disclosed compounds can effectively inhibit cell proliferation of one or more types of cancer cells, including: multiple myeloma cells, such as MM.1S cells; leukemia cells, such as CCRF-CEM, HL-60 (TB), MOLT-4, RPMI-8226, and SR; non-small lung cancer cells, such as A549 / ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, and NCI-H522; colon cancer cells, such as COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, and SW-620; CNS: SF-268, SF-295, SF-539, SNB-19, SNB-75, and U251; melanoma cancer cells, such as LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, and UACC-62; ovarian cancer cells such as IGR-OV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI / ADR-RES, and SK-OV-3; renal cancer cells such as 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, and UO-31; prostate cancer cells such as DU-145 and PC-3; and breast cancer cells such as MCF7, MDA-MB-231 / ATCC, MDA-MB-468, HS 578T, BT-549, and T-47D.

[0254] Cell proliferation and its inhibition by the presently disclosed compounds can be assessed by cell viability methods disclosed in the art, including colorimetric assays utilizing dyes such as MTT, XTT, and MTS to assess cell viability. Preferably, the disclosed compounds have an IC of less than about 10 μM, 5 μM, 1 μM, 0.5 μM, 0.01 μM, 0.005 μM, 0.001 μM, or less in the selected assay. 50 .

[0255] The disclosed compounds can be formulated as anti-cancer therapeutics, including hematological malignancies, breast, lung, pancreatic and prostate malignancies. The disclosed compounds can also be formulated as anti-inflammatory therapeutics.

[0256] The compounds utilized in the methods disclosed herein can be formulated as pharmaceutical compositions comprising: (a) a therapeutically effective amount of one or more compounds disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition can include the compound in the range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition can be administered to provide a daily dose of the compound of about 0.1 to 100 mg / kg body weight (preferably about 0.5 to 20 mg / kg body weight, more preferably about 0.1 to 10 mg / kg body weight). In certain embodiments, after administration of the pharmaceutical composition to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours after administration), the concentration of the compound at the site of action can be within a concentration range bounded by endpoints selected from 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM (e.g., 0.1 μM-1.0 μM).

[0257] The disclosed compounds and pharmaceutical compositions comprising the disclosed compounds can be administered in methods of treating subjects in need thereof. For example, in methods of treatment, subjects in need thereof can include subjects having a cell proliferative disease, disorder, or condition such as cancer (e.g., cancers such as multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and breast cancer).

[0258] In certain embodiments of the disclosed methods of treatment, as little as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg can be administered to a subject once a day, twice a day, three times a day, four times a day, once a week, twice a week, or three times a week. , 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg or 2000 mg of the compound to treat the disease or disorder in the subject. In certain embodiments, up to 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg may be administered to a subject once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times weekly. The compound of the dosage of 1,2,4,5-dihydrotestosterone-1,4-drug, 1,2 ...

[0259] In certain embodiments, the minimum dose level of the compound to achieve treatment in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000 or 20000 ng / kg body weight of the subject. In certain embodiments, the maximum dose level of the compound to achieve treatment in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000 or 20000 ng / kg body weight of the subject. Minimum and / or maximum dosage levels of the compounds to achieve treatment in the disclosed methods of treatment may include dosage levels that fall within a range having any of the disclosed dosage levels as endpoints (e.g., 500-2000 ng / kg subject body weight).

[0260] The compounds utilized in the methods disclosed herein can be formulated as pharmaceutical compositions in solid dosage forms, although any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast-melt dosage form, a controlled-release dosage form, a lyophilized dosage form, a delayed-release dosage form, an extended-release dosage form, a pulsed-release dosage form, a mixed immediate-release and controlled-release dosage form, or a combination thereof.

[0261] The compounds utilized in the methods disclosed herein can be formulated as pharmaceutical compositions comprising a carrier. For example, the carrier can be selected from proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0262] The compounds utilized in the methods disclosed herein can be formulated as pharmaceutical compositions comprising one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Fillers can include lactose monohydrate, anhydrous lactose, and various starches; examples of binders are various celluloses and cross-linked polyvinyl pyrrolidone, microcrystalline cellulose such as PH101 and PH102, microcrystalline cellulose and silicified microcrystalline cellulose (ProSolv SMCC TM Suitable lubricants (including agents that affect the flow properties of the powder to be compressed) may include colloidal silicon dioxide, such as 200, talc, stearic acid, magnesium stearate, calcium stearate and silica gel. Examples of sweeteners may include any natural or artificial sweeteners such as sucrose, xylitol, saccharin sodium, sodium cyclamate, aspartame and acesulfame. Examples of flavoring agents are (a trademark of MAFCO), bubble gum flavoring, fruit flavoring, etc. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid (such as butylparaben), alcohols (such as ethyl or benzyl alcohol), phenolic compounds (such as phenol), or quaternary ammonium compounds (such as benzalkonium chloride).

[0263] Suitable diluents may include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, calcium hydrogen phosphate, sugars, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as PH101 and PH102; lactose such as lactose monohydrate, anhydrous lactose and DCL21; calcium hydrogen phosphate such as Mannitol; starch; sorbitol; sucrose; and glucose.

[0264] Suitable disintegrants include lightly cross-linked polyvinyl pyrrolidone, corn starch, potato starch, maize starch and modified starches, cross-linked sodium carboxymethylcellulose, crospovidone, sodium starch glycolate, and mixtures thereof.

[0265] The example of effervescent agent is effervescent partner such as organic acid and carbonate or bicarbonate.Suitable organic acid comprises for example citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, succinic acid and alginic acid and its anhydride and acid salt.Suitable carbonate and bicarbonate comprise for example sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, glycine carbonate sodium salt (sodium glycine carbonate), L-lysine carbonate and arginine carbonate.Alternatively, can only have the sodium bicarbonate component of effervescent partner.

[0266] The compounds utilized in the methods disclosed herein can be formulated as pharmaceutical compositions for delivery by any suitable route. For example, the pharmaceutical compositions can be administered orally, intravenously, intramuscularly, subcutaneously, topically, and by pulmonary routes. Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders, and granules.

[0267] The compounds utilized in the methods disclosed herein can be administered in conventional dosage forms prepared by combining the active ingredients with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating, and compressing or dissolving the ingredients to meet the desired preparation requirements.

[0268] The pharmaceutical composition comprising the compound can be suitable for administration by any appropriate route, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes. Such formulations can be prepared by any method known in the pharmaceutical art, for example, by combining the active ingredient with a carrier or excipient.

[0269] Pharmaceutical compositions suitable for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0270] Pharmaceutical compositions suitable for transdermal administration can be presented as discrete patches intended to remain in close contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient can be delivered from the patch by iontophoresis.

[0271] Pharmaceutical compositions suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration, and, in the case of ointments and creams, emollients.

[0272] For application to the eye or other external tissues (e.g., oral cavity and skin), the pharmaceutical composition is preferably applied as a topical ointment or cream. When formulated as an ointment, the compound can be used with a paraffinic or water-miscible ointment base. Alternatively, the compound can be formulated into a cream together with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions suitable for topical application to the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent.

[0273] Pharmaceutical compositions suitable for nasal administration (wherein the carrier is a solid) include coarse powders having a particle size (e.g., in the range of 20 to 500 microns) that are administered by snorting (i.e., by rapidly inhaling the powdered container close to the nose into the nasal passages). Formulations suitable for administration as nasal sprays or as nasal drops (wherein the carrier is a liquid) include aqueous or oily solutions of the active ingredient.

[0274] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents and solutes to make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be present in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and may be stored in freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier, such as water for injection, prior to use. Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0275] Tablets and capsules for oral administration may be in unit dose presentation and may contain conventional excipients such as binders, for example, syrups, gum arabic, gelatin, sorbitol, gum tragacanth, or polyvinyl pyrrolidone; fillers, for example, lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tableting lubricants, for example, magnesium stearate, talc, polyethylene glycol, or silicon dioxide; disintegrants, for example, potato starch; or acceptable wetting agents such as sodium lauryl sulfate. Tablets may be coated according to methods well known in ordinary pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product to be reconstituted with water or other suitable vehicles before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, emulsifiers, for example, lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, oily esters such as glycerol, propylene glycol or ethanol; preservatives, for example, methyl or propylparaben or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0276] The disclosed compounds or pharmaceutical compositions comprising the disclosed compounds can be administered in therapeutic methods. For example, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds can be administered in methods for treating cell proliferative diseases and disorders. Cell proliferative diseases and disorders treatable by the disclosed methods can include, but are not limited to, cancers selected from the group consisting of: multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer.

[0277] Optionally, the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds can be administered with additional therapeutic agents, optionally in combination, to treat cell proliferative diseases and disorders. In certain embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds, wherein the additional therapeutic agents are administered before, simultaneously with, or after administration of the disclosed compounds or pharmaceutical compositions comprising the disclosed compounds. In certain embodiments, the disclosed pharmaceutical compositions are formulated to comprise the disclosed compounds and further comprise one or more additional therapeutic agents, e.g., one or more additional therapeutic agents for treating cell proliferative diseases and disorders.

[0278] In certain embodiments, additional therapeutic agents may include, but are not limited to, therapeutic agents used to treat leukemias and lymphomas, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), and non-Hodgkin's lymphoma.

[0279] In certain embodiments, additional therapeutic agents may include, but are not limited to, antimetabolite antitumor agents that inhibit DNA synthesis. Suitable antimetabolite antitumor agents that inhibit DNA synthesis may include, but are not limited to, nucleoside and / or nucleotide derivatives. Suitable nucleoside and / or nucleotide derivatives may include, but are not limited to, cytosine arabinoside (ara-C), also known as cytosine arabinoside. Example

[0280] The following examples are exemplary and are not intended to limit the scope of the claimed subject matter.

[0281] Example 1

[0282] Synthesis of A4BC1R1 (NUCC-0226301), A4BC1R3 (NUCC-0226276), A4BC1R4 (NUCC-0226302), A4BC1R5 (NUCC-0226277), and A4BC1_2Ts (NUCC-0226263)

[0283] Synthesis scheme

[0284] Scheme 1: Preparation of BC1_DHP

[0285]

[0286] Scheme 2: Preparation of BC1_Pht

[0287]

[0288] Scheme 3: Preparation of A4BC1_2Ts (NUCC-0226263)

[0289]

[0290] Scheme 4: Preparation of target B (A4BC1_NH2)

[0291]

[0292] Scheme 5: Preparation of A4BC1R1 (NUCC-0226301)

[0293]

[0294] Scheme 6: Preparation of A4BC1R3 (NUCC-0226276)

[0295]

[0296] Scheme 7: Preparation of A4BC1R4 (NUCC-0226302)

[0297]

[0298]

[0299] Scheme 8: Preparation of A4BC1R5 (NUCC-0226277)

[0300]

[0301] Chemical synthesis

[0302] Unless otherwise noted, all chemical reagents were obtained from commercial suppliers and used without further purification. Unless otherwise noted, no precautions were taken to exclude air or moisture during the reaction. Normal phase column chromatography was performed using silica gel columns and ACS grade solvents. Analytical TLC was performed on EM reagent 0.25 mm silica gel 60 F254 plates and visualized by UV light. 1 The identity of the compound was confirmed by H and F (NMR) spectroscopy, which was recorded on a Bruker 400 MHz spectrometer using the corresponding residual solvent peaks (CDCl3, 1H δ = 7.27; CD3OD, 1H δ = 3.31; DMSO-d6, 1H δ = 2.50) as internal standards. 1 H-NMR chemical shifts are reported to the second decimal place. Proton coupling constants are expressed in Hertz (Hz). Standard abbreviations are used. 1 Spin multiplicity of H NMR data.

[0303] LCMS method:

[0304] Method 1: 5_95AB_6min-220-254-ELSD

[0305] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C1850*2.1 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0306] Method 2:5_95CD_6min-220-254-ELSD

[0307] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0308] Method 3: 5-95AB_2min

[0309] LC / MS (column used for chromatography was Kinetex 5 μm EVO C18 100A 2.1*30 mm. Detection method was diode array (DAD). MS mode was positive electrospray ionization. MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B was maintained for 0.46 min. 95-5% B (1.61-1.50 min), wherein 0.11 min was maintained at 5% B. The flow rate was 1.5 mL / min.

[0310] Method 4: 5_95CD_6min_MS1500-220-254-ELSD

[0311] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O + 10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1500.

[0312] Method 5: 5_95AB_6min_MS1500-220-254-ELSD

[0313] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1500.

[0314] For the largest experiments run:

[0315] General procedure for the preparation of compound 2-ET37412-62.

[0316]

[0317] To a solution of DHP (2.3 g, 27.29 mmol, 1.7 eq) in DCM (20 mL) was added compound 1 (2 g, 16.06 mmol, 1 eq) and TsOH (276 mg, 1.61 mmol, 0.1 eq) in one portion at 25° C. under N 2 . The mixture was stirred at 25° C. for 12 h. TLC indicated the reaction was complete. The reaction was concentrated to produce a residue, which was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to provide compound 2 (2 g, 59.69% yield) as a colorless oil.

[0318] 1 H NMR (ET37412-62-1, 400 MHz, chloroform-d) δ 1.47-1.66 (m, 6H), 1.70-1.77 (m, 1H), 1.80-1.90 (m, 1H), 3.47-3.55 (m, 1H), 3.58-3.67 (m, 3H), 3.69-3.74 (m, 2H), 3.76-3.81 (m, 2H), 3.84-3.92 (m, 2H), 4.65 (t, J = 3.64 Hz, 1H)

[0319] General procedure for the preparation of compound 3-ET37412-.

[0320]

[0321] To a solution of 4-hydroxybenzaldehyde (900 mg, 7.37 mmol, 1 eq) in DMA (10 mL) was added compound 2 (1.85 g, 8.84 mmol, 1.2 eq) and KCO (2.04 g, 14.74 mmol, 2 eq) in one portion at 25°C under N2. The mixture was stirred at 90°C for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was poured into ice water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to yield compound 3 (1.5 g, 69.15% yield) as a colorless oil.

[0322] LCMS(ESI+):RT=0.824min,m / z 211.0(M-DHP) + .

[0323] 5-95AB_2min

[0324] LC / MS (column used for chromatography was Kinetex 5 μm EVO C18 100A 2.1*30 mm. Detection method was diode array (DAD). MS mode was positive electrospray ionization. MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B was maintained for 0.46 min. 95-5% B (1.61-1.50 min), wherein 0.11 min was maintained at 5% B. The flow rate was 1.5 mL / min.

[0325] General procedure for the preparation of BC1_DHP-ET37412-71.

[0326]

[0327] To a solution of compound 3 (1.4 g, 23.78 mmol, 1 eq) in MeOH (14 mL) was added NaBH4 (269 mg, 7.13 mmol, 1.5 eq) in one portion at 25 °C under N2. The mixture was stirred at 25 °C for 12 h. The mixture was poured into ice water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to produce BC1-DHP (1 g, 71% yield) as a colorless oil.

[0328] 1 H NMR (ET37412-71-1 400 MHz, chloroform-d) δ 1.48-1.65 (m, 5H), 1.70-1.78 (m, 1H), 1.79-1.91 (m, 1H), 3.48-3.57 (m, 1H), 3.62-3.69 (m, 1H), 3.77 (t, J = 4.83 Hz, 2H), 3.84-3.95 (m, 4H), 4.08-4.24 (m, 2H), 4.59-4.71 (m, 3H), 6.93 (d, J = 8.56 Hz, 2H), 7.19-7.37 (m, 3H)

[0329] General procedure for the preparation of compound 7 - ET43259-3.

[0330]

[0331] To a solution of compound 3 (2 g, 6.79 mmol, 1 eq) in MeOH (20 mL) was added TsOH (2.34 g, 13.59 mmol, 2 eq) in one portion at 25° C. under N 2 . The mixture was stirred at 25° C. for 12 h. The reaction was concentrated to compound 7 (1.2 g, 84% yield) as a colorless oil.

[0332] 1 H NMR (ET37412-92-1,400MHz, chloroform-d) δ3.63-3.71(m,1H),3.63-3.71(m,1H),3.78(br d,J=3.79Hz,2H),3.88-3.97(m,2H),4.19-4.26(m,2H),7.03(br d,J=8.44Hz,2H),7.70-7.96(m,2H),9.77-9.99(m,1H)

[0333] General procedure for the preparation of compound 8 - ET37412-103.

[0334]

[0335] To a solution of compound 7 (1 g, 4.76 mmol, 1 eq) in DCM (10 mL) was added TEA (1.93 g, 19.03 mmol, 4 eq) and TosCl (1.36 g, 7.14 mmol, 1.5 eq) in one portion at 25 °C under N2. The mixture was stirred at 25 °C for 12 hours. The residue was poured into ice water (10 mL). The aqueous phase was extracted with DCM (10 mL). The organic phase was washed with brine (5 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to produce compound 8 (1 g, 58% yield) obtained as a colorless oil.

[0336] LCMS(ESI+):RT=0.931min,m / z 365.0(M+1) + .

[0337] 5-95AB_2min

[0338] LC / MS (column used for chromatography was Kinetex 5 μm EVO C18 100A 2.1*30 mm. Detection method was diode array (DAD). MS mode was positive electrospray ionization. MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B was maintained for 0.46 min. 95-5% B (1.61-1.50 min), wherein 0.11 min was maintained at 5% B. The flow rate was 1.5 mL / min.

[0339] General procedure for the preparation of compound 9 - ET37412-105.

[0340]

[0341] To a solution of compound 8 (905.77 mg, 2.49 mmol, 1 eq) in MeOH (2 mL) was added NaBH4 (103.43 mg, 2.73 mmol, 1.1 eq) in one portion at 25 ° C under N2. The mixture was stirred at 25 ° C for 1 h. LCMS indicated that the reaction was complete. The residue was poured into ice water (10 mL). The aqueous phase was extracted with ethyl acetate (20 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated to produce compound 9 (0.3 g, 33% yield) obtained as a white solid.

[0342] 1 H NMR (ET37412-105-1, 400 MHz, chloroform-d) δ 2.43 (s, 3H), 2.66 (dd, J = 6.72, 2.57 Hz, 1H), 3.70-3.81 (m, 4H), 4.00-4.23 (m, 5H), 4.63 (s, 2H), 6.85-6.92 (m, 2H), 7.28-7.41 (m, 5H), 7.80 (d, J = 8.31 Hz, 2H)

[0343] General procedure ET37412-115 for the preparation of BC1_Pht.

[0344]

[0345] To a solution of compound 9 (230 mg, 627.68 μmol, 1 eq) in DMF (1 mL) was added (1,3-dioxoisoindolin-2-yl)-potassium (127.89 mg, 690.45 μmol, 1.1 eq) at 0°C under N2. The mixture was stirred at 90°C for 12 h. LCMS indicated the reaction was complete. The residue was poured into ice water (2 mL). The aqueous phase was extracted with ethyl acetate (5 mL). The combined organic phases were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered, and concentrated to yield a residue, which was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to yield BC1-Pht (100 mg, 47% yield) as a yellow solid.

[0346] 1H NMR (ET37412-115-1,400MHz, chloroform-d) δ3.79-3.87(m,5H),3.89-3.95(m,2H),4.03-4.09(m,2H),4.59(s,2H),6.77-6.83(m,2H),7.22(br d,J=7.06Hz,2H),7.64-7.74(m,2H),7.78-7.87(m,2H)

[0347] General procedure for the preparation of A4BC1_2-DHP-ET37412-96.

[0348]

[0349] To a solution of core A6 (1 g, 2.09 mmol, 1 eq) and BC1-DHP (928.5 mg, 3.13 mmol, 1.5 eq) in THF (10 mL) was added PPh (2.47 g, 9.4 mmol, 4.5 eq) and DEAD (1.9 g, 9.4 mmol, 4.5 eq) at 0°C under N2. The mixture was stirred at 25°C for 12 h. The mixture was added to ice water (20 mL). The aqueous phase was extracted with ethyl acetate (30 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative TLC to produce A4BC1-2DHP (800 mg, 50% yield) as a yellow oil.

[0350] LCMS(ESI+):RT=0.976min,m / z 779.3(M+23) + .

[0351] 5-95AB_2min

[0352] LC / MS (column used for chromatography was Kinetex 5 μm EVO C18 100A 2.1*30 mm. Detection method was diode array (DAD). MS mode was positive electrospray ionization. MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B was maintained for 0.46 min. 95-5% B (1.61-1.50 min), wherein 0.11 min was maintained at 5% B. The flow rate was 1.5 mL / min.

[0353] General procedure for the preparation of A4BC1_2 - ET37412-106.

[0354]

[0355] To a solution of A4BC1_2DHP (800 mg, 1.06 mmol, 1 eq) in MeOH (10 mL) was added TsOH (363 mg, 2.11 mmol, 2 eq) in one portion at 25°C under N2. The mixture was stirred at 25°C for 12 h. TLC indicated the reaction was complete. The mixture was concentrated to produce A4BC1_2 (500 mg, 794 μmol, 70% yield) as a white oil. The residue was used in the next step without any purification.

[0356] General procedure for the preparation of A4BC1_2Ts (NUCC-0226263) - ET37412-104.

[0357]

[0358] To a solution of A4BC1_2 (500 mg, 742 μmol, 1 eq) in DCM (5 mL) was added Py (587 mg, 7.43 mmol, 10 eq) and TosCl (1.42 g, 7.43 mmol, 10 eq) in one portion at 25°C under N2. The mixture was stirred at 25°C for 12 h. LCMS indicated that the reaction was complete. The mixture was added to ice water (10 mL). The aqueous phase was extracted with DCM (20 mL). The combined organic phases were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated to produce a residue, which was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to produce A4BC1_2Ts (320 mg, 52% yield) as a white solid.

[0359] LCMS(ESI+):RT=0.969min,m / z 827.2(M+1) + .

[0360] 5-95AB_2min

[0361] LC / MS (column used for chromatography was Kinetex 5 μm EVO C18 100A 2.1*30 mm. Detection method was diode array (DAD). MS mode was positive electrospray ionization. MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B was maintained for 0.46 min. 95-5% B (1.61-1.50 min), wherein 0.11 min was maintained at 5% B. The flow rate was 1.5 mL / min.

[0362] General procedure for the preparation of A4BC1_2Pht-ET37412-117.

[0363]

[0364] To a solution of core A6 (60 mg, 125.32 μmol, 1 eq) and BC1_Pht (64.17 mg, 187.98 μmol, 1.5 eq) in THF (2 mL) at 0°C under N2 was added PPh3 (164.35 mg, 626.60 μmol, 5 eq) and DIAD (109.13 mg, 626.60 μmol, 113.91 μL, 5 eq) in one portion. The mixture was stirred at 25°C for 12 h. TLC indicated the reaction was complete. The mixture was added to ice water (5 mL). The aqueous phase was extracted with ethyl acetate (5 mL). The combined organic phases were washed with brine (5 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to yield A4BC1_2Pht (100.5 mg, 99.98% yield, crude purity) as a yellow oil.

[0365] General procedure for the preparation of A4BC1_NH2-ET37412-128.

[0366]

[0367] To a solution of A4BC1_2-Pht (400 mg, 498.68 μmol, 1 eq) in EtOH (5 mL) at 25°C under N2 was added NH2NH2.H2O (312.05 mg, 4.99 mmol, 302.96 μL, 80% purity, 10 eq). The mixture was stirred at 25°C for 1 hour. LCMS indicated the reaction was complete. The mixture was concentrated to yield a residue. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 50%-75%, 8 min) to yield A4BC1_NH2 (200 mg, 317.47 μmol, 63.66% yield) as a white solid.

[0368] 1 H NMR (ET37412-128-1,400MHz, chloroform-d) δ2.96 (br s,2H),3.55-3.67(m,3H),3.77-3.86(m,6H),4.07-4.16(m,2H),5.02(s ,2H),6.51-6.59(m,1H),6.57(s,1H),6.76(brd,J=8.68Hz,1H),6.88(br d,J=8.44Hz,2H),7.15(br d,J=8.44Hz,2H),7.20(br d,J=8.56Hz,1H),7.49-7.54(m,1H),7.56-7.61(m,1H),7.78(s,1H)

[0369] General procedure for the preparation of A4BC1R1 (NUCC-0226301) - ET37412-130.

[0370]

[0371] To a solution of A4BC1-NH2 (100 mg, 148.81 μmol, 1 eq) in DMSO (2 mL) was added DIEA (57.70 mg, 446.42 μmol, 77.76 μL, 3 eq) and R1 (61.65 mg, 223.21 μmol, 1.5 eq) at 25°C under N2. The mixture was stirred at 60°C for 12 h. LCMS indicated the reaction was complete. The mixture was filtered to yield a residue, which was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 um; mobile phase: [water (0.05% NH3H2O ​​+ 10 mM NH4HCO3)-ACN]; B%: 50%-85%, 8 min) to yield A4BC1R1 (5 mg, 4% yield) as a yellow solid.

[0372] 1 H NMR (ET37412-130-11, 400 MHz, chloroform-d) δ 2.08-2.15 (m, 1H), 2.66-2.98 (m, 3H), 3.51 (t, J = 5.32 Hz, 2H), 3.80 (t, J = 5.38 Hz, 2H), 3.84 (s, 3H), 3.86-3.89 (m, 2H), 4.11-4.16 (m, 2H), 4.91 (dd, J = 12.04, 5.32 Hz, 1H), 5.02 (s, 2H), 5.16-5.27 (m, 1H), 5.16-5.27 (m, 1H) ,6.57(s,1H),6.78(d,J=8.56Hz,1H),6.85-6.90(m,2H),6.93(d,J=8.56Hz,1H),7.09(d,J=6.97Hz,1H),7.15(d,J=8.68Hz,2H), 7.21(d,J=8.56Hz,1H),7.47(dd,J=8.50,7.15Hz,1H),7.51-7.55(m,1H),7.58-7.62(m,1H),7.78(d,J=1.83Hz,1H),8.01(s,1H)

[0373] LCMS(ESI+):RT=3.602min,m / z 886.2(M+1) + .

[0374] 5_95CD_6min_MS1500-220-254-ELSD:

[0375] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O + 10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1500.

[0376] General procedure for the preparation of A4BC1R3 (NUCC-0226276) - ET37412-113.

[0377]

[0378] To a solution of A4BC1_2Ts (100 mg, 120.89 μmol, 1 eq) and R3 (104.10 mg, 241.78 μmol, 2 eq) in MeCN (1 mL) was added K2CO3 (33.42 mg, 241.78 μmol, 2 eq) and KI (2.01 mg, 12.09 μmol, 0.1 eq). The mixture was stirred at 70°C for 12 h. LCMS indicated the reaction was complete. The mixture was filtered and concentrated to yield a residue, which was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30 mm*10 um; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 50%-80%, 10 min) to yield A4BC1R3 (5 mg, 4% yield) as a white solid.

[0379] 1H NMR (ET37412-113-1,400MHz, methanol-d4) δ0.99(s,9H),1.95-2.27(m,2H),2.45(s,3H),2.55-2.82(m,2H),3.39(br s,2H),3.58-3.69(m,2H),3.72-3.91(m,5H),4.12(brd,J=4.16Hz,1H),4.29-4.46(m,2H),4.50-4.66(m,4H),5.01(br s,1H),6.58(s,1H),6.80-6.94(m,3H),7.13-7.19(m,2H),7.23(d,J=8.56H z,1H),7.34-7.49(m,3H),7.59(q,J=8.15Hz,2H),7.74(s,1H),8.85(s,1H)

[0380] LCMS(ESI+):RT=2.662min,m / z 1043.3(M+1) + .

[0381] 5_95AB_6min_MS1500-220-254-ELSD:

[0382] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1500.

[0383] General procedure for the preparation of A4BC1R4 (NUCC-0226302) - ET37412-131.

[0384]

[0385]

[0386] To a solution of A4BC1_NH2 (100 mg, 158.74 μmol, 1 eq) in DMF (2 mL) was added R4 (79.11 mg, 238.10 μmol, 1.5 eq), DIEA (61.55 mg, 476.21 μmol, 82.94 μL, 3 eq), and HATU (90.53 mg, 238.10 μmol, 1.5 eq) at 25°C under N2. The reaction was stirred at 25°C for 12 h. LCMS indicated the reaction was complete. The reaction was filtered to give a residue, which was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75*30mm*3um; mobile phase: [water (0.05% NH3H2O+10mM NH4HCO3)-ACN]; B%: 40%-75%, 8 min) to give A4BC1R4 (9 mg, 6% yield) as a white solid.

[0387] 1 H NMR (ET37412-131-yl, 400MHz, chloroform-d) δ1.94-1.99(m,1H),2.40-2.55(m,1H),2.60-2.72(m,2H),3.49-3.58(m,2H),3.61 -3.69(m,2H),3.76-3.80(m,5H),4.02-4.12(m,2H),4.54(s,2H),4.77(dd,J=12.59,5.38Hz,1H),4.91(s,2H),5.23(br s,1H),6.49(s,1H),6.69(d,J=8.68Hz,1H),6.75-6.81(m,2H),7.05(dd,J=12.41, 8.62Hz,3H),7.14(d,J=8.56Hz,1H),7.42-7.48(m,2H),7.50-7.54(m,1H),7.57(br t,J=5.26Hz,1H),7.63(dd,J=8.31,7.46Hz,1H),7.70(d,J=1.83Hz,1H),7.88(s,1H)

[0388] LCMS(ESI+):RT=3.346min,m / z 944.1(M+1) + .

[0389] 5_95CD_6min_MS1500-220-254-ELSD:

[0390] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O + 10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1500.

[0391] General procedure for the preparation of A4BC1R5 (NUCC-0226277) - ET37412-112.

[0392]

[0393] To a solution of A4BC1_2Ts (100 mg, 120.89 μmol, 1 eq) and R5 (128.78 mg, 241.78 μmol, 2 eq) in DMSO (1 mL) was added K2CO3 (33.42 mg, 241.78 μmol, 2 eq) and KI (2.01 mg, 12.09 μmol, 0.1 eq). The mixture was stirred at 70 ° C for 12 h. LCMS indicated that the reaction was complete. The mixture was added to ice water (1 mL). The aqueous phase was extracted with ethyl acetate (2 mL). The organic phase was concentrated in vacuo to give a residue, which was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 55%-85%, 10min) to give A4BC1R5 (8mg, 6% yield) as a white solid.

[0394] 1H NMR (ET37412-112-1,400MHz, methanol-d4) δ1.01(s,9H),1.20-1.41(m,4H),1.98-2.24(m,2H),2.46(s,3H),3.72-3.86(m,5H),3.95(br d,J=12.35Hz,4H),4.14(br s,2H),4.25(br s,2H),4.34-4.44(m,1H),4.46(br s,1H),4.56-4.66(m,2H),4.73(s,1H),5.01(s,2H),6.58(s,1H),6.85(br d,J=8.16Hz,3H),6.96-7.09(m,2H),7.15(br d,J=7.94Hz,2H),7.23(br d,J=8.60Hz,1H),7.45(d,J=7.72Hz,1H),7.53-7.64(m,2H),7.74(s,1H),8.80(s,1H)

[0395] LCMS(ESI+):RT=3.067min,m / z 1145.3(M+1) + .

[0396] 5_95AB_6min_MS1500-220-254-ELSD:

[0397] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1500.

[0398] Synthesis of NUCC-0226258, NUCC-0226259, NUCC-0226260, and NUCC-0226261

[0399]

[0400] Synthesis scheme:

[0401]

[0402] The largest experiment ever run:

[0403] A suspension of compound 1 (10 g, 65.73 mmol, 8.47 mL, 1 eq) in TFAA (37 mL) was placed in a high-pressure tube (100 mL). Sodium trifluoroacetate (19.67 g, 144.60 mmol, 2.2 eq) was added and the system was capped and stirred at 130 ° C for 24 h. A total of 30 batches were set up as parallel reactions. LCMS showed that all starting materials were consumed. The reactions were cooled to 25 ° C and combined, and then diluted with EtOAc (1 L). The mixture was neutralized with saturated aqueous K2CO3 until no more bubbling was observed. The organic layer was separated and the aqueous portion was extracted with EtOAc (3×500 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, concentrated to 1 / 3 the volume of EtOAc, and the flask was allowed to stand at 25 ° C for 8 hours. Compound 2 (180 g, 793.0 mmol, 40.0% yield) was collected as a white solid.

[0404] Reaction LCMS:

[0405] LCMS method:

[0406] LCMS (ESI+): m / z=231.1(M+H)+, RT: 0.806min.

[0407] 5_95AB_2min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0408] General procedure for the preparation of compound 3-ET32240-1059

[0409]

[0410] A solution of compound 2 (180 g, 782.13 mmol, 1 eq), iodine (794.05 g, 3.13 mol, 630.20 mL, 4 eq), and pyridine (247.47 g, 3.13 mol, 252.52 mL, 4 eq) in chloroform (1 L) was stirred at 25°C for 8 h. LCMS indicated the reaction was complete. The mixture was poured into water (500 mL) and triturated with petroleum ether:ethyl acetate (10:1, 800 mL) to produce compound 3 (210 g, 589.83 mmol, 75.41% yield) as a slightly yellow solid.

[0411] Reaction LCMS:

[0412] LCMS method:

[0413] LCMS (ESI+): m / z=356.9(M+H)+, RT: 0.890min.

[0414] 5_95AB_2min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0415] 1 H NMR (400 MHz, methanol-d4) δ ppm 6.79 (s, 1H) 7.02 (d, J = 8.82 Hz, 1H) 7.95-7.99 (m, 1H)

[0416] General procedure for the preparation of compound 4-ET32240-1078

[0417]

[0418] To a solution of compound 3 (130 g, 365.13 mmol, 1 eq), bromomethylbenzene (74.94 g, 438.16 mmol, 52.04 mL, 1.2 eq), and bromomethylbenzene (74.94 g, 438.16 mmol, 52.04 mL, 1.2 eq) in acetone (1 L) was added potassium carbonate (100.93 g, 730.26 mmol, 2 eq). The mixture was stirred at 80°C for 8 h. TLC indicated the reaction was complete. The mixture was poured into water (500 mL) and extracted with ethyl acetate (3 x 500 mL). The organic layer was dried over Na2SO4 and concentrated to produce the crude product, which was purified by chromatography on silica gel, eluting with petroleum ether:ethyl acetate = 10:1 to 5:1, to produce the desired product, compound 4 (120 g, 268.96 mmol, 73.66% yield), as a slightly yellow solid.

[0419] 1 H NMR (400 MHz, chloroform-d) δ ppm 5.27 (s, 2H) 6.63 (s, 1H) 6.96 (d, J = 8.93 Hz, 1H) 7.10-7.39 (m, 8H) 7.40-7.46 (m, 2H) 8.08 (d, J = 8.93 Hz, 1H)

[0420] General procedure for the preparation of compound 5-ET32240-1083

[0421]

[0422] To a solution of compound 4 (100 g, 22.41 mmol, 1 eq), [4-chloro-3-(trifluoromethyl)phenyl]boronic acid (5.03 g, 22.41 mmol, 1 eq), and cesium carbonate (14.61 g, 44.83 mmol, 2 eq) in toluene (2 L), ethanol (400 mL), and water (80 mL) was added Pd(dppf)Cl (1.64 g, 2.24 mmol, 0.1 eq) under a N atmosphere. The mixture was stirred at 80°C for 8 h. TLC indicated the reaction was complete. The mixture was poured into water (500 mL) and extracted with ethyl acetate (3 x 500 mL). The organic layer was dried over NaSO and concentrated to yield the crude product, which was triturated with ethyl acetate:petroleum ether (1:10, 500 mL) to yield the desired product, compound 5 (93 g, 18.64 mmol, 83.18% yield), as a slightly yellow solid.

[0423] 1H NMR (400 MHz, chloroform-d) δ ppm 5.26 (s, 2H) 6.71 (s, 1H) 7.27 (d, J = 2.93 Hz, 2H) 7.34-7.41 (m, 3H) 7.55-7.66 (m, 2H) 7.85 (s, 1H) 8.26 (d, J = 8.93 Hz, 1H)

[0424] General procedure for the preparation of compound 6-ET32240-1131

[0425]

[0426] Compound 5 (100 g, 200.48 mmol, 1 equivalent), methylhydrazine (27.71 g, 601.44

[0427] A suspension of 4-[4-[[4-[[4-( ...4-(4-4-(4-4-(4-4-(4-4-(4-4-(4-4-(4-4-(4-4

[0428] LCMS (ESI+): m / z=527.2(M+H)+, RT: 1.203min.

[0429] 5_95AB_2min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0430] General procedure for the preparation of compound 7-ET32240-1147

[0431]

[0432] To a solution of compound 6 (42 g, 79.72 mmol, 1 eq) in DCM (500 mL) was added acetyl chloride (7.51 g, 95.66 mmol, 6.83 mL, 1.2 eq) and TEA (9.68 g, 95.66 mmol, 13.31 mL, 1.2 eq). The mixture was stirred at 25 ° C for 8 hours. TLC indicated that all the starting material was consumed. Once complete, the mixture was poured into water and extracted with DCM (2×100 mL). The organic layer was dried over Na2SO4 and concentrated to produce compound 7 (45 g, 79.10 mmol, 99.23% yield) as a white solid, which was used directly in the next step.

[0433] 1 H NMR (400 MHz, chloroform-d) δ 1.72-1.81 (m, 3H), 3.75-3.84 (m, 3H), 5.10-5.16 (m, 2H), 6.45-6.52 (m, 1H), 7.07 (d, J = 8.60 Hz, 1H), 7.21-7.27 (m, 3H), 7.38 (s, 2H), 7.44-7.49 (m, 1H), 7.51-7.57 (m, 1H), 7.72-7.76 (m, 1H)

[0434] General procedure for the preparation of compound core A6 (NUCC-0226261) - ET32240-1158

[0435]

[0436] A solution of compound 7 (50 g, 87.89 mmol, 1 eq) in DCM (200 mL) was cooled to 0°C. BCl3 (1 M, 175.78 mL, 2 eq) was added dropwise to the mixture while maintaining the temperature below 0°C. The mixture was stirred at 0°C for 4 hours. TLC indicated that all the starting material was consumed. After completion, the mixture was added to ice (100 mL) and the organic layer was separated. The aqueous layer was extracted with EtOAc (3 x 100 mL). The organic layer was dried over Mg2SO4 and concentrated to produce the product, which was purified by chromatography on silica gel eluting with PE:EA = 10:1 to 1:1 to produce compound A6 (25 g, 52.22 mmol, 59.41% yield) as a white solid.

[0437] 1H NMR (400 MHz, chloroform-d) δ 1.78 (s, 3H), 3.76-3.84 (m, 3H), 5.95 (s, 1H), 6.48-6.53 (m, 1H), 6.96-7.02 (m, 1H), 7.23-7.28 (m, 1H), 7.48-7.53 (m, 1H), 7.62 (d, J = 8.16 Hz, 1H), 7.70-7.74 (m, 1H)

[0438] Synthesis of Target D (NUCC-0226219) and Target E (NUCC-0226223)

[0439]

[0440] Synthesis Scheme 9: Preparation of Target D

[0441]

[0442] Scheme 10: Preparation of BC1_Pht

[0443]

[0444] Chemical synthesis

[0445] The largest experiment ever run:

[0446] General procedure for preparation of target D (NUCC-0226219) - ET37412-4.

[0447]

[0448] To a mixture of Core A6_1 (55 mg, 97.53 μmol, 1 eq) and (2S)-5-[bis(tert-butoxycarbonylamino)methyleneamino]-2-(tert-butoxycarbonylamino)pentanoic acid (55.54 mg, 117.04 μmol, 1.2 eq) in DMF (1 mL) were added HATU (44.50 mg, 117.04 μmol, 1.2 eq) and DIEA (18.91 mg, 146.30 μmol, 1.5 eq) in one portion. The reaction was stirred at 25°C for 12 h. KCO (50 mg) was then added to the reaction mixture, which was then stirred at 25°C for 2 h. LCMS indicated the reaction was complete. The reaction was filtered to give a residue, which was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75*30mm*3um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 65%-95%, 8min) to give target D (11 mg, 11% yield) as a white solid.

[0449] 1 H NMR(ET37412-4-1,400MHz,DMSO-d6)δ0.78-0.87(m,2H),1.02(br s,4H),1.36(d,J=12.28Hz,19H),1.45(s,12H),2.77-2.91(m,2H),3.07(br s,2H),3.24(br s,2H),3.78(s,4H),6.56-6.71(m,1H),6.61-6.68(m,1H),6.61-6.68(m,1H),6.66(s,1H),6.76(brd,J=8.23Hz,1H),7.07(br s,1H),7.64(br s,1H),7.68-7.79(m,2H),7.90(s,1H),8.26(br t,J=5.60Hz,1H),11.48(br s,1H)

[0450] LCMS(ESI+):RT=3.051min,m / z 978.3(M+1) + .

[0451] 5_95AB_6min-220-254-ELSD

[0452] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C1850*2.1 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0453] General procedure for the preparation of compound 6 (NUCC-0226260) -ET37412-.

[0454]

[0455] To a mixture of compound 5 (5 g, 24.97 mmol, 1 eq) in DMF (60 mL) was added NaH (1.50 g, 37.46 mmol, 60% purity, 1.5 eq). After stirring for 1 h, 1,5-dibromopentane (6.89 g, 29.96 mmol, 4.05 mL, 1.2 eq) was added to the mixture. The reaction was stirred at 25°C for 12 h. The residue was poured into ice water (100 mL). The aqueous phase was extracted with ethyl acetate (100 ml). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to produce a residue, which was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to provide compound 6 (6 g, 69% yield) as a colorless oil.

[0456] 1H NMR (ET37412-34-1, 400 MHz, chloroform-d) δ 1.49 (s, 8H), 1.56-1.65 (m, 4H), 1.77-2.01 (m, 2H), 3.27-3.53 (m, 5H), 3.57-3.73 (m, 2H), 3.96-4.22 (m, 3H)

[0457] General procedure for the preparation of compound 7 - ET37412-71.

[0458]

[0459] To a mixture of Core A6 (830 mg, 1.73 mmol, 1 eq) and Compound 6 (1.14 g, 2.60 mmol, 80% purity, 1.5 eq) in DMF (5 mL) was added KCO (359.40 mg, 2.60 mmol, 1.5 eq) in one portion at 25°C. The reaction was stirred at 25°C for 12 h. LCMS indicated the reaction was complete. The reaction was poured into water (10 mL) and extracted with ethyl acetate (20 mL). The organic layer was concentrated to produce a residue, which was purified by silica gel chromatography eluting with petroleum ether / ethyl acetate = 1:0 to 0:1 to produce Compound 7 (400 mg, 31% yield) as a colorless oil.

[0460] LCMS(ESI+):RT=0.948min,m / z 691.3(M+1) + .

[0461] 5-95AB_2min

[0462] LC / MS (column used for chromatography was Kinetex 5 μm EVO C18 100A 2.1*30 mm. Detection method was diode array (DAD). MS mode was positive electrospray ionization. MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B was maintained for 0.46 min. 95-5% B (1.61-1.50 min), wherein 0.11 min was maintained at 5% B. The flow rate was 1.5 mL / min.

[0463] General procedure for the preparation of compound 8 - ET37412-48.

[0464]

[0465] To a mixture of compound 7 (400 mg, 1 equivalent) in dioxane (0.5 mL) was added HCl / dioxane (1 mL). The reaction mixture was stirred at 25 ° C for 12 h. LCMS showed that the reaction was complete. The reaction was concentrated to produce a residue, which was used in the next step without any purification to produce compound 8 (330 mg, 91% yield, HCl) as a yellow oil.

[0466] LCMS(ESI+):RT=0.773min,m / z 647.7(M+1) + .

[0467] 5-95AB_2min:

[0468] LC / MS (column used for chromatography was Kinetex 5 μm EVO C18 100A 2.1*30 mm. Detection method was diode array (DAD). MS mode was positive electrospray ionization. MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B was maintained for 0.46 min. 95-5% B (1.61-1.50 min), wherein 0.11 min was maintained at 5% B. The flow rate was 1.5 mL / min.

[0469] General procedure for preparation of target E (NUCC-0226223) - ET37412-52.

[0470]

[0471] To a mixture of compound 8 (300.21 mg, 549.00 μmol, 1 eq) and compound 2 (250 mg, 549.00 μmol, 1 eq) in DCM (5 mL) was added triphosgene (407.29 mg, 1.37 mmol, 2.5 eq) and TEA (833.29 mg, 8.23 ​​mmol, 1.15 mL, 15 eq) at 25 ° C. The reaction mixture was stirred at 25 ° C for 12 h. Thereafter, K2CO3 (50 mg) was added to the mixture and the reaction was stirred at 25 ° C for 2 h. LCMS indicated that the reaction was complete. The reaction was poured into ice water (10 mL). The mixture was extracted with DCM (20 mL). The organic layer was concentrated to give a residue, which was purified by preparative HPLC (column: Phenomenex Gemini-NX 150*30mm*5um; mobile phase: [water (0.1% TFA)-ACN]; B%: 50%-80%, 9 min) to give target E (0.045 g, 7% yield) as a white solid.

[0472] 1 H NMR (ET37412-52-P1B, 400 MHz, methanol-d4) δ 1.17-1.28 (m, 2H), 1.31-1.39 (m, 2H), 1.45 (d, J = 5.95 Hz, 6H), 1.61-1.71 (m, 2H), 2.97 (br s,2H),3.02-3.12(m,1H),3.18-3.27(m,1H),3.41-3.53(m,1H),3.56-3.68(m,1H),3.79(s,3H),3.88 (s,2H),3.91(s,3H),3.96-4.02(m,2H),4.91(dd,J=12.24,6.06Hz,1H),5.98(d,J=11.03Hz,1H),6.2 4(d,J=11.03Hz,1H),6.58(s,1H),6.74-6.84(m,3H),6.99(d,J=8.38Hz,2H),7.17(dd,J=16.21,8.49 Hz,4H),7.26(dd,J=17.42,8.60Hz,3H),7.56-7.61(m,1H),7.63-7.70(m,2H),7.75(d,J=1.76Hz,1H)

[0473] LCMS(ESI+):RT=2.966min,m / z 1085.0(M+1) + .

[0474] 5_95AB_6min_MS1500-220-254-ELSD

[0475] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1500.

[0476] Synthesis of R5 (NUCC-0226278)

[0477]

[0478] Synthesis scheme

[0479] Scheme 11: Preparation of R5 (NUCC-0226278)

[0480]

[0481]

[0482] The largest experiment ever run:

[0483] General procedure for the preparation of 2-hydroxy-4-(4-methylthiazol-5-yl)benzonitrile (D2) - ET39710-105

[0484]

[0485] To a mixture of 4-bromo-2-hydroxy-benzonitrile (D1) (120 g, 606.01 mmol) in DMF (1 L) was added 4-methylthiazole (180.26 g, 1.82 mol), KOAc (118.95 g, 1.21 mol) and Pd(OAc) (6.80 g, 30.30 mmol) at 20 ° C under N2. The reaction mixture was stirred at 120 ° C for 12 h. LCMS showed that the reaction was complete. The mixture was cooled to 20 ° C and filtered on celite. The filtrate was poured into water (1 L) and extracted with EA (3×1.5 L). The combined organic layers were concentrated under reduced pressure to produce a 1 L solution. The solution was washed with brine (3×500 mL), dried over Na 2 SO 4 , filtered and the filtrate was concentrated under reduced pressure to give a residue, which was triturated with PE / EA=10:1 (500 mL) and the solid was collected by suction filtration to give 2-hydroxy-4-(4-methylthiazol-5-yl)benzonitrile (D2) (80 g, 61.04% yield) as a yellow solid.

[0486] 1 H NMR (400MHz, DMSO-d6) δ=11.32(s,1H),9.07(s,1H),7.70(d,J=7.8Hz,1H),7.13(d,J=1.5Hz,1H),7.07(dd,J=1.7,8.1Hz,1H),2.49(s,3H)

[0487] General Procedure for the Preparation of 2-(Aminomethyl)-5-(4-methylthiazol-5-yl)phenol (D3) - ET39710-106

[0488]

[0489] To a stirred solution of 2-hydroxy-4-(4-methylthiazol-5-yl)benzonitrile (D2) (80 g, 369.93 mmol) in THF (1 L) was added LAH (35.10 g, 924.82 mmol) in several portions at 10°C under a nitrogen atmosphere. The resulting mixture was heated at 50°C for 1 h. LCMS indicated the reaction was complete. The mixture was cooled to 0°C and quenched with H2O (35 mL, added slowly and dropwise), 15% NaOH (aq.) (35 mL), and H2O (105 mL). The precipitated solid was removed by suction filtration, and the filtrate was concentrated under reduced pressure to produce 2-(aminomethyl)-5-(4-methylthiazol-5-yl)phenol (D3) (45 g, 55.22% yield) as a yellow solid.

[0490] 1H NMR (400MHz, DMSO-d6) δ = 8.80 (s, 1H), 6.84 (d, J = 7.3Hz, 1H), 6.37 (s, 1H), 6.12 (dd, J = 1.5, 7.3Hz, 1H), 3.53 (s, 2H), 2.42 (s, 3H)

[0491] General procedure for the preparation of tert-butyl (2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate (D4) - ET39710-107

[0492]

[0493] To a solution of 2-(aminomethyl)-5-(4-methylthiazol-5-yl)phenol (D3) (45 g, 204.28 mmol) in DMF (500 mL) was added (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (47.24 g, 204.28 mmol), DIEA (52.80 g, 408.55 mmol), HOBt (41.40 g, 306.41 mmol) and EDCI (47.57 g, 306.41 mmol) at 0°C under N2. The mixture was stirred at 20°C for 0.5 h. LCMS indicated that the reaction was complete. The reaction mixture was poured into water (500 mL) and stirred for 5 min. The mixture was extracted with EA (3×800 mL). The combined organic layers were washed with brine (1 L), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to yield a residue. The residue was purified by column chromatography on silica gel (eluted with PE / EA=20:1 to 0:1) to give tert-butyl (2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate (D4) (23.4 g, yield 26.42%) as a yellow solid.

[0494] 1 H NMR (400MHz, DMSO-d6) δ = 9.92 (s, 1H), 8.96 (s, 1H), 8.54-8.30 (m, 1H), 7.27-7.15 (m, 1H), 6.91 (s, 1H), 6.89-6.81 (m, 1H) ,4.32-4.11(m,4H),3.48-3.34(m,3H),2.44(s,3H),2.13-1.98(m,1H),1.93-1.81(m,1H),1.41(s,3H),1.26-1.20(m,6H)

[0495] General procedure for the preparation of (2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (D5) - ET39710-112

[0496]

[0497] A mixture of tert-butyl (2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carboxylate (D4) (23.4 g, 53.98 mmol) in HCl / dioxane (250 mL, 4 M) was stirred at 20° C. for 0.5 h. LCMS indicated the reaction was complete. The mixture was concentrated under reduced pressure to yield (2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (D5) (17.80 g, 89.16% yield, HCl salt) as a white solid.

[0498] 1 H NMR (400MHz, DMSO-d6) δ = 10.17-9.86 (m, 2H), 9.05-8.96 (m, 2H), 8.66 (br d,J=1.9Hz,1H),7.20(d,J=7.6Hz,1H),7.02(d,J=1.4Hz,1H),6.91(dd,J=1.4,7.6Hz,1H),4.44(br s,1H),4.41-4.34(m,1H),4.31(t,J=5.5Hz,2H),3.40-3.28(m,1H),3.15-3.03(m,1H),2.46(s,3H),2.37-2.27(m,1H),1.96-1.87(m,1H)

[0499] General procedure for the preparation of tert-butyl ((S)-1-((2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (D6) - ET39710-113

[0500] To a solution of (2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (D5) (16 g, 43.26 mmol) in DMF (160 mL) at 0°C was added (2S)-2-(tert-butoxycarbonylamino)-3,3-dimethyl-butanoic acid (10.01 g, 43.26 mmol), DIEA (116.77 g, 129.78 mmol), and HATU (19.74 g, 51.91 mmol). The mixture was stirred at 20°C for 1 h. LCMS indicated the reaction was complete. The mixture was poured into ice water (100 mL) and stirred for 5 min. The mixture was extracted with EA (3 x 150 mL).

[0501] The combined organic layers were washed with brine (300 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluted with PE / EA=50:1 to 0:1) to produce tert-butyl ((S)-1-((2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (D6) (12.1 g, 51.17% yield) as a yellow solid.

[0502] 1 H NMR (400MHz, MeOD-d4) δ=8.85(s,1H),8.74(dd,J=1.4,4.3Hz,1H),8.43(dd,J=1.2,8.3Hz,1H ),7.52(dd,J=4.8,8.6Hz,1H),7.36(d,J=8.1Hz,1H),6.92-6.87(m,2H),4.60(s,1H),4.50(br s,1H),4.45-4.32(m,2H),4.29(s,1H),3.91-3.76(m,2H),2.49-2.47(m,3H),2.25-2.06(m,2H),1.48-1.42(m,9H),1.00(s,9H)

[0503] General procedure for the preparation of (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[[2-hydroxy-4-(4-methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (D7) - ET39710-115

[0504]

[0505] A mixture of tert-butyl ((S)-1-((2S,4R)-4-hydroxy-2-((2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (D6) (12 g, 21.95 mmol) in HCl / dioxane (120 mL, 4 M) was stirred at 20 ° C for 20 min. LCMS indicated that the reaction was complete. The mixture was concentrated under reduced pressure to produce (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[[2-hydroxy-4-(4-methylthiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide (D7) (9.95 g, 93.84% yield, HCl salt) as a white solid.

[0506] 1 H NMR (400MHz, DMSO-d6) δ = 9.05 (s, 1H), 8.63 (t, J = 6.1Hz, 1H), 8.15 (br d,J=3.9Hz,3H),7.32(d,J=7.8Hz,1H),6.98(d,J=2.0Hz,1H),6.81(dd,J=2.0,7.8Hz,1H),4.57(t,J=8.3Hz,1H),4.37(br s,1H),4.33-4.23(m,1H),4.20-4.10(m,1H),3.90(br d,J=4.9Hz,1H),3.77(br d,J=10.8Hz,1H),3.64-3.51(m,1H),2.12(br dd,J=7.8,12.7Hz,1H),1.91(s,1H),1.59(s,4H),1.01(s,9H)

[0507] General procedure for the preparation of (2S,4R)-1-((S)-2-(1-fluorocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (NUCC-0226278) - ET39710-118

[0508]

[0509] To a solution of 1-fluorocyclopropanecarboxylic acid (861.90 mg, 8.28 mmol) in DMF (40 mL) was added (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (D7) (4 g, 8.28 mmol), HOBt (1.68 g, 12.42 mmol), DIEA (3.21 g, 24.84 mmol) and EDCI (1.93 g, 12.42 mmol) at 0°C. The mixture was stirred at 20°C for 1 h. LCMS indicated that the reaction was complete. The reaction mixture was poured into water (40 mL) and stirred for 5 min. The mixture was extracted with EA (3 x 50 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to yield a residue. The residue was purified by preparative HPLC (HCl conditions) to yield R5 (0.9 g, 20.40% yield) as a white solid.

[0510] LCMS (ESI+): m / z=533.0 (M+H) + ,RT:0.832min.

[0511] 1 H NMR (400MHz, DMSO-d6) δ = 9.97-9.71 (m, 1H), 9.09-8.94 (m, 1H), 8.66-8.4 5(m,1H),7.36-7.23(m,2H),7.01-6.87(m,1H),6.87-6.80(m,1H),4.70- 4.43(m,6H),4.32-4.03(m,3H),3.69-3.56(m,2H),2.13-2.03(m,1H),1. 96-1.87(m,1H),1.44-1.30(m,2H),1.27-1.17(m,2H),1.01-0.89(m,9H)

[0512] Preparative HPLC method:

[0513] Instrument: Shimadzu LC-8A preparative HPLC

[0514] Column: Phenomenex luna C18 250*50mm*10um

[0515] Mobile phase: A represents H2O=(0.04% HCl) and B represents CH3CN

[0516] Gradient: from 25% to 45% B in 20 min

[0517] Flow rate: 80mL / min

[0518] Wavelength: 220 and 254 nm

[0519] SFC method:

[0520] Column: Chiralpak AD-3, 50×4.6mm ID, 3um

[0521] Mobile phase: A: CO2 B: EtOH (0.1% IPAm, v / v)

[0522] gradient:

[0523]

[0524] Flow rate: 3.4 mL / min

[0525] Column temperature: 35°C

[0526] ABPR:1800psi

[0527] Synthesis of A04B01C01D01 (NUCC-0226545)

[0528]

[0529] Synthesis scheme

[0530] Scheme 12: Preparation of A04B01C01D01 (NUCC-0226545)

[0531]

[0532]

[0533] Chemical synthesis

[0534] LCMS method:

[0535] Method 1: 5_95AB_2min

[0536] LC / MS (The column used for chromatography was a Chromolith RP-18e 25-2 mm. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95-100% B (0.70 -1.15 min), 5% B in 1.16 min, with a hold at 5% B for 0.34 min. The flow rate was 1.5 mL / min.

[0537] Method 2: 50_100CD_6min-220-254-ELSD

[0538] LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0539] The largest experiment ever run:

[0540] General procedure for the preparation of compound 2-ET42365-46.

[0541]

[0542] To a solution of AlCl3 (5.12 g, 38.42 mmol, 2.10 mL, 1.2 equivalents) in DCM (50 mL) was added dropwise acetyl chloride (3.77 g, 48.03 mmol, 3.43 mL, 1.5 equivalents) at 5-10 ° C. The reactants were stirred at 5 ° C for 15 minutes, then compound 1 (5 g, 32.02 mmol, 1 equivalent) was added dropwise at 0-10 ° C., and the reactants were stirred at 20 ° C for 6 hours. TLC (petroleum ether / ethyl acetate=3 / 1) showed that the starting material was exhausted and two new peaks were produced. The reactants were poured into 200 mL of ice water. The aqueous layer was extracted with dichloromethane (2×100 mL). The combined organic phases were washed with water (100 mL), dried over sodium sulfate and concentrated under reduced pressure to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give Compound 2 (4.5 g, yield 67.37%) as a colorless oil.

[0543] 1 H NMR (ET42365-46-P1A, 400 MHz, chloroform-d) δ 2.52 (d, J = 1.63 Hz, 3H), 3.82 (s, 3H), 3.84 (s, 3H), 6.22-6.28 (m, 2H)

[0544] 19 F NMR (ET42365-46-P1A, 400 MHz, chloroform-d) δ -112.015

[0545] General procedure for the preparation of compound 3 - ET42365-68.

[0546]

[0547] To a solution of compound 2 (4 g, 20.18 mmol, 1 equivalent) in DCM (80 mL) was added dropwise BBr (20.22 g, 80.73 mmol, 7.78 mL, 4 equivalents) at -30 ° C, and the reactants were stirred at 20 ° C for 24 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reactants were poured into 100 mL of ice water. The aqueous layer was extracted with dichloromethane (2 × 50 mL). The combined organic phases were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 30 / 1 to 10 / 1) to produce compound 3 (2.3 g, 63.63% yield) as a white solid.

[0548] 1H NMR (ET42365-68-P1H, 400MHz, DMSO-d6) δ2.53(d,J=6.80Hz,3H),6.11(d,J=2.19Hz,1H),6.19(dd,J=14.03,2.19Hz,1H),11.06(s,1H),13.05(s,1H)

[0549] General procedure for the preparation of compound 4-ET43588-7.

[0550]

[0551] A suspension of compound 3 (2 g, 11.76 mmol, 1 eq) in TFAA (20 mL) was placed in a high-pressure tube (30 mL). Sodium trifluoroacetate (3.52 g, 25.86 mmol, 2.2 eq) was added and the system was capped and stirred at 130 ° C for 24 hours. LCMS showed that all starting materials were consumed. The reactants were cooled to 25 ° C and neutralized with saturated K2CO3 aqueous solution until no more bubbling was observed. The organic layer was separated and the aqueous portion was extracted with ethyl acetate (3×150 mL). The combined organic layers were washed with brine (100 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel, eluting with petroleum ether / ethyl acetate=50 / 1 to 5 / 1 to produce compound 4 (0.48 g, yield 10.5%) as a light yellow solid.

[0552] LCMS(ESI+):RT:0.773min,m / z=249.0(M+H) + .

[0553] 5-95AB_2min: LC / MS (The column used for chromatography was a Chromolith RP-18e 25-2 mm. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95-100% B (0.70 -1.15 min), 5% B in 1.16 min, with a hold of 0.34 min at 5% B. The flow rate was 1.5 mL / min.

[0554] General procedure for the preparation of compound 6 - ET43588-20.

[0555]

[0556] To a solution of compound 4 (0.43 g, 1.73 mmol, 1 eq) in CHCl (9 mL) was added I (1.76 g, 6.93 mmol, 4 eq) and pyridine (548.31 mg, 6.93 mmol, 559.50 μL, 4 eq) at 25 ° C. The reaction was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction mixture was poured into water (20 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel, eluting with petroleum ether / ethyl acetate = 10 / 1 to 2 / 1 to produce compound 6 (0.38 g, yield 45.5%) as a brown solid.

[0557] 1 H NMR(ET43588-20-P1H,400MHz,MeOD)δ6.72-6.80(m,2H)

[0558] General procedure for the preparation of compound 7 - ET43588-21.

[0559]

[0560] A solution of compound 6 (0.38 g, 1.02 mmol) in DMF (3.5 mL) was added to 1-(bromomethyl)-4-chloro-benzene (250.51 mg, 1.22 mmol) and K2CO3 (280.83 mg, 2.03 mmol, 2 equivalents) at 25 ° C. The reaction mixture was stirred and heated at 80 ° C for 2 hours. LCMS showed that the reaction was complete. After cooling to room temperature, the mixture was poured into water (20 mL) and extracted with ethyl acetate (3×20 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel, eluting with petroleum ether / ethyl acetate = 5 / 1 to 3 / 1 to produce compound 7 (0.5 g, yield 80.0%) as a brown solid.

[0561] 1 H NMR (ET43588-20-P1H, 400MHz, CDCl3) δ5.25 (s, 2H) 6.66 (s, 1H) 6.77 (d, J = 12.17Hz, 1H) 7.39-7.48 (m, 4H).

[0562] General procedure for the preparation of compound 8 - ET43588-38.

[0563] Reactions were performed in parallel but pooled for purification.

[0564]

[0565] To a solution of compound 7 (0.28 g, 748.61 μmol) in toluene (67.2 mL), ethanol (13.44 mL) and water (2.688 mL) was added [4-chloro-3-(trifluoromethyl)phenyl]boronic acid (167.97 mg, 748.61 μmol), Na2CO3 (487.82 mg, 1.50 mmol) and Pd(dppf)Cl2 (54.78 mg, 74.86 μmol) at 25 ° C under N2 atmosphere. The reaction mixture was stirred and heated at 80 ° C under N2 atmosphere for 2 hours. LCMS showed that the reaction was complete. Two more reactions were set up as described above and combined for purification. The reaction mixture was poured into water (30 mL) and extracted with ethyl acetate (3×30 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was recrystallized from ethyl acetate / petroleum ether (10 / 1, 3 mL) to give compound 8 (0.21 g, yield 26.7%) as a brown solid.

[0566] 1 H NMR(ET43588-38-PH1,400MHz,CDCl3)δppm 5.15(s,2H)6.63(s,1H)6.89(d,J=12.13Hz,1H)7.19(d,J=8.38Hz,2H)7.35(d,J=8.5 0Hz, 2H) 7.51 (dd, J = 8.38, 1.88Hz, 1H) 7.61 ( d, J = 8.25Hz, 1H) 7.77 ( d, J = 1.88Hz, 1H).

[0567] General procedure for the preparation of A04B01C01D01 - ET43588-57.

[0568]

[0569] To a suspension of compound 8 (0.16 g, 290.26 μmol) in ethanol (1.6 mL) was added CH 3 NH NH 2 (200.59 mg, 1.74 mmol, 229.24 μL, 40% purity) at 25 ° C. The reaction mixture was then stirred and heated at 80 ° C for 4 hours. LCMS indicated that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to produce A04B01C01D01 (17.7 mg, 10.5% yield) as a white solid.

[0570] 1H NMR (ET43588-57-P1H1, 400MHz, CDCl3) δ3.85 (s, 3H) 5.04 (s, 2H) 5.27 (s, 1H) 6.53 (d, J = 11.21Hz, 1H) 6. 65(s,1H)7.17(d,J=8.23Hz,2H)7.34(d,J=8.34Hz,2H)7.48-7.55(m,1H)7.57-7.64(m,1H)7.77(s,1H).

[0571] Preparative HPLC method:

[0572] Instrument: Gilson 281 semi-preparative HPLC system

[0573] Mobile phase: A: 10 mM NH4HCO3 solution in H2O; B: CAN

[0574] Column: Waters Xbridge BEH C18 100*25mm*5um

[0575] Flow rate: 25mL / min

[0576] Monitoring wavelength: 220 and 254nm

[0577]

[0578] LCMS(ESI+): RT: 2.919min, m / z=579.1(M+H) + .

[0579] 50_100CD_6min-220-254-ELSD

[0580] LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0581] Synthesis of NUCC-0226530 (ET42365-98-1), NUCC-0226528 (ET42365-86-1), and NUCC-0226520 (ET42365-63-1)

[0582]

[0583] Scheme 13: Preparation of A02B01C09D01 (NUCC-0226530)

[0584]

[0585] Scheme 14: Preparation of A02B01C04D01 (NUCC-0226528)

[0586]

[0587] Scheme 3: Preparation of A01B01C04D01 (NUCC-0226520)

[0588]

[0589] Chemical synthesis

[0590] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0591] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0592] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0593] The largest experiment ever run:

[0594] General procedure for the preparation of compound 2-ET42365-29.

[0595]

[0596] To a solution of compound 1 (10 g, 43.45 mmol, 1 equivalent) and pyridine (13.75 g, 173.81 mmol, 14.03 mL, 4 equivalents) in DCM (150 mL) was added dropwise Tf2O (18.39 g, 65.18 mmol, 10.75 mL, 1.5 equivalents) at 0 ° C. The reactants were stirred at 20 ° C for 12 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reactants were diluted with water (300 mL) and extracted with DCM (3 × 100 mL). The organic layer was separated and the combined organic layers were washed with brine (150 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to produce compound 2 (12 g, yield 72.43%) as a white solid.

[0597] 1 H NMR (ET42365-29-P1A, 400 MHz, chloroform-d) δ 6.79 (s, 1H), 7.42 (dd, J = 8.88, 2.25 Hz, 1H), 7.56 (d, J = 2.25 Hz, 1H), 8.34 (d, J = 8.88 Hz, 1H)

[0598] General procedure for the preparation of compound 3-ET42365-43.

[0599]

[0600] To a solution of compound 2 (12 g, 33.13 mmol, 1 eq), Cs2CO3 (21.59 g, 66.26 mmol, 2 eq) and diphenylmethylamine (9.11 g, 49.70 mmol, 8.59 mL, 1.5 eq) in THF (300 mL) was added Pd(OAc)2 (743.81 mg, 3.31 mmol, 0.1 eq) and BINAP (4.13 g, 6.63 mmol, 0.2 eq) under nitrogen, and the reaction was stirred at 60 ° C for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was filtered and the filtrate was concentrated to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 5 / 1) to produce compound 3 (13 g, yield 79.4%) as a yellow solid.

[0601] LCMS(ESI+):RT=0.827min,m / z 396.2(M+H) + .

[0602] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0603] General procedure for the preparation of compound 4-ET42365-49.

[0604]

[0605] To a solution of compound 3 (13 g, 26.30 mmol, 80% purity, 1 equivalent) in DMF (150 mL) was added NBS (5.62 g, 31.57 mmol, 1.2 equivalents) portionwise at 0 ° C. The reactants were stirred at 25 ° C. for 16 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reactants were diluted with water (300 mL) and extracted with ethyl acetate (3 × 150 mL). The organic layer was separated and the combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 60 / 1 to 10 / 1) to produce compound 4 (9 g, yield 68.54%) as a yellow solid.

[0606] 1 H NMR (ET42365-49-P1A, 400 MHz, chloroform-d) δ 5.70 (br d, J = 5.04 Hz, 1H), 5.77 (d, J = 5.48 Hz, 1H), 6.60-6.66 (m, 2H), 7.29-7.43 (m, 10H), 7.88 (d, J = 8.99 Hz, 1H)

[0607] General procedure for the preparation of compound 5-ET42365-65.

[0608]

[0609] To a solution of compound 4 (1 g, 2.11 mmol, 1 eq), [4-fluoro-3-(trifluoromethyl)-phenyl]boronic acid (613.75 mg, 2.95 mmol, 1.4 eq) and NaCO (446.96 mg, 4.22 mmol, 2 eq) in a mixture of toluene (20 mL), EtOH (5 mL) and HO (1.2 mL) was added Pd(dppf)Cl.CHCl (172.19 mg, 210.85 μmol, 0.1 eq) under nitrogen and the reaction was stirred at 100° C. for 40 hours. LCMS indicated that the starting material was consumed and a new peak with the desired Ms was detected. The reaction was filtered and the filtrate was concentrated to yield the crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give compound 5 (1.5 g, yield 37.19%) as a white solid.

[0610] LCMS(ESI+):RT=0.943min,m / z 574.3(M+H) + .

[0611] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0612] General procedure for the preparation of compound 6 - ET42365-70.

[0613]

[0614] To a solution of compound 5 (1.5 g, 2.61 mmol, 1 eq) in EtOH (30 mL)

[0615] To the 4-nitro-2-oxo-1-oxo-4-oxo-2 ...

[0616] LCMS(ESI+):RT=0.943min,m / z 602.3(M+H) + .

[0617] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0618] General procedure for the preparation of A01B01C04D01 - ET42365-75.

[0619]

[0620] To a solution of compound 6 (0.5 g, 830.61 μmol, 1 equivalent) in MeOH (10 mL) was added concentrated HCl (2.52 g, 24.92 mmol, 36% purity, 30 equivalents) and the reactants were stirred at 80 ° C for 12 hours. LCMS showed that about 18% of the starting material remained and a new peak with the desired product Ms was detected. The reactants were concentrated to produce a crude product. The crude product was purified by chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 5 / 1) to produce A01B01C04D01 (550 mg, 68.38% yield) obtained as a yellow solid.

[0621] LCMS(ESI+):RT=0.800min,m / z 436.2(M+H) + .

[0622] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0623] General procedure for the preparation of A02B01C04D01 - ET42365-88.

[0624]

[0625] To a solution of A01B01C04D01 (200 mg, 458.98 μmol, 1 eq) in CHCl 3 (8 mL) was added NCS (73.55 mg, 550.77 μmol, 1.2 eq). The mixture was stirred at 20 ° C for 12 hours. TLC (petroleum ether / ethyl acetate = 3 / 1) showed that the starting material was consumed and new spots were produced. The reactants were concentrated to produce a crude product. The crude product was purified by preparative TLC (petroleum ether / ethyl acetate = 3 / 1) to produce A02B01C04D01 (90 mg, 40.7% yield) as a yellow solid.

[0626] LCMS(ESI+):RT=0.833min,m / z 470.1(M+H) + .

[0627] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0628] General procedure for the preparation of A02B01C09D01 - ET42365-98.

[0629]

[0630] To a solution of A02B01C04D01 (90 mg, 191.41 μmol, 1 equivalent) in AcOH (2 mL) was added paraformaldehyde (57.47 mg, 1.91 mmol, 10 equivalents). The mixture was stirred at 20 ° C for 1.5 hours, then NaBH3CN (60.14 mg, 957.05 μmol, 5 equivalents) was added and the mixture was stirred at 25 ° C for 12 hours. TLC (petroleum ether / ethyl acetate = 4 / 1) showed that the starting material was consumed and new spots were produced. The reactants were concentrated to produce a crude product. The crude product was purified by preparative TLC (petroleum ether / ethyl acetate = 4 / 1) to produce A02B01C09D01 (41.9 mg, 42.57% yield) as an off-white solid.

[0631] 1 H NMR (ET42365-98-P1, 400 MHz, chloroform-d) δ 2.53 (s, 6H), 3.79 (s, 3H), 4.86 (s, 1H), 6.50 (s, 1H), 7.20 (s, 1H), 7.37 (dd, J = 8.19, 1.94 Hz, 1H), 7.55-7.61 (m, 2H)

[0632] LCMS(ESI+):RT=2.940min,m / z 498.1(M+H) + .

[0633] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0634] General procedure for the preparation of A02B01C04D01 - ET42365-86.

[0635]

[0636] To a solution of A01B01C04D01 (150 mg, 344.23 μmol, 1 equivalent) in CHCl 3 (6 mL) was added NCS (68.95 mg, 516.35 μmol, 1.5 equivalents) at 0° C. The reactants were stirred at 20° C. for 48 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reactants were concentrated to produce a crude product. The crude product was purified by preparative TLC (petroleum ether / ethyl acetate=3 / 1) to produce A02B01C04D01 (26.5 mg, 16.27% yield) as a yellow solid.

[0637] 1H NMR (ET42365-86-P1B, 400 MHz, chloroform-d) δ 3.86 (s, 3H), 4.17 (br s, 2H), 4.82 (s, 1H), 6.56 (s, 1H), 7.20 (s, 1H), 7.53-7.57 (m, 1H), 7.73 (d, J = 8.28 Hz, 1H), 7.76 (d, J = 1.63 Hz, 1H)

[0638] LCMS(ESI+):RT=3.527min,m / z 470.1(M+H) + .

[0639] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0640] General procedure for the preparation of A01B01C04D01 - ET42365-63.

[0641]

[0642] To a solution of compound 6 (100 mg, 166.12 μmol, 1 eq) in MeOH (1 mL) was added concentrated HCl (504.75 mg, 4.98 mmol, 36% purity, 30 eq), and the reaction was stirred at 80°C for 8 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was concentrated to yield a crude product. The crude product was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 45%-65%, 8 min) to yield A01B01C04D01 (21.5 mg, 29.61% yield) as a pink solid.

[0643] 1H NMR (ET42365-63-P1B, 400MHz, chloroform-d) δ3.76 (br s,2H),3.85(s,3H),4.87(s,1H),6.48(d,J=8.25Hz,1H),6.55(s,1H),7.05(d,J=8.25 Hz,1H),7.56(dd,J=8.19,1.81Hz,1H),7.69(d,J=8.25Hz,1H),7.78(d,J=1.63Hz,1H)

[0644] LCMS(ESI+):RT=3.311min,m / z 436.1(M+H) + .

[0645] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0646] Synthesis of NUCC-0226529 (ET42365-96-1) and NUCC-0226527 (ET42365-83-1)

[0647]

[0648] Synthesis scheme

[0649] Scheme 14: Preparation of A02B01C05D01 (NUCC-0226529)

[0650]

[0651] Scheme 15: Preparation of A01B01C03D01 (NUCC-0226527)

[0652] Chemical synthesis

[0653] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0654] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0655] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0656] The largest experiment ever run:

[0657] General procedure for the preparation of compound 7-ET42365-79.

[0658]

[0659] To a solution of compound 6 (500 mg, 830.61 μmol, 1 eq) in AcOH (10 mL) was added paraformaldehyde (249.40 mg, 8.31 mmol, 10 eq). After stirring at 20 ° C for 1.5 hours, NaBH3CN (260.99 mg, 4.15 mmol, 5 eq) was added. The mixture was stirred at 25 ° C for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reactants were concentrated to produce a crude product, which was diluted with water (50 mL) and extracted with ethyl acetate (3×50 mL). The organic layer was separated and the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give crude compound 7 (750 mg, yield 58.63%) obtained as a yellow solid. The crude product was used directly in the next step without further purification.

[0660] LCMS(ESI+):RT=0.969min,m / z 616.3(M+H) + .

[0661] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0662] General procedure for the preparation of A01B01C03D01 - ET42365-89.

[0663]

[0664] To a solution of compound 7 (550 mg, 892.86 μmol, 1 eq) in MeOH (10 mL) was added concentrated HCl (2.64 g, 26.79 mmol, 2.59 mL, 37% purity, 30 eq). The mixture was stirred at 80 ° C for 24 hours. LCMS indicated that approximately 16% of the starting material remained and a new peak with the desired product Ms was detected. The reaction was concentrated to produce a crude product, which was diluted with water (50 mL) and extracted with ethyl acetate (3×50 mL). The organic layer was separated and the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to produce A01B01C03D01 (330 mg, yield 69.85%) as a yellow solid.

[0665] LCMS(ESI+):RT=0.852min,m / z 450.2(M+H) + .

[0666] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0667] General procedure for the preparation of A02B01C05D01 - ET42365-96.

[0668]

[0669] To a solution of A01B01C03D01 (300 mg, 566.95 μmol, 85% purity, 1 eq) in CHCl₃ (12 mL) was added NCS (227.12 mg, 1.70 mmol, 3 eq). The mixture was stirred at 20°C for 12 hours. LCMS indicated approximately 45% of the starting material remained and 26% of the desired product was detected. The reaction was concentrated to yield a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 um; mobile phase [water (10 mM NH₄HCO₃)-ACN]; B%: 50%-80%, 8 min) to yield A02B01C05D01 (65.4 mg, 23.82% yield) as a white solid.

[0670] 1 H NMR (ET42365-96-P1A, 400 MHz, chloroform-d) δ 2.39 (s, 3H), 3.86 (s, 3H), 4.89 (s, 1H), 6.55 (s, 1H), 7.22 (s, 1H), 7.55-7.60 (m, 1H), 7.64-7.69 (m, 1H), 7.78 (d, J = 1.88 Hz, 1H)

[0671] LCMS(ESI+):RT=2.520min,m / z 484.1(M+H) + .

[0672] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0673] General procedure for the preparation of A01B01C03D01 - ET42365-83.

[0674]

[0675] To a solution of compound 7 (200 mg, 324.68 μmol, 1 eq) in MeOH (2 mL) was added HCl (959.84 mg, 9.74 mmol, 37% purity, 30 eq). The mixture was stirred at 80°C for 12 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was concentrated to produce a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 55%-75%, 8 min) to produce A01B01C03D01 (25.7 mg, 17.19% yield) as a pink solid.

[0676] 1 H NMR (ET42365-83-P1A, 400 MHz, chloroform-d) δ 2.84 (s, 3H), 3.85 (s, 3H), 4.78 (s, 1H), 6.42 (br d, J = 8.63 Hz, 1H), 6.55 (s, 1H), 7.16 (d, J = 8.38 Hz, 1H), 7.52 (br d, J = 7.88 Hz, 1H), 7.66-7.76 (m, 2H)

[0677] LCMS(ESI+):RT=3.551min,m / z 450.1(M+H) + .

[0678] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0679] Synthesis of NUCC-0226501 (ET42365-34-1)

[0680]

[0681] Synthesis scheme: Preparation of A02B01C02D01_isomer (NUCC-0226501)

[0682]

[0683] Chemical synthesis

[0684] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0685] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0686] The largest experiment ever run:

[0687] General procedure for the preparation of core A3_2-ET43365-14.

[0688]

[0689] To a solution of Core A3_1 (200 mg, 417.74 μmol, 1 eq) in DMF (4 mL) was added KCO (115.47 mg, 835.47 μmol, 2 eq) and MeI (118.59 mg, 835.47 μmol, 2 eq), and the reaction was stirred at 20°C for 12 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (3 x 20 mL). The organic layer was separated and the combined organic layers were washed with brine (30 mL), dried over NaSO, filtered, and concentrated under reduced pressure to produce Core A3_2 (180 mg, 78.69% yield) as a yellow oil. The crude product was used directly in the next step without further purification.

[0690] LCMS(ESI+):RT=0.862min,m / z 493.2(M+H) + .

[0691] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0692] General procedure for the preparation of core A3_5-ET42365-22.

[0693]

[0694] To a solution of core A3_2 (180 mg, 365.26 μmol, 1 eq) in CH 3 CN (4 mL) was added portionwise NCS (53.65 mg, 401.79 μmol, 1.1 eq) at 20° C. The reaction was stirred at 80° C. for 12 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was concentrated under high vacuum to produce a crude product. The crude product was purified by preparative TLC (petroleum ether / ethyl acetate = 2 / 1) to produce core A3_5 (110 mg, 51.41% yield) as a white solid.

[0695] 1 H NMR (ET42365-22-P1A, 400 MHz, chloroform-d) δ 1.78 (s, 3H), 3.78 (s, 3H), 3.87 (s, 3H), 7.06 (d, J = 8.63 Hz, 1H), 7.37 (d, J = 8.63 Hz, 1H), 7.46 (dd, J = 8.25, 1.75 Hz, 1H), 7.56 (d, J = 8.25 Hz, 1H), 7.67 (d, J = 1.63 Hz, 1H)

[0696] General procedure for the preparation of A02B01C02D01_isomers-ET42365-34.

[0697]

[0698] To a solution of core A3_5 (100 mg, 189.67 μmol, 1 eq) in MeOH (5 mL) was added K2CO3 (52.43 mg, 379.33 μmol, 2 eq) and the reaction was stirred at 20 ° C for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was diluted with water (30 mL), adjusted to pH = 6-7, and extracted with ethyl acetate (3×15 mL). The organic layer was separated and the combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18150*40mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 65%-95%, 8min) to give A02B01C02D01_isomer (84.5mg, yield 90.99%) as a white solid.

[0699] 1 H NMR (ET42365-34-P1A, 400 MHz, chloroform-d) δ 3.82 (d, J = 4.75 Hz, 6H), 4.97 (s, 1H), 6.76 (d, J = 8.76 Hz, 1H), 7.29 (d, J = 8.63 Hz, 1H), 7.53 (dd, J = 8.13, 2.00 Hz, 1H), 7.66 (d, J = 8.25 Hz, 1H), 7.75 (d, J = 1.88 Hz, 1H)

[0700] LCMS(ESI+):RT=2.439min,m / z 485.1(M+H) + .

[0701] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0702] Synthesis of NUCC-0226500 (ET42365-41-1)

[0703]

[0704] Synthesis scheme: Preparation of A02B01C02D01 (NUCC-0226500)

[0705]

[0706] Chemical synthesis

[0707] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0708] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0709] The largest experiment ever run:

[0710] General procedure for the preparation of core A3_4-ET43365-31.

[0711]

[0712] To a solution of Core A3_1 (700 mg, 1.46 mmol, 1 eq) and N-isopropylpropane-2-amine (295.89 mg, 2.92 mmol, 413.26 μL, 2 eq) in CHCl 3 (25 mL) was added NCS (214.76 mg, 1.61 mmol, 1.1 eq) at 0° C. The reaction was stirred at 25° C. for 24 hours. LCMS indicated that approximately 28% of the starting material remained and 64% of the desired product was detected. The reaction was concentrated under high vacuum to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to produce Core A3_4 (200 mg, 25.32% yield) as a white solid.

[0713] 1 H NMR (ET42365-31-P1A, 400 MHz, chloroform-d) δ 1.78 (s, 3H), 3.83 (s, 3H), 5.92 (s, 1H), 6.51 (s, 1H), 7.42 (s, 1H), 7.50 (dd, J = 8.25, 1.50 Hz, 1H), 7.61 (d, J = 8.38 Hz, 1H), 7.72 (d, J = 1.38 Hz, 1H)

[0714] General procedure for the preparation of core A3_3-ET42365-40.

[0715]

[0716] To a solution of Core A3_4 (200 mg, 389.70 μmol, 1 eq) and K2CO3 (107.72 mg, 779.40 μmol, 2 eq) in DMF (5 mL) was added MeI (110.63 mg, 779.40 μmol, 2 eq) and the reaction was stirred at 25°C for 2 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (3×15 mL). The organic layer was separated and the combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce Core A3_3 (150 mg, 69.35% yield) as a yellow oil. The crude product was used directly in the next step without further purification.

[0717] LCMS(ESI+):RT=0.892min,m / z 527.2(M+H) + .

[0718] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0719] General procedure for the preparation of A02B01C02D01 - ET42365-41.

[0720]

[0721] To a solution of core A3_3 (150 mg, 284.50 μmol, 1 eq) in MeOH (5 mL) was added K2CO3 (78.64 mg, 569.00 μmol, 2 eq) and the reaction was stirred at 20 ° C for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was diluted with water (30 mL), adjusted to pH = 6-7, and extracted with ethyl acetate (3×15 mL). The organic layer was separated and the combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18150*40mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 65%-95%, 8min) to give A02B01C02D01 (106.5mg, yield 75.69%) as an off-white solid.

[0722] 1 H NMR (ET42365-41-P1A, 400 MHz, chloroform-d) δ 3.59 (s, 3H), 3.87 (s, 3H), 5.06 (s, 1H), 6.61 (s, 1H), 7.34 (s, 1H), 7.56-7.62 (m, 1H), 7.65-7.71 (m, 1H), 7.80 (s, 1H)

[0723] LCMS(ESI+):RT=2.501min,m / z 485.1(M+H) + .

[0724] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0725] Synthesis of Series 10-D, E, F, M, K, L

[0726]

[0727] Scheme 16: Pathway to the synthesis of Series 10-D, Series 10-E, Series 10-F, Series 10-M, Series 10-K-P1, Series 10-K-P2, Series 10-L-P1 and Series 10-L-P2.

[0728] Let's use Series 10-E as an example.

[0729]

[0730] Scheme 17: Synthesis of Compound 2

[0731]

[0732] Scheme 18: Synthesis of Compound 2

[0733]

[0734] Scheme 19: Synthesis of Compound 2

[0735]

[0736] Scheme 20: Synthesis of compound 3a

[0737]

[0738] Scheme 21: Synthesis of series 10-K-P1 and series 10-K-P2

[0739]

[0740] The largest experiment ever run:

[0741] General procedure for the preparation of compound 2-ET37412-154

[0742]

[0743] A mixture of Core A6 (100 mg, 208.87 μmol), Compound 2 (55.85 mg, 313.30 μmol), diisopropyl azodicarboxylate (84.47 mg, 417.74 μmol, 81.22 μL), and triphenylphosphine (109.57 mg, 417.74 μmol) in tetrahydrofuran (2 mL) was degassed and purged with N₂ three times, and the reaction was stirred at 25°C for 12 h. K₂CO₃ (57.73 mg, 417.74 μmol) was then added to the reaction, and the mixture was stirred at 25°C for 1 h. LCMS indicated the reaction was complete. The reaction was poured into water (2 mL) and extracted with ethyl acetate (5 mL). The organic layer was concentrated to give a residue, which was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (10mM NH4HCO3)-acetonitrile]; B%: 85%-98%, 10 min) to give series 10-E (9 mg, yield 7%) as a white solid.

[0744] 1 H NMR: ET37412-154-HNMRP1 (400MHz, CDCl3) δ0.90 (d, J=6.6Hz, 6H), 1.50 (br d,J=6.3Hz,3H),1.85(quind,J=6.7,13.4Hz,1H),2.46(d,J=7.2Hz,2H),5.08(s,1H),3.81(s,3H),5.32(q,J=6.3 Hz,1H),6.53(s,1H),6.60(d,J=8.7Hz,1H),7.15-7.05(m,5H),7.60-7.53(m,1H),7.67-7.62(m,1H),7.83(s,1H)

[0745] LCMS method:

[0746] LCMS (ESI+): m / z = 597.3 (M+H) + ,RT:3.534min.

[0747] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0748] General procedure for the preparation of compound 2 - Notebook page: ET37412-181.

[0749]

[0750] To a solution of compound 1 (1 g, 5.67 mmol) in tetrahydrofuran (10 mL) at -40 °C

[0751] Borane tetrahydrofuran complex (6.81 mL, 6.81 mmol) was added to the reaction mixture. Chloro-bis[(1R,2S,3R,5R)-2,6,6-trimethylnorpinan-3-yl]borane (2.18 g, 6.81 mmol) in tetrahydrofuran (5 mL) was added at -40 ° C, and the reaction was stirred at 25 ° C for 2 h. The reaction was poured into water (20 mL). The organic phase was separated and the aqueous phase was extracted 3 times with ethyl acetate (10 mL). The organic phase was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under vacuum to produce a crude product. The crude product was purified by column chromatography on silica gel (petroleum ether / ethyl acetate = 1 / 0 to 1 / 2) to produce compound 2 (0.5 g, yield 45%) as a colorless oil.

[0752] 1 H NMR (15017259-181-P1 400 MHz, chloroform-d)

[0753] δ0.92(d,J=6.62Hz,6H),1.50(d,J=6.62Hz,3H),1.83-1.92(m,2H),2.48(d,J=7. 28Hz,2H),4.88(q,J=6.39Hz,1H),7.14(d,J=7.94Hz,2H),7.29(d,J=7.94Hz,2H)

[0754] General procedure for the preparation of Compound_2-ET37412-153.

[0755]

[0756] To a solution of compound 1 (0.5 g, 2.84 mmol) in methanol (10 mL) was added NaBH4 (214.63 mg, 5.67 mmol). The reaction mixture was stirred at 25 ° C for 12 h under N2 atmosphere. The reaction was poured into water (20 mL) and extracted with ethyl acetate (15 mL). The organic layer was concentrated to produce compound 2 (300 mg, 59% yield) as a colorless oil.

[0757] 1 H NMR(ET37412-153-HNMRP1,400MHz,DMSO-d6)

[0758] δ0.85(d,J=6.63Hz,6H),1.29(d,J=6.38Hz,3H),1.73-1.85(m,1H),2.41(d,J=7.13Hz,2H),7.07(d,J=7.88Hz,2H),7.23(d,J=7.88Hz,2H)

[0759] General procedure for the preparation of compound 2-ET37412-165

[0760]

[0761] To a solution of compound 1 (0.5 g, 2.81 mmol) in tetrahydrofuran (5 mL) was added BH 3 .THF (1 M, 5.61 mL). The mixture was stirred at 25 ° C for 12 hours. TLC indicated that the reaction was complete. The reactants were quenched with methanol (5 mL), and the mixture was then concentrated to produce a residue, which was purified by preparative TLC to produce compound 2 (0.3 g, 65% yield) as a colorless oil.

[0762] General procedure for the preparation of compound 5a-ET37412-264

[0763]

[0764] To a solution of compound 4a (5 g, 37.26 mmol) in tetrahydrofuran (50 mL) was added lithium diisopropylamide (2 M, 20.50 mL) and trimethylsilyl chloride (4.45 g, 40.99 mmol) at 0 ° C. The reaction was stirred at 25 ° C for 12 h. TLC showed that the reaction was complete. The reaction was poured into water (50 mL) and extracted with ethyl acetate (60 mL). The organic layer was dried and concentrated to produce compound 5a (5 g, yield 65%) as a colorless oil, which was used directly in the next step.

[0765] 1 H NMR(ET37412-264-1,400MHz, CDCl3-d6)

[0766] δ0.08-0.14(m,9H),2.19(s,3H),4.23(d,J=1.47Hz,1H),4.61-4.82(m,1 H),4.71(d,J=1.59Hz,1H),6.97(d,J=8.07Hz,2H),7.33(d,J=8.19Hz,2H)

[0767] General procedure for the preparation of compound 6a - Notebook page: ET37412-267.

[0768]

[0769] To a solution of compound 5a (4 g, 19.38 mmol) in dichloromethane (10 mL) was added CH2I2 (7.79 g, 29.08 mmol) and diethylzinc (1 M, 29.08 mL) at 0 ° C. The reaction mixture was stirred at 25 ° C for 12 h. TLC showed that the reaction was complete. The reactant was poured into water (10 mL) and extracted with ethyl acetate (20 mL). The organic layer was dried and concentrated to produce compound 6a (1 g, yield 23%) as a yellow oil, which was used directly in the next step.

[0770] 1 H NMR(ET37412-267-11,400MHz,CDCl3-d6)δ0.00(s,9H),0.85-0.95(m,2H),1.06-1.16(m,2H),2.26(s,3H),7.15-7.23(m,4H)

[0771] General procedure for the preparation of compound 3a - Notebook page: ET37412-281.

[0772]

[0773] To a solution of compound 6a (1 g, 12.71 mmol) in tetrahydrofuran (10 mL) was added trimethylchlorosilane (0.41 g, 15.25 mmol) at 0 ° C. The reaction mixture was stirred at 25 ° C for 1 h. TLC showed that the reaction was complete. The reactant was poured into water (10 mL) and extracted with ethyl acetate (20 mL). The organic layer was dried and concentrated to produce compound 3a (1 g, yield 53%) as a yellow oil, which was used directly in the next step.

[0774] 1H NMR(ET37412-281-2,400MHz,CDCl3-d6)

[0775] δ0.95-1.05(m,2H),1.18-1.25(m,2H),2.34(s,3H),7.11-7.25(m,4H)

[0776] General procedure for the preparation of series 10-K-P1 and series 10-K-P2 - ET37412-175.

[0777]

[0778] 40 mg of series 10-K-1 was separated by SFC (column: DAICEL CHIRALCEL OJ (250 mm×30 mm, 10 μm); mobile phase: [0.1% NH 3 H 2 OMEOH]; B%: 50%-50%, 15 min column: Chiralcel OJ-3, 50×4.6 mm ID, 3 μm, mobile phase: A: CO 2 B: MeOH (0.05% IPAm, v / v), flow rate: 3.4 mL / min, column temperature: 35° C., ABPR: 1800 psi). The two fractions were concentrated to produce series 10-K-P1 (24 mg, yield 64%) and series 10-K-P2 (12 mg, yield 33%).

[0779] 1 H NMR(ET37412-175-1,400MHz,MeOD)

[0780] δ1.45(d,J=6.17Hz,3H),3.76(s,3H),5.43(d,J=6.39Hz,1H),6.54(s,1H),6.50-6.58(m,1H),6.66(d,J= 8.60Hz,1H),7.10(d,J=8.60Hz,1H),7.18-7.33(m,5H),7.59-7.65(m,1H),7.66-7.72(m,1H),7.78(s,1H)

[0781] LCMS (ESI+): m / z=541.1 (M+H) + ,RT:3.187min

[0782] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0783] SFC method:

[0784] Column: Chiralcel OJ-3, 50×4.6mm ID, 3um

[0785] Mobile phase: A: CO2 B: MeOH (0.05% IPAm, v / v)

[0786] gradient:

[0787]

[0788] Flow rate: 3.4 mL / min

[0789] Column temperature: 35°C

[0790] ABPR:1800psi

[0791] 1 H NMR (ET37412-175-2, 400MHz, MeOD) δ1.45 (d, J=6.17Hz, 3H), 3.76 (s, 3H), 5.43 (q, J=6.84Hz, 1H), 6.54 (s, 1H), 6. 65(d,J=8.82Hz,1H),7.10(d,J=8.60Hz,1H),7.17-7.36(m,5H),7.60-7.66(m,1H),7.67-7.71(m,1H),7.78(s,1H)

[0792] LCMS (ESI+): m / z=541.1 (M+H) + ,RT:3.187min

[0793] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0794] SFC method:

[0795] Column: Chiralcel OJ-3, 50×4.6mm ID, 3um

[0796] Mobile phase: A: CO2 B: MeOH (0.05% IPAm, v / v)

[0797] gradient:

[0798]

[0799] Flow rate: 3.4 mL / min

[0800] Column temperature: 35°C

[0801] ABPR:1800psi

[0802] Synthesis of Series 10-A (NUCC-0226303)

[0803]

[0804] Synthesis scheme

[0805] Scheme 22: Synthesis of Compound 5

[0806]

[0807] Scheme 23: Synthesis of Series 10-A

[0808]

[0809] The largest experiment ever run:

[0810] General procedure for the preparation of compound 2-ET37412-122

[0811]

[0812] To a solution of compound 1 (10 g, 78.02 mmol) and propane-1,3-diol (29.69 g, 390.11 mmol) in acetonitrile (100 mL) was added benzyl(trimethyl)ammonium hydroxide (1.21 g, 2.89 mmol). The reaction mixture was stirred at 25°C for 72 h. TLC (petroleum ether / ethyl acetate = 3:1, Rf = 0.5) indicated the reaction was complete. The solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to produce compound 2 (11 g, 69% yield) as a colorless oil.

[0813] General procedure for the preparation of compound 3-ET37412-126

[0814]

[0815] Under inert atmosphere conditions (argon), I2 (5.59 g, 22.03 mmol), triphenylphosphine (4.62 g, 17.62 mmol) and imidazole (1.20 g, 17.62 mmol) were dissolved in tetrahydrofuran (30 mL). A solution of compound 2 (3 g, 14.69 mmol) in tetrahydrofuran (30 mL) was added to the reactants and the reactants were stirred at 25 ° C for 12 h. LCMS showed that the reaction was complete. The reaction mixture was filtered to remove the white precipitate and the solvent was evaporated. The crude product was purified by silica gel chromatography, eluting with petroleum ether / ethyl acetate = 1 / 0, 0 / 1 to produce compound 3 (2.4 g, 52% yield) as a brown oil.

[0816] 1 H NMR: ET37412-126-2 (400MHz, CDCl3)

[0817] δ1.45(s,9H),2.00-2.06(m,2H),2.47(t,2H),3.29(br t,J=6.17Hz,2H),3.49(t,2H),3.65(t,2H)

[0818] General procedure for the preparation of compound 4-ET37412-107

[0819]

[0820] To a mixture of Core A6 (500 mg, 1.04 mmol) and Compound 3 (394 mg, 1.25 mmol) in N,N-dimethylformamide (1 mL) was added KCO (173 mg, 1.25 mmol). The reaction was stirred at 25°C for 12 h. LCMS indicated that the reaction was complete. The reaction was poured into water (20 mL) and extracted with ethyl acetate (20 mL). The organic layer was concentrated to produce a residue, which was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to produce Compound 4 (400 mg, 58% yield) as a colorless oil.

[0821] LCMS method:

[0822] LCMS (ESI+): m / z=665.3(M+H)+, RT: 0.996min.

[0823] 5-95AB_2min: LC / MS (the column used for chromatography was a Kinetex 5μm EVO C18100A. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 2.20 min. 5% B in 0.01 min, 5-95% B (0.01-1.00 min), 95-100% B (1.00 -1.80 min), 5% B in 1.81 min, with a hold of 0.39 min at 5% B. The flow rate was 1.0 mL / min (0.01-1.80) 1.2 mL (1.81-2.20).

[0824] General procedure for the preparation of compound 5 - ET37412-140

[0825]

[0826] A solution of compound 4 (380 mg, 395.71 mmol) in HCl / dioxane (5 mL, 5 N) was stirred at 25° C. for 12 h. TLC (petroleum ether / ethyl acetate = 1:1, Rf = 0.05) indicated the reaction was complete. The reaction was concentrated to produce compound 5 (250 mg, 70% yield) as a colorless oil, which was used in the next step without further purification.

[0827] General procedure for the preparation of compound 7-ET37412-141

[0828]

[0829] To a solution of compound 6 (122.20 mg, 557.41 mmol) in N,N-dimethylformamide (2 mL) was added o-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (264 mg, 697 mmol), N-ethyl-N-isopropylpropane-2-amine (180.10 mg, 1.39 mmol), and R3 (200 mg, 464.51 mmol). The reaction was stirred at 25°C for 12 h. TLC (petroleum ether / ethyl acetate = 1:1, Rf = 0.49) indicated the reaction was complete. The reaction was poured into water (20 mL) and extracted with ethyl acetate (20 mL). The organic layer was concentrated to give a residue, which was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to give Compound 7 (250 mg, 85% yield) as a yellow solid.

[0830] General procedure for the preparation of compound 8-ET37412-143

[0831]

[0832] A solution of compound 7 (250 mg, 395.71 μmol) in HCl / dioxane (5 mL, 4 N) was stirred at 25° C. for 12 h. LCMS indicated the reaction was complete. The reaction was concentrated to yield compound 8 (250 mg, 83.18% yield) as a colorless oil, which was used in the next step without further purification.

[0833] LCMS (ESI+): m / z=532.3(M+H)+, RT: 0.637min.

[0834] 5-95AB_2min: LC / MS (the column used for chromatography was a Kinetex 5μm EVO C18100A. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 2.20 min. 5% B in 0.01 min, 5-95% B (0.01-1.00 min), 95-100% B (1.00 -1.80 min), 5% B in 1.81 min, with a hold of 0.39 min at 5% B. The flow rate was 1.0 mL / min (0.01-1.80) 1.2 mL (1.81-2.20).

[0835] General Procedure for Preparation of Series 10-A - ET37412-147

[0836]

[0837] To a mixture of compound 5 (100 mg, 164 mmol) and compound 8 (174.6 mg, 328 mmol) in N,N-dimethylformamide (1 mL) were added N-ethyl-N-isopropylpropane-2-amine (106 mg, 821 mmol) and o-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (124 mg, 328 mmol). The mixture was stirred at 25°C for 12 h. Afterwards, KCO (45.39 mg, 328.45 μmol) was added to the mixture, and the reaction was stirred at 25°C for 1 h. LCMS indicated the reaction was complete. The reaction was filtered to give a residue, which was purified by preparative HPLC (column: Phenomenex luna C18100*40mm*5um; mobile phase: [water (0.1% 2,2,2-trifluoroacetic acid)-acetonitrile]; B%: 40%-74%, 8 min) to give series 10-A (5 mg, 3% yield) as a white solid.

[0838] 1 H NMR: ET37412-147-P1A1 (400MHz, DMSO-d6)

[0839] δ0.88-0.97(m,9H),1.74(br t,J=6.05Hz,2H),1.90(ddd,J=12.81,8.71,4.52Hz,1H),2.00-2.10(m,1H),2.29(br t,J=6.36Hz,2H),2.41-2.46(m,3H),3.15-3.24(m,2H),3.29(br t,J=6.17Hz,2H),3.43-3.52(m,4H),3.56-3.67(m,4H),3.73-3.74(m,3H),3.94(br d,J=1.47Hz,2H),3.99(br t,J=5.99Hz,3H),4.19-4.28(m,1H),4.37(brd,J=6.60Hz,1H),4.40-4.47(m,1H),4.55(d,J=9 .66Hz,1H),6.71(s,1H),6.76(d,J=8.68Hz,1H),7.25(d,J=8.56Hz,1H),7.39(s,4H),7.44(br d,J=9.41Hz,1H),7.64(br d,J=8.31Hz,1H),7.75(dd,J=4.95,3.24Hz,2H),7.96(br t,J=5.50Hz,1H),8.57(t,J=5.93Hz,1H),8.97(s,1H)

[0840] LCMS (ESI+): m / z=1080.3(M+H)+, RT: 2.723min.

[0841] 5_95AB_6min_MS1500-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1500.

[0842] Synthesis of Series 10-B (NUCC-0226304)

[0843]

[0844] Synthesis scheme:

[0845] Scheme 24: Synthesis of Series 10-B (NUCC-0226304)

[0846]

[0847] General procedure for the preparation of compound 2-ET37412-142

[0848]

[0849] To a solution of compound 1 (215.76 mg, 868.87 mmol) in 1-methyl-2-pyrrolidone (2 mL) was added N-ethyl-N-isopropylpropane-2-amine (187.16 mg, 1.45 mmol) and 2-(2,6-dioxo-3-piperidinyl)-4-fluoro-isoindoline-1,3-dione (200 mg, 724.06 mmol). The reaction was stirred at 90°C for 12 h. TLC (petroleum ether / ethyl acetate = 1:1, RF = 0.23) indicated the reaction was complete. The reaction was poured into water (20 mL) and extracted with ethyl acetate (20 mL). The organic layer was concentrated to give a residue, which was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to give Compound 2 (150 mg, 41% yield) as a colorless oil.

[0850] General procedure for the preparation of compound 3-ET37412-144

[0851]

[0852] A solution of compound 2 (200 mg, 396.41 mmol) in HCl / dioxane (2 mL, 4 M) was stirred at 25° C. for 0.2 h. LCMS indicated the reaction was complete. The reaction was concentrated to yield compound 3 (120 mg, 75% yield) as a colorless oil, which was used in the next step without further purification.

[0853] LCMS method:

[0854] LCMS (ESI+): m / z=405.1(M+H)+, RT: 0.605min.

[0855] 5-95AB_2min: LC / MS (the column used for chromatography was a Kinetex 5μm EVO C18100A. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 2.20 min. 5% B in 0.01 min, 5-95% B (0.01-1.00 min), 95-100% B (1.00 -1.80 min), 5% B in 1.81 min, with a hold of 0.39 min at 5% B. The flow rate was 1.0 mL / min (0.01-1.80) 1.2 mL (1.81-2.20).

[0856] General procedure for the preparation of compound 4-ET37412-158

[0857]

[0858] To a solution of compound 5 (100 mg, 164.23 mmol) and compound 3 (79.70 mg, 197.07 mmol) in N, N-dimethylformamide (1 mL) was added N-ethyl-N-isopropylpropane-2-amine (106.13 mg, 821 mmol) and o-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (74.93 mg, 197.07 mmol). The reactants were stirred at 25 ° C for 12 h. LCMS showed that the reaction was complete. The reactants were poured into water (5 mL) and extracted with ethyl acetate (5 mL). The organic layer was concentrated to produce a residue, which was purified by preparative TLC to produce compound 4 (50 mg, 31% yield) as a yellow solid.

[0859] 1 H NMR: ET37412-158-g1 (400MHz, DMSO-d6)

[0860] δ1.71-1.76(m,3H),1.76-1.84(m,2H),1.94-2.06(m,1H),2.27(t,J=6.36Hz,2H),2.53-2.65(m,3H),2.8 2-2.94(m,1H),3.16(q,J=5.71Hz,2H),3.28-3.39(m,6H),3.53-3.63(m,6H),3.74-3.83(m,3H),4.10(br t,J=5.99Hz,2H),5.05(dd,J=12.90,5.44Hz,1H),6.59(br s,1H),6.68(s,1H),7.03(d,J=7.09Hz,1H),7.13(d,J=8.56Hz,1H),7.23(d,J=8.68 Hz,1H),7.54-7.64(m,3H),7.72(d,J=1.47Hz,1H),7.79-7.88(m,2H),11.08(s,1H)

[0861] General Procedure for Preparation of Series 10-B - ET37412-162

[0862]

[0863] To a solution of compound 4 (30 mg, 30.14 μmol, 1 eq) in dimethyl sulfoxide (1 mL) was added KCO (8.33 mg, 60.28 mmol). The reaction was stirred at 80° C. for 12 h. The reaction was purified by preparative HPLC (column: [0.1% 2,2,2-trifluoroacetic acid)-acetonitrile]; mobile phase: [water (0.1% 2,2,2-trifluoroacetic acid)-acetonitrile]; B%: 50%-80%, 9 min) to produce series 10-B (20 mg, 60% yield) as a white solid.

[0864] 1 H NMR: ET37412-162-1 (400MHz, CDCl3-d6)

[0865] δ1.87-1.96(m,2H),2.08-2.19(m,1H),2.44-2.57(m,2H),2.68-2.77(m,2H),2.82-2.9 1(m,1H),3.41-3.48(m,6H),3.55-3.67(m,9H),3.68-3.74(m,3H),3.84(s,3H),4.04(br t,J=6.02Hz,2H),4.89(br dd,J=12.04,5.36Hz,1H),6.57(s,1H),6.69(d,J=8.58Hz,1H),6.90(br d,J=8.46Hz,2H),7.09-7.14(m,1H),7.21(d,J=8.46Hz,1H),7.47-7.54(m,2H),7.60-7.64(m,1H),7.75(s,1H),8.63(br s,1H)

[0866] LCMS (ESI+): m / z = 953.3 (M+H) + ,RT:2.831min.

[0867] 5_95AB_6min_MS1500-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1500.

[0868] Final reports for NUCC-0226504 (ET42365-21-1), NUCC-0226503 (ET42365-19-1), NUCC-0226499 (ET42365-26-1), NUCC-0226498 (ET42365-25-1), NUCC-0226497 (ET42365-23-1), and NUCC-0226496 (ET42365-20-1)

[0869]

[0870] Synthesis scheme: Preparation of A01B02C01D01 (NUCC-0226504), A01B07C01D01 (NUCC-0226503), A01B04C01D01 (NUCC-0226499), A01B06C01D01 (NUCC-0226498), A01B08C01D01 (NUCC-0226497), and A01B03C01D01 (NUCC-0226496)

[0871]

[0872] Take A01B03C01D01 (NUCC-0226496) as an example.

[0873]

[0874]

[0875] Chemical synthesis

[0876] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0877] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0878] General procedure for the preparation of compound 4-ET42365-6

[0879]

[0880] To a solution of compound 3 (2.5 g, 7.02 mmol, 1 equivalent) in DMF (50 mL) was added K2CO3 (1.94 g, 14.04 mmol, 2 equivalents) and 1-(bromomethyl)-4-chloro-benzene (1.73 g, 8.43 mmol, 1.2 equivalents) and the reactants were stirred at 30 ° C for 8 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reactant was diluted with water (150 mL), the precipitate was filtered and dried under high vacuum to produce compound 4 (2 g, 56.3% yield) as a white solid. The product was used directly in the next step without further purification.

[0881] LCMS(ESI+):RT:1.144min,m / z=480.8(M+H) + .

[0882] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0883] General procedure for the preparation of compound 5-ET42365-10

[0884]

[0885] To a solution of compound 4 (200 mg, 416.14 μmol, 1 eq), Na2CO3 (88.21 mg, 832.29 μmol, 2 eq) and (3,4-dichlorophenyl)boronic acid (95.29 mg, 499.37 μmol, 1.2 eq) in a mixture of toluene (6 mL), EtOH (1.2 mL) and H2O (0.3 mL) was added Pd(dppf)Cl2.CH2Cl2 (33.98 mg, 41.61 μmol, 0.1 eq) under nitrogen and the reactants were stirred at 80 ° C for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reactants were filtered and the filtrate was concentrated to produce a crude product. The crude product was purified by preparative TLC (petroleum ether / ethyl acetate=2 / 1) to produce compound 5 (150 mg, 64.92% yield) as a yellow solid.

[0886] LCMS(ESI+):RT:0.936min,m / z=499.1(M+H) + .

[0887] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0888] General procedure for the preparation of A01B03C01D01-ET42365-20

[0889]

[0890] To a solution of compound 5 (150 mg, 300.18 μmol, 1 eq) in EtOH (2.5 mL) was added CH 3 NH NH 2 (150 mg, 1.30 mmol, 171.43 μL, 40% purity, 4.34 eq) and the reaction was stirred at 80° C. for 16 hours. LCMS indicated that the starting material was consumed and two new peaks with the desired product Ms were detected. The reaction was concentrated to yield the crude product. The crude product was purified by preparative HPLC (column: Phenomenex luna C18 100*40mm*5um; mobile phase: [water (0.1% TFA)-ACN]; B%: 55%-85%, 8min) and then (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 60%-90%, 8min) to produce A01B03C01D01 (42.2mg, yield 26.64%) as a white solid.

[0891] 1 H NMR: (ET42365-20-P1X, 400MHz, chloroform-d) δ3.84(s,3H),5.07(s,2H),5.15(s,1H),6.55(s,1H),6.70(d,J=8.51Hz,1H),7 .19(t,J=8.88Hz,3H),7.26(d,J=1.88Hz,1H),7.33(d,J=8.38Hz,2H),7.55(d,J=1.88Hz,1H),7.59(d,J=8.25Hz,1H)

[0892] LCMS(ESI+):RT:2.889min,m / z=527.1,529.0(M+H,M+2+H) + .

[0893] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0894] Synthesis of Series 10-C (NUCC-0226502)

[0895]

[0896] Synthesis Scheme: Synthesis of Series 10-C (NUCC-0226502)

[0897]

[0898] The largest experiment ever run:

[0899] General procedure for the preparation of compound 2-ET37412-173

[0900]

[0901] To a solution of compound 1 (1 g, 4.35 mmol) in pyridine (10 mL) was added trifluoromethanesulfonic anhydride (2.45 g, 8.69 mmol). The mixture was stirred at 25 ° C for 1 h. LCMS showed that the reaction was complete. The reactant was poured into water (10 mL) and extracted with ethyl acetate (10 mL). The organic layer was concentrated to produce compound 2 (1.1 g, yield 70%) as a yellow oil, which was used directly in the next step.

[0902] LCMS method:

[0903] LCMS (ESI+): m / z=363.1(M+H)+, RT: 0.837 min.

[0904] 5-95AB_2min: LC / MS (the column used for chromatography was a Kinetex 5μm EVO C18100A. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 2.20 min. 5% B in 0.01 min, 5-95% B (0.01-1.00 min), 95-100% B (1.00 -1.80 min), 5% B in 1.81 min, with a hold of 0.39 min at 5% B. The flow rate was 1.0 mL / min (0.01-1.80) 1.2 mL (1.81-2.20).

[0905] General procedure for the preparation of compound 3-ET37412-183

[0906]

[0907] To a solution of compound 2 (1 g, 2.76 mmol) in tetrahydrofuran (10 mL) under N₂ was added palladium(II) acetate (61.98 mg, 276.09 μmol), diphenylmethylamine (1.01 g, 5.52 mmol), Cs₂CO₃ (1.80 g, 5.52 mmol), and (±)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (343.83 mg, 552.18 μmol, 0.2 equiv). The mixture was stirred at 60°C for 12 h. LCMS indicated the reaction was complete. The reaction was concentrated to yield a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to yield compound 3 (0.4 g, 37% yield) as a yellow solid.

[0908] 1 H NMR: ET37412-183-1 (400MHz, CDCl3)

[0909] δ5.00(br s,1H),5.65(br d,J=3.30Hz,1H),6.39(d,J=2.20Hz,1H),6.57(s,1H),6.71(dd,J=8.80,2.20Hz,1H),7.31-7.41(m,10H),7.94(d,J=8.80Hz,1H)

[0910] General procedure for the preparation of compound 4-ET37412-214

[0911]

[0912] To a solution of compound 3 (330 mg, 834.65 μmol) in N,N-dimethylformamide (3 mL) was added N-bromosuccinimide (163.41 mg, 918.12 μmol). The reaction was stirred at 80 ° C for 12 h. LCMS showed that the reaction was complete. The reaction was poured into water (10 mL) and extracted with ethyl acetate (10 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to provide compound 4 (200 mg, yield 51%) as a white solid.

[0913] LCMS (ESI+): m / z = 476.2 (M+H) + ,RT:0.935min.

[0914] General procedure for the preparation of compound 5. Notebook page: ET37412-234.

[0915]

[0916] To a solution of compound 4 (0.2 g, 0.421 μmol) in tetrahydrofuran (2 mL) was added (4-chloro-3-(trifluoromethyl)phenyl)boronic acid (0.113 g, 0.506 μmol), [2-(2-aminophenyl)phenyl]-methylsulfonyloxy-palladium; dicyclohexyl-[3,6-dimethoxy-2-(2,4,6-triisopropylphenyl)phenyl]phosphane (38 mg, 0.042 μmol) and potassium phosphate (179 mg, 0.843 μmol) at 25°C. The reaction was stirred at 80°C for 12 h. The reaction was poured into water (5 mL). The organic phase was separated and the aqueous phase was extracted three times with ethyl acetate (5 mL). The organic phase was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under vacuum to produce the crude product. The crude product was purified by column chromatography on silica gel (petroleum ether / ethyl acetate = 1 / 0 to 1 / 2) to give compound 5 (0.1 g, yield 41%) as a colorless oil.

[0917] LCMS: m / z=574.4 (M+H) + ,RT:1.211min

[0918] 5-95AB_2min: LC / MS (the column used for chromatography was a Kinetex 5μm EVO C18100A 2.1*30mm. The detection method was a diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B was held for 0.46 min. 95-5% B (1.61 -1.50 min), wherein 5% B was held for 0.11 min. The flow rate was 1.5 mL / min.

[0919] General procedure for the preparation of series 10-C. Notebook page: ET37412-246.

[0920]

[0921] To a solution of compound 5 (0.1 g, 0.174 μmol) in ethanol (1 mL) was added diazomethane (60 mg, 0.34 μmol) at 25°C. The reaction was stirred at 80°C for 12 h. The reaction was poured into water (5 mL). The mixture was extracted three times with ethyl acetate (5 mL). The organic phase was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under vacuum to produce a crude product, which was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 um; mobile phase: [water (10 mM NH4HCO3)-acetonitrile]; B%: 60%-90%, 8 min) to produce series 10-C (7 mg, 15% yield) as a white solid.

[0922] 1 H NMR(ET37412-246-P1A5,400MHz,DMSO-d6)

[0923] δ3.71(s,3H),4.86(d,J=5.95Hz,1H),5.67(d,J=5.73Hz,1H),6.27(d,J=8.60Hz,1H),6.60(s,1H),6.99(d,J=8.38Hz,1H), 7.22(td,J=5.68,2.54Hz,2H),7.26-7.34(m,8H),7.72(dd,J=8.38,1.54Hz,1H),7.81(dd,J=4.85,3.31Hz,2H),8.53(s,1H)

[0924] LCMS: m / z=602.2 (M+H) + ,RT:3.023min

[0925] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0926] Synthesis of NUCC-0226595 (ET42365-226-1)

[0927]

[0928] Synthesis scheme: Preparation of A03B01D01 (NUCC-0226595)

[0929]

[0930] Chemical synthesis

[0931] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0932] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0933] The largest experiment ever run:

[0934] General procedure for the preparation of compound 2 - ET43365-193.

[0935]

[0936] To a solution of compound 1 (1 g, 5.75 mmol, 1 eq), [4-chloro-3-(trifluoromethyl)phenyl]-boronic acid (1.94 g, 8.63 mmol, 1.5 eq) and Na2CO3 (1.22 g, 11.50 mmol, 2 eq) in a mixture of toluene (30 mL), EtOH (6 mL) and H2O (1.2 mL) was added Pd(dppf)Cl2.CH2Cl2 (469.35 mg, 575.00 μmol, 0.1 eq) under nitrogen and the reaction was stirred at 100 ° C for 2 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was filtered and the filtrate was concentrated to give the crude product. The crude product was diluted with water (60 mL) and extracted with ethyl acetate (3×50 mL). The organic layer was washed with aqueous K2CO3 (2×60 mL) and brine (60 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to provide compound 2 as a yellow solid (0.5 g, yield 28.6%). The crude product was used directly in the next step without further purification.

[0937] 1 H NMR (ET42365-193-P1A, 400 MHz, chloroform-d) δ 7.21-7.25 (m, 1H), 7.28-7.31 (m, 1H), 7.60 (d, J = 8.25 Hz, 1H), 8.08 (dd, J = 8.32, 1.69 Hz, 1H), 8.33 (d, J = 2.25 Hz, 2H)

[0938] General procedure for the preparation of compound 3 - ET42365-207.

[0939]

[0940] To a solution of compound 2 (400 mg, 1.46 mmol, 1 eq) in DMF (4 mL) was added NBS (312.21 mg, 1.75 mmol, 1.2 eq) portionwise at 0°C, and the reaction was stirred at 20°C for 3 hours. LCMS indicated that approximately 30% of the starting material remained and a new peak with the desired product Ms was detected (approximately 35%). The reaction was diluted with water (20 mL) and extracted with ethyl acetate (3×15 mL). The combined organic layers were washed with brine (20 mL), dried over Na 2 SO 4 and concentrated to produce a crude product. The crude product was purified by preparative HPLC (column: Phenomenex Gemini-NX 80*40 mm*3 um; mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B%: 30%-60%, 8 min) to produce compound 3 (120 mg, yield 23.29%) as a yellow solid.

[0941] LCMS(ESI+):RT=0.833min,m / z 352.0,354.0(M+H) + .

[0942] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0943] General procedure for the preparation of A03B01D01 - ET42365-226.

[0944]

[0945] To a solution of compound 3 (120 mg, 340.39 μmol, 1 eq), [2-methyl-5-(trifluoromethyl)-pyrazol-3-yl]boronic acid (85.81 mg, 442.51 μmol, 1.3 eq) and K 3 PO 4 (144.51 mg, 680.79 μmol, 2 eq) in a mixture of H 2 O (1 mL) and THF (5 mL) was added di-tert-butyl(cyclopentyl)phosphane; palladium dichloride; iron (22.18 mg, 34.04 μmol, 0.1 eq) under a nitrogen atmosphere, and the reaction was stirred at 80° C. for 12 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was filtered and the filtrate was concentrated to yield a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (10mMNH4HCO3)-ACN]; B%: 65%-95%, 8min) to give A03B01D01 (26.8mg, yield 18.37%) as a white solid.

[0946] 1H NMR (ET42365-257-P1A, 400 MHz, chloroform-d) δ 4.28 (s, 3H), 5.50 (br s, 1H), 6.81 (s, 1H), 7.36 (d, J = 8.38 Hz, 1H), 7.53 (d, J = 8.38 Hz, 1H), 7.65 (d, J = 8.50 Hz, 1H), 8.14-8.21 (m, 1H), 8.43 (s, 1H)

[0947] LCMS(ESI+):RT=3.535min,m / z 422.1(M+H) + .

[0948] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0949] Synthesis of NUCC-0226574 (ET42365-172-1)

[0950]

[0951] Synthesis scheme

[0952] Scheme 25: Preparation of A01B01C06D01 (NUCC-0226574)

[0953]

[0954] Scheme 26: Preparation of Compound 12A

[0955]

[0956] Chemical synthesis

[0957] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0958] 50_100AB_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.00 min, held at 100% B for 1.00 min, and then 100-50% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0959] The largest experiment ever run:

[0960] General procedure for the preparation of compound 7 - ET43365-132.

[0961]

[0962] To a solution of core A3_1 (600 mg, 1.25 mmol, 1 eq) and pyridine (396.51 mg, 5.01 mmol, 404.61 μL, 4 eq) in DCM (20 mL) was added Tf2O (530.37 mg, 1.88 mmol, 310.16 μL, 1.5 eq) dropwise at 0 ° C. The reaction was stirred at 20 ° C for 12 hours. TLC (petroleum ether / ethyl acetate = 2 / 1) showed that the starting material was consumed and a new peak was generated. The reaction was diluted with water (60 mL) and extracted with DCM (3×30 mL). The organic layer was separated and the combined organic layer was washed with brine (60 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give Compound 7 (600 mg, yield 74.46%) as a white solid.

[0963] 1 H NMR (ET42365-132-P1A, 400 MHz, chloroform-d) δ 1.80 (s, 3H), 3.85 (s, 3H), 6.57 (s, 1H), 7.47 (dd, J = 8.25, 1.75 Hz, 1H), 7.49-7.56 (m, 2H), 7.64 (d, J = 8.25 Hz, 1H), 7.69 (d, J = 1.63 Hz, 1H)

[0964] General procedure for the preparation of compound 12A - ET42365-142.

[0965]

[0966] To a solution of compound 11 (500 mg, 3.66 mmol, 1 eq), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolan (1.02 g, 4.03 mmol, 1.1 eq) and NaOtBu (703.65 mg, 7.32 mmol, 2 eq) in THF (10 mL) was added MeOH (234.60 mg, 7.32 mmol, 296.29 μL, 2 eq), Xantphos (317.74 mg, 549.14 μmol, 0.15 eq) and CuCl (10.87 mg, 109.83 μmol, 2.63 μL, 0.03 eq) under nitrogen. The mixture was stirred at 70° C. for 1 hour. HPLC shows that remaining about 19% of starting material and detects new peak. Reactant is diluted with water (60mL) and extracted with ethyl acetate (3×50mL).By the combined organic layer salt water (60mL) washing, through Na SO Dry and concentrate to produce crude product, it is passed through column chromatography purification on silica gel (eluting with petroleum ether / ethyl acetate=100 / 1 to 20 / 1) to produce compound 12A (270mg, yield 22.3%) as yellow solid.

[0967] 1 H NMR (ET42365-142-P1A, 400 MHz, chloroform-d) δ 1.22 (s, 12H), 6.04 (d, J = 18.39 Hz, 1H), 7.16-7.24 (m, 3H), 7.29-7.36 (m, 2H)

[0968] General procedure for the preparation of compound 12 - ET42365-151.

[0969]

[0970] To a solution of compound 7 (200 mg, 327.42 μmol, 1 eq), compound 12A (216.55 mg, 654.84 μmol, 80% purity, 2 eq) and Na2CO3 (69.41 mg, 654.84 μmol, 2 eq) in a mixture of dioxane (5 mL) and H2O (1 mL) was added Pd(dppf)Cl2.CH2Cl2 (26.74 mg, 32.74 μmol, 0.1 eq) under nitrogen and the reactants were stirred at 85 ° C for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reactants were filtered and the filtrate was concentrated to produce a crude product. The crude product was purified by preparative TLC (petroleum ether / ethyl acetate=2 / 1) to produce compound 12 (35 mg, 14.27% yield) as a yellow solid.

[0971] LCMS(ESI+):RT=0.971min,m / z 599.2(M+H) + .

[0972] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0973] General procedure for the preparation of compound 13 - ET42365-154.

[0974]

[0975] To a solution of compound 12 (35 mg, 46.72 μmol, 80% purity, 1 eq) in MeOH (2 mL) was added K2CO3 (12.91 mg, 93.43 μmol, 2 eq) and the reaction was stirred at 20 ° C for 3 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reactant was diluted with water (10 mL) and extracted with ethyl acetate (3×10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated to produce crude compound 13 (18 mg, 62.22% yield) as a yellow solid. The crude product was used directly in the next step without further purification.

[0976] LCMS(ESI+):RT=0.969min,m / z 557.2(M+H) + .

[0977] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0978] General procedure for the preparation of compound 13 - ET42365-154.

[0979]

[0980] To a solution of compound 13 (30 mg, 53.83 μmol, 1 equivalent) in THF (15 mL) was added Rh / C (30.00 mg) under argon. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred at 20°C under a H2 balloon for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reactants were filtered and the filtrate was concentrated to produce a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 70%-95%, 8 min) to produce A01B01C06D01 (21.7 mg, 69.12% yield) as a white solid.

[0981] 1 H NMR (ET42365-172-P1A, 400MHz, chloroform-d) δ2.65-2.79(m,4H),3.86(s,3H),4.82(s,1H),6.61(s,1H),6.83(d,J=8.38H z,2H),7.03(d,J=7.88Hz,1H),7.17-7.21(m,2H),7.22-7.26(m,2H),7.52(d,J=2.00Hz,1H),7.63(d,J=8.13Hz,1H)

[0982] LCMS(ESI+):RT=3.135min,m / z 559.1(M+H) + .

[0983] 50_100AB_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.00 min, held at 100% B for 1.00 min, and then 100-50% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[0984] Synthesis of NUCC-0226566 (ET42365-147-1)

[0985]

[0986] Synthesis scheme: Preparation of A01B01C08D01 (NUCC-0226566)

[0987]

[0988] Chemical synthesis

[0989] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0990] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[0991] The largest experiment ever run:

[0992] General procedure for the preparation of compound 10 - ET43365-145.

[0993]

[0994] To a solution of compound 7 (200 mg, 327.42 μmol, 1 eq), 2-[(4-chlorophenyl)methyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (124.03 mg, 491.13 μmol, 1.5 eq) and NaCO (69.41 mg, 654.84 μmol, 2 eq) in a mixture of dioxane (6 mL) and H O (1.5 mL) was added Pd(dppf)Cl.CHCl (26.74 mg, 32.74 μmol, 0.1 eq) under nitrogen and the reaction was stirred at 85 ° C for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was filtered and the filtrate was concentrated to produce crude compound 10 (120 mg, 49.92% yield) as a brown solid. The crude product was used in the next step without further purification.

[0995] LCMS(ESI+):RT=0.959min,m / z 587.2(M+H) + .

[0996] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[0997] General procedure for the preparation of A01B01C08D01 - ET42365-147.

[0998]

[0999] To a solution of compound 10 (120 mg, ~163.45 μmol, crude) in MeOH (5 mL) was added K CO (45.18 mg, 326.90 μmol, 2 equivalents) and the reactants were stirred at 20 ° C for 3 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3×30 mL). The organic layer was separated and the combined organic layer was washed with brine (30 mL), dried over Na SO , filtered and concentrated under reduced pressure to produce a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 70%-95%, 8min) to give A01B01C08D01 (62.8mg, yield 70.46%) as an off-white solid.

[1000] 1 H NMR (ET42365-147-P1A, 400MHz, chloroform-d) δ3.73 (d, J = 3.13Hz, 2H), 3.87 (s, 3H), 4.86 (br s,1H),6.61(s,1H),6.80(d,J=8.25Hz,2H),6.99(d,J=7.88Hz,1H),7.19(d,J=8.38Hz,2H),7.25(s,1H),7.30(br d,J=1.50Hz,1H),7.41(d,J=1.50Hz,1H),7.60(d,J=8.25Hz,1H)

[1001] LCMS(ESI+):RT=3.026min,m / z 545.1(M+H) + .

[1002] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1003] Synthesis of NUCC-0226548 (ET42365-124-1)

[1004]

[1005] Synthesis scheme

[1006] Scheme 27: Preparation of A4BC1R1A (NUCC-0226548)

[1007]

[1008] Scheme 28: Preparation of BC1_Pht

[1009]

[1010] Scheme 29: Preparation of Compound 7

[1011]

[1012] Chemical synthesis

[1013] 5-95AB_2min: LC / MS (the column used for chromatography was Kinetex EVO C18 2.1*30mm, 5um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70 -1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1014] NEG50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was negative electrospray ionization. The MS range was 100-1000.

[1015] The largest experiment ever run:

[1016] General procedure for the preparation of compound 2-ET43365-97.

[1017]

[1018] To a solution of compound 1 (5 g, 47.56 mmol, 4.76 mL, 1 equivalent) in toluene (60 mL) was added isobenzofuran-1,3-dione (7.04 g, 47.56 mmol, 1 equivalent). The mixture was stirred at 120 ° C for 12 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reactants were concentrated to produce compound 2 (11 g, 88.49% yield) as a yellow solid. The product was used directly in the next step without further purification.

[1019] 1 H NMR (ET42365-97-P1A, 400 MHz, chloroform-d) δ 2.66 (br s, 1H), 3.59-3.63 (m, 2H), 3.67-3.71 (m, 2H), 3.73-3.78 (m, 2H), 3.89-3.95 (m, 2H), 7.70-7.76 (m, 2H), 7.83-7.89 (m, 2H)

[1020] General procedure for the preparation of compound 3 - ET42365-103.

[1021]

[1022] To a solution of compound 2 (3 g, 12.75 mmol, 1 equivalent) and Et3N (3.23 g, 31.88 mmol, 4.44 mL, 2.5 equivalents) in DCM (60 mL) was added dropwise MsCl (2.19 g, 19.13 mmol, 1.48 mL, 1.5 equivalents) at 0 ° C. The reactants were stirred at 20 ° C for 12 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reaction mixture was diluted with water (100 mL) and extracted with DCM (2 × 100 mL). The organic layer was separated and the combined organic layer was washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to a crude product. The crude product was purified by column chromatography on silica gel (petroleum ether / ethyl acetate = 20 / 1 to 2 / 1) to produce compound 3 (3.7 g, yield 87.97%) as a yellow solid.

[1023] 1H NMR (ET42365-103-P1A, 400 MHz, chloroform-d) δ 3.00 (s, 3H), 3.71-3.82 (m, 4H), 3.88-3.95 (m, 2H), 4.28-4.36 (m, 2H), 7.70-7.78 (m, 2H), 7.82-7.91 (m, 2H)

[1024] General procedure for the preparation of compound 4-ET43587-64.

[1025]

[1026] To a solution of compound 3 (3 g, 9.57 mmol, 8.47 mL, 1 equivalent) in acetone (45 mL) was added NaI (7.18 g, 47.87 mmol, 5 equivalents). The mixture was stirred at 65 ° C for 12 hours. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that the starting material was consumed and new spots were produced. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (3×20 mL). The organic layer was separated and the combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue. The crude product was purified by chromatography on silica gel (eluting with petroleum ether / ethyl acetate = 50 / 1 to 3 / 1) to produce compound 4 (3 g, yield 81.71%) obtained as a red oil.

[1027] LCMS(ESI+):RT=0.734min,m / z 346.0(M+H) + .

[1028] 5-95AB_2min: LC / MS (the column used for chromatography was Kinetex EVO C18 2.1*30mm, 5um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70 -1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1029] General procedure for the preparation of compound BC1_Pht-ET42365-118.

[1030]

[1031] To a solution of compound 5 (431.62 mg, 3.48 mmol, 1.2 eq) and K2CO3 (600.67 mg, 4.35 mmol, 1.5 eq) in CH3CN (15 mL) was added compound 4 (1 g, 2.90 mmol, 1 eq) and the reaction was stirred at 90°C for 12 hours. LCMS indicated that approximately 10% of the starting material remained and a new peak with the desired product Ms was detected. The reaction mixture was diluted with water (60 mL) and extracted with ethyl acetate (3×50 mL). The organic layer was separated and the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to produce BC1-Pht (0.6 g, 36.4% yield) as a colorless oil.

[1032] LCMS(ESI+):RT=0.656min,m / z 324.2(M+H) + .

[1033] 5-95AB_2min: LC / MS (the column used for chromatography was Kinetex EVO C18 2.1*30mm, 5um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70 -1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1034] General procedure for the preparation of A4BC1_2-Pht-ET42365-119.

[1035]

[1036] To a solution of Core A3-1 (400 mg, 835.47 μmol, 1 eq), BC1_Pht (342.23 mg, 1.00 mmol, 1.2 eq) and PPh3 (876.54 mg, 3.34 mmol, 4 eq) in THF (10 mL) was added DIAD (675.76 mg, 3.34 mmol, 649.77 μL, 4 eq) dropwise at 0°C and the reaction was stirred at 25°C for 12 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction mixture was diluted with water (60 mL) and extracted with ethyl acetate (3×50 mL). The organic layer was separated and the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 3 / 1) to give A4BC1_2-Pht (0.6 g, yield 71.63%) as a colorless oil.

[1037] LCMS(ESI+):RT=0.951min,m / z 824.3(M+H) + .

[1038] 5-95AB_2min: LC / MS (the column used for chromatography was Kinetex EVO C18 2.1*30mm, 5um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70 -1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1039] General procedure for the preparation of compound 7 - ET42365-114.

[1040]

[1041] To a solution of compound 6 (1g, 3.62mmol, 1 equivalent) in DMF (15mL) was added K2CO3 (750.52mg, 5.43mmol, 1.5 equivalents) and iodomethane (1.03g, 7.24mmol, 450.76μL, 2 equivalents) at 0°C and the reactants were stirred at 20°C for 12 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reactant was diluted with water (60mL) and stirred for 1 hour. The solid was filtered and dried under high vacuum to obtain compound 7 (0.8g, 76.13% yield) as a pink solid. The product was used directly in the next step without further purification.

[1042] 1 H NMR (ET42365-114-P1A, 400 MHz, chloroform-d) δ 2.05-2.21 (m, 1H), 2.71-2.90 (m, 2H), 2.93-3.08 (m, 1H), 3.22 (s, 3H), 4.99 (br dd, J = 12.67, 5.40 Hz, 1H), 7.43 (t, J = 8.41 Hz, 1H), 7.69-7.74 (m, 1H), 7.74-7.82 (m, 1H)

[1043] General procedure for the preparation of A4BC1_NH2-ET43259-123.

[1044]

[1045] To a solution of A4BC1_2-Pht (600 mg, 598.42 μmol, 80% purity, 1 eq) in EtOH (10 mL) was added NH2NH2.H2O (299.57 mg, 5.98 mmol, 290.84 μL, 10 eq) and the reaction was stirred at 25 °C for 1 hour. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction mixture was diluted with water (60 mL) and extracted with ethyl acetate (3×50 mL). The organic layer was separated and the combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to produce crude A4BC1_NH2 (300 mg, 71.62% yield) as a yellow solid. The crude product was used directly in the next step without further purification.

[1046] LCMS(ESI+):RT=0.736min,m / z 630.3(M+H) + .

[1047] 5-95AB_2min: LC / MS (the column used for chromatography was Kinetex EVO C18 2.1*30mm, 5um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70 -1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1048] General procedure for the preparation of A4BC1R1A - ET42365-124.

[1049]

[1050] To a solution of A4BC1-NH2 (300 mg, 401.78 μmol, 90% purity, 1 eq) in DMSO (6 mL) was added DIEA (155.78 mg, 1.21 mmol, 209.95 μL, 3 eq) and compound 7 (233.23 mg, 803.56 μmol, 2 eq), and the reaction was stirred at 60°C for 12 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 60%-80%, 8 min) and lyophilized to produce A4BC1R1A (108.9 mg, 30.08% yield) as a yellow solid.

[1051] 1H NMR (ET42365-124-P1A, 400 MHz, chloroform-d) δ 2.04-2.13 (m, 1H), 2.68-2.83 (m, 2H), 2.90-3.04 (m, 1H), 3.21 (s, 3H), 3.51 (t, J = 5.38 Hz, 2H), 3.80 (t, J = 5.44 Hz, 2H), 3.83-3.90 (m, 5H), 4.10-4.18 (m, 2H), 4.86-4.95 (m, 1H), 5.02 (s, 2H), 5.11 (s, 1H), 6.5 7(s,1H),6.78(d,J=8.63Hz,1H),6.88(d,J=8.76Hz,2H),6.93(d,J=8.50Hz,1H),7.09(d,J=7.13Hz,1H),7.15(d,J=8.63H z,2H),7.21(d,J=8.51Hz,1H),7.47(dd,J=8.51,7.25Hz,1H),7.51-7.55(m,1H),7.57-7.63(m,1H),7.79(d,J=1.75Hz,1H)

[1052] LCMS(ESI+):RT=2.569min,m / z 898.2(MH) + .

[1053] NEG50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was negative electrospray ionization. The MS range was 100-1000.

[1054] Synthesis of Series A5-3B (NUCC-0226604) and Series A5-3C (NUCC-0226603)

[1055]

[1056] Synthesis scheme

[1057] Scheme 30: Preparation of C-4A

[1058]

[1059] Scheme 31: Preparation of Series A5-3B (NUCC-0226604)

[1060]

[1061] Scheme 32: Preparation of series (NUCC-0226603)

[1062]

[1063] Chemical synthesis

[1064] LCMS method:

[1065] Method 1: 5_95AB_6min-220-254-ELSD

[1066] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C1850*2.1 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1067] Method 2:5_95CD_6min-220-254-ELSD

[1068] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1069] The largest experiment ever run:

[1070] General procedure for the preparation of C-4A - ET43318-125.

[1071]

[1072] To a mixture of C-4B (2 g, 11.29 mmol, 1.82 mL) and HCl (12 M, 940.89 μL, 1 equivalent) in EtOH (30 mL) was added PtO 2 (256.38 mg, 1.13 mmol) at 20° C. The mixture was stirred under 15 psi of hydrogen for 12 hours. TLC (ethyl acetate / MeOH=5 / 1) indicated that the starting material was consumed and a new spot was generated. The product with the desired Ms was detected by LCMS. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to produce C-4A (2.2 g, 71.63% yield) as a yellow solid, which was used directly in the next step without further purification.

[1073] 1 H NMR (400 MHz, chloroform-d) δ 1.34 (t, J = 7.07 Hz, 6H), 2.41 (dt, J = 18.51, 7.75 Hz, 2H), 3.15-3.38 (m, 2H), 4.03-4.26 (m, 4H), 7.70-8.72 (m, 2H)

[1074] General procedure for the preparation of compound 3-1 - ET43318-157.

[1075]

[1076] To a solution of C (161.45 mg, 1.04 mmol) and Core A6 (500 mg, 1.04 mmol) in DMF (5 mL) was added Cs2CO3 (340.27 mg, 1.04 mmol) at 20° C. The mixture was stirred for 12 hours.

[1077] The product of 3-1 (250mg, 24.06% yield) was obtained by column chromatography on silica gel (eluting with petroleum ether / ethyl acetate=20 / 1 to 3 / 1).

[1078] 1H NMR (400MHz, chloroform-d) δ1.79(s,3H),3.82(s,3H),5.22(s,2H),6.51(s,1H),6.79(d,J=8.00Hz,1H),7. 06(d,J=8.63Hz,1H),7.32-7.36(m,1H),7.38-7.43(m,3H),7.49(dd,J=8.25,1.75Hz,1H),7.57(br d,J=8.50Hz,1H),7.68(d,J=8.13Hz,1H),7.76(d,J=1.75Hz,1H),10.02(s,1H)

[1079] General procedure for the preparation of compound 3-2A - ET43318-165.

[1080]

[1081] To a solution of C-4A (47.07 mg, 216.27 μmol) in THF (2 mL) was added TEA (54.71 mg, 540.67 μmol) at 20 ° C. The mixture was stirred at 20 ° C for 0.5 hours, and then 3-1 (100 mg, 180.22 μmol) was added. After stirring for 2 hours, AcOH (32.47 mg, 540.67 μmol) and NaBH (OAc) (76.39 mg, 360.45 μmol) were added. The mixture was stirred for another 2.5 hours. LCMS showed that the starting material was exhausted and 52.8% of the peak with the desired Ms was detected (Rt = 0.750 min). TLC (petroleum ether / ethyl acetate = 2: 1) showed that a new spot was produced. The reaction mixture was poured into saturated NaHCO (60 mL) and extracted with ethyl acetate (60 mL × 3). The organic layers were combined, washed with brine (2×10 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (ethyl acetate / MeOH=5 / 1) to give 3-2A (80 mg, 42.52% yield) as a colorless oil.

[1082] 1H NMR (400MHz, chloroform-d) δ1.31 (t, J = 7.07Hz, 6H), 1.61 (br s,4H),1.78(s,3H),2.00(dt,J=18.17,7.18Hz,2H),2.87-2.99(m,2H),3.75-3.84(m,5H),4.05-4.16(m,4H),5.12(s,2H),6.50(s, 1H),7.07(d,J=8.63Hz,1H),7.20(d,J=8.00Hz,2H),7.28-7.35(m,3H),7.44-7.50(m,1H),7.51-7.57(m,1H),7.73(d,J=1.75Hz,1H)

[1083] General procedure for the preparation of compound series A5-3B - ET43318-168.

[1084]

[1085] To a solution of 3-2A (80 mg, 104.98 μmol) in MeOH (1 mL) was added KCO (43.53 mg, 314.93 μmol) at 20 ° C. The mixture was stirred for 12 hours. LCMS showed that the starting material was exhausted and 86% of the peak with the desired Ms was detected (Rt = 0.737 min). The mixture was filtered to produce a filtrate. The filtrate was purified by preparative HPLC to produce series A5-3B (30.6 mg, 40.48% yield) as an off-white solid.

[1086] Preparative HPLC method:

[1087] Instrument: Gilson 281 semi-preparative HPLC system

[1088] Mobile phase: A: 10 mM NH4HCO3 solution in H2O; B: acetonitrile

[1089] Column: Waters Xbridge BEH C18 100*30mm*10um

[1090] Flow rate: 25mL / min

[1091] Monitoring wavelength: 220 and 254nm

[1092]

[1093] 1H NMR (400 MHz, chloroform-d) δ 1.30 (t, J = 7.03 Hz, 6H), 1.95 (t, J = 7.15 Hz, 1H), 1.99 (t, J = 7.15 Hz, 1H), 2.89 (dt, J = 15.83, 7.18 Hz, 2H), 3.76 (s, 2H), 3.84 (s, 3H), 4.01-4.13 (m,4H),5.08(s,2H),6.56(s,1H),6.75(d,J=8.68Hz,1H),7.19(t,J=7.83Hz,3H) ,7.27-7.30(m,2H),7.51-7.56(m,1H),7.57-7.63(m,1H),7.80(d,J=1.83Hz,1H)

[1094] LCMS(ESI+):RT=2.476min,m / z 720.1(M+H) + .

[1095] 5_95AB_6min-220-254-ELSD

[1096] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1097] General procedure for the preparation of series A5-3C (NUCC-0226603) - ET43318-197.

[1098]

[1099] To a solution of series A5-3B (25 mg, 34.72 μmol) and 2,6-lutidine (148.82 mg, 1.39 mmol) in DCM (1 mL) was added TMSBr (106.31 mg, 694.41 μmol) dropwise at 0 ° C. The mixture was warmed to 20 ° C and stirred for 12 hours. LCMS showed that the starting material was exhausted and 60.3% of the product with the desired Ms was detected (Rt = 0.788 min). The mixture was filtered to produce a filtrate. The filtrate was purified by preparative HPLC to produce series A5-3C (7.5 mg, 32.34% yield) as a white solid.

[1100] Preparative HPLC method:

[1101] Instrument: Gilson 281 semi-preparative HPLC system

[1102] Mobile phase: A: 10 mM NH4HCO3 solution in H2O; B: acetonitrile

[1103] Column: Waters Xbridge BEH C18 100*30mm*10um

[1104] Flow rate: 25mL / min

[1105] Monitoring wavelength: 220 and 254nm

[1106]

[1107] 1 H NMR (400MHz, methanol-d4) δ1.79-2.02(m,2H),3.26(br dd,J=14.57,7.57Hz,2H),3.79(s,3H),4.18(s,2H),5.14(s,2H),6.58(s,1H), 6.86(d,J=8.75Hz,1H),7.25(d,J=8.63Hz,1H),7.34(d,J=8.13Hz,2H),7.44(br d,J=7.88Hz,2H),7.58-7.67(m,2H),7.78(d,J=1.50Hz,1H)

[1108] LCMS(ESI+):RT=2.741min,m / z 664.2(M+H) + .

[1109] 5_95CD_6min-220-254-ELSD

[1110] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1111] Synthesis of A4-3 (NUCC-0226596)

[1112]

[1113] Synthesis scheme

[1114] Scheme 33: Preparation of A4-3 (NUCC-0226596)

[1115]

[1116] Chemical synthesis

[1117] LCMS method:

[1118] Method 1:5_95CD_6min-220-254-ELSD

[1119] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1120] The largest experiment ever run:

[1121] General procedure for the preparation of series A4-3 (NUCC-0226596) - ET43318-189.

[1122]

[1123] To a solution of D-7 (25 mg, 28.70 μmol) and 2,6-lutidine (61.50 mg, 0.574 mmol) in DCM (0.5 mL) was added TMSBr (43.93 mg, 0.287 mmol) dropwise at 0 ° C. The mixture was warmed to 20 ° C and stirred for 12 hours. LCMS showed that the starting material was exhausted and 18.3% of the product with the desired Ms was detected. The mixture was filtered to produce a filtrate. The filtrate was purified by preparative HPLC to produce series A4-3 (3.4 mg, 15.36% yield) as a white solid.

[1124] Preparative HPLC method:

[1125] Instrument: Gilson 281 semi-preparative HPLC system

[1126] Mobile phase: A: 10 mM NH4HCO3 solution in H2O; B: acetonitrile

[1127] Column: Waters Xbridge BEH C18 100*30mm*10um

[1128] Flow rate: 25mL / min

[1129] Monitoring wavelength: 220 and 254nm

[1130]

[1131] 1 H NMR (400MHz, methanol-d4) δ2.10(br s,5H),3.79(s,3H),3.99(br t,J=5.13Hz,2H),5.02(s,2H),6.59(s,1H),6.86(dd,J=8.57,5.94Hz,3H),7.15(d,J=8.50H z,2H),7.23(d,J=8.50Hz,1H),7.54-7.59(m,1H),7.60-7.66(m,1H),7.74(d,J=1.38Hz,1H)

[1132] LCMS(ESI+):RT=2.575min,m / z 759.1(M+H) + .

[1133] 5_95CD_6min-220-254-ELSD

[1134] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1135] Synthesis of A4-3 (NUCC-0226596)

[1136]

[1137] Synthesis scheme

[1138] Scheme 34: Preparation of L1-M1 (NUCC-022706)

[1139]

[1140]

[1141] Chemical synthesis

[1142] LCMS method:

[1143] Method 1:5_95CD_6min-220-254-ELSD

[1144] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1145] The largest experiment ever run:

[1146] General procedure for the preparation of series A4-3 - ET43318-196.

[1147]

[1148]

[1149] To a solution of D-7 (60 mg, 68.88 μmol) and 2,6-lutidine (295.21 mg, 2.76 mmol) in DCM (2 mL) was added TMSBr (210.89 mg, 1.38 mmol) dropwise at 0 ° C. The mixture was warmed to 20 ° C and stirred for 2 hours. LCMS showed that the starting material was exhausted and 18.6% of the product with the desired quality was detected. The mixture was filtered to produce a filtrate. The filtrate was purified by preparative HPLC to produce series A4-3 (11.2 mg, 21.43% yield) as a white solid.

[1150] Preparative HPLC method:

[1151] Instrument: Gilson 281 semi-preparative HPLC system

[1152] Mobile phase: A: 10 mM NH4HCO3 solution in H2O; B: acetonitrile

[1153] Column: Waters Xbridge BEH C18 100*30mm*10um

[1154] Flow rate: 25mL / min

[1155] Monitoring wavelength: 220 and 254nm

[1156]

[1157] 1 H NMR(400MHz, methanol-d4)δ2.11(br s,5H),3.79(s,3H),3.93-4.03(m,2H),5.02(s,2H),6.58(s,1H),6.85(dd,J=8.57,6.44Hz,3H),7.14( d,J=8.63Hz,2H),7.23(d,J=8.63Hz,1H),7.52-7.59(m,1H),7.60-7.65(m,1H),7.74(d,J=1.75Hz,1H)

[1158] LCMS(ESI+):RT=2.571min,m / z 759.1(M+H) + .

[1159] 5_95CD_6min-220-254-ELSD

[1160] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1161] Synthesis of D-7 (NUCC-0226597)

[1162]

[1163] Synthesis scheme

[1164] Preparation of D-7 (NUCC-0226597):

[1165]

[1166]

[1167] Chemical synthesis

[1168] LCMS method:

[1169] Method 1: 5_95AB_6min-220-254-ELSD

[1170] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C1850*2.1 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1171] The largest experiment ever run:

[1172] General procedure for the preparation of compound D-3 - ET43318-118.

[1173]

[1174] To a solution of 1-[diethoxyphosphorylmethyl(ethoxy)phosphoryl]oxyethane (10 g, 34.70 mmol) in THF (100 mL) was added portionwise NaH (1.53 g, 38.17 mmol) at 0°C under nitrogen. After stirring for 30 min, D-2 (9.29 g, 41.64 mmol) was added. The mixture was heated to 70°C and stirred for 12 hours. TLC (ethyl acetate / MeOH=5 / 1, Rf=0.55) showed that the starting material was consumed and new spots were generated. The reaction mixture was poured into NH4Cl (300 mL) and extracted with ethyl acetate (3×100 mL). The organic layers were combined, washed with brine (2×50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce D-3 (12 g, crude) as a yellow oil, which was used directly in the next step without further purification.

[1175] 1 H NMR (400 MHz, chloroform-d) δ 1.32-1.37 (m, 12H), 1.55-1.61 (m, 4H), 1.73 (br dd, J = 6.50, 3.00 Hz, 1H), 1.81-1.92 (m, 3H), 1.96-2.09 (m, 2H), 2.26-2.48 (m, 1H), 3.52-3.55 (m, 2H), 3.85-3.90 (m, 2H), 4.15-4.23 (m, 8H), 4.58-4.61 (m, 1H)

[1176] General procedure for the preparation of compound D-4-ET43318-137.

[1177]

[1178] To a solution of D-3 (5 g, 11.62 mmol) in MeOH (50 mL) was added 4-methylbenzenesulfonic acid (100.02 mg, 580.84 μmol) at 20 ° C. The mixture was stirred for 12 hours. TLC (ethyl acetate / MeOH = 5:1, Rf = 0.4) showed that the starting material was consumed and a new spot was generated. The mixture was concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluted with ethyl acetate / methanol = 20 / 1 to 5 / 1) to produce D-4 (1.2 g, 26.85% yield) as a colorless oil.

[1179] 1H NMR (400 MHz, chloroform-d) δ 1.35 (t, J = 7.00 Hz, 12H), 1.75-1.89 (m, 2H), 2.00-2.13 (m, 2H), 2.38-2.47 (m, 1H), 3.67 (t, J = 5.94 Hz, 2H), 4.19 (dq, J = 13.80, 7.03 Hz, 8H)

[1180] General procedure for the preparation of compound D-5 - ET43318-152.

[1181]

[1182] At 0 ℃ under nitrogen to PPh3 (908.91mg, 3.47mmol) in THF (10mL) solution was dropwise added DIAD (700.71mg, 3.47mmol). After stirring for 30min, D-4 (800mg, 2.31mmol) and methyl 4-hydroxybenzoate (527.23mg, 3.47mmol) were added. The mixture was warmed to 20 ℃ and stirred for 12 hours. LCMS showed that the starting material was exhausted and 31.9% of the product with the desired Ms was detected. TLC (ethyl acetate / MeOH=5 / 1, Rf=0.65) showed that a new spot was produced. The reaction mixture was poured into water (100mL) and extracted with ethyl acetate (3×30mL). The organic layers were combined, washed with brine (2×10mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluted with ethyl acetate / methanol = 1 / 0 to 10 / 1) to give D-5 (450 mg, 40.55% yield) as a colorless oil.

[1183] 1 H NMR (400 MHz, chloroform-d) δ 1.34 (td, J = 7.04, 1.44 Hz, 12H), 2.07-2.20 (m, 4H), 2.29-2.45 (m, 1H), 3.88 (s, 3H), 4.03 (t, J = 5.50 Hz, 2H), 4.15-4.23 (m, 8H), 6.84-6.94 (m, 2H), 7.93-8.03 (m, 2H)

[1184] General procedure for the preparation of compound linker D3 - ET43318-158.

[1185]

[1186] To a solution of D-5 (600 mg, 1.25 mmol) in THF (20 mL) was added LiAlH4 (142.20 mg, 3.75 mmol) at 0°C. The mixture was warmed to 20°C and stirred for 2 hours. LCMS indicated that the starting material was consumed and 31.2% of the product with the desired Ms was detected. The mixture was cooled to 0°C, H2O (0.142 mL) was slowly added dropwise, followed by a NaOH solution (0.142 mL, 15%) and stirred for 30 min, followed by H2O (0.426 mL). The mixture was warmed to 25°C and stirred for 1 hour. The mixture was filtered and the filter cake was washed with THF (2×25 mL). The filtrate was concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluted with ethyl acetate / methanol = 1 / 0 to 10 / 1) to produce connector D3 (200 mg, 28.32% yield) as a colorless oil.

[1187] 1 H NMR (400 MHz, chloroform-d) δ 1.34 (td, J = 7.07, 1.50 Hz, 12H), 2.06-2.21 (m, 4H), 2.30-2.46 (m, 1H), 3.98 (t, J = 5.69 Hz, 2H), 4.14-4.24 (m, 8H), 4.62 (s, 2H), 6.84-6.90 (m, 2H), 7.27-7.31 (m, 2H)

[1188] General procedure for the preparation of compound D-6 - ET43318-163.

[1189]

[1190] To a solution of connector D3 (180 mg, 397.87 μmol) and core A6 (209.54 mg, 437.65 μmol) in toluene (3 mL) was added CMBP (144.04 mg, 596.80 μmol) dropwise at 20°C under nitrogen. The mixture was heated to 80°C and stirred for 12 hours. LCMS indicated that the starting material remained and 41.9% of the product with the desired Ms was detected. The reaction mixture was poured into water (60 mL) and extracted with ethyl acetate (2×20 mL). The organic layers were combined, washed with brine (2×10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluting with ethyl acetate / methanol = 1 / 0 to 10 / 1) to produce D-6 (300 mg, 33.03% yield) as a yellow oil.

[1191] 1H NMR (400 MHz, chloroform-d) δ 0.94 (s, 12H), 1.77 (s, 3H), 2.06-2.18 (m, 4H), 2.29-2.46 (m, 1H), 3.79-3.86 (m, 3H), 3.97 (br t,J=5.44Hz,2H),4.14-4.24(m,8H),4.99-5.08(m,2H),6.45-6.57(m,1H),6.80-6.88(m,2H),7.08(d,J=8.63H z,1H),7.12-7.19(m,2H),7.32(d,J=8.63Hz,1H),7.42-7.48(m,1H),7.49-7.56(m,1H),7.71(d,J=1.38Hz,1H)

[1192] General procedure for the preparation of compound D-7 (NUCC-0226597) - ET43318-169.

[1193]

[1194] To a solution of D-6 (200 mg, 219.02 μmol) in MeOH (0.5 mL) and H₂O (0.5 mL) was added K₂CO₃ (90.81 mg, 657.05 μmol) at 20°C. The mixture was stirred for 12 hours. LCMS indicated that the starting material was consumed and 93.3% of the product with the desired Ms was detected. The mixture was filtered to produce a filtrate. The filtrate was purified by preparative HPLC to produce D-7 (87.4 mg, 45.21% yield) as an off-white solid.

[1195] Preparative HPLC method:

[1196] Instrument: Gilson 281 semi-preparative HPLC system

[1197] Mobile phase: A: 10 mM NH4HCO3 solution in H2O; B: acetonitrile

[1198] Column: Waters Xbridge BEH C18 100*30mm*10um

[1199] Flow rate: 25mL / min

[1200] Monitoring wavelength: 220 and 254nm

[1201]

[1202] 1H NMR (400 MHz, chloroform-d) δ 1.33 (td, J = 7.06, 1.41 Hz, 12H), 2.02-2.19 (m, 4H), 2.26-2.44 (m, 1H), 3.84 (s, 3H), 3.96 (t, J = 5.62 Hz, 2H), 4.11-4.22 (m, 8H), 5.02 (s, 2H), 5.40 (s, 1H ),6.56(s,1H),6.76(d,J=8.56Hz,1H),6.83(d,J=8.68Hz,2H),7.14(d,J=8.68Hz,2H) ,7.20(d,J=8.56Hz,1H),7.49-7.55(m,1H),7.56-7.61(m,1H),7.78(d,J=1.83Hz,1H)

[1203] LCMS(ESI+):RT=3.072min,m / z 871.1(M+H) + .

[1204] 5_95AB_6min-220-254-ELSD

[1205] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1206] Synthesis of NUCC-0226914 (ET43587-416-1)

[1207]

[1208] Scheme 35: Preparation of Series 11-3-12 (NUCC-0226914)

[1209]

[1210] Chemical synthesis

[1211] LCMS method:

[1212] Method 1: 5_95AB_2min

[1213] LC / MS (column used for chromatography was Kinetex EVO C18 2.1*30 mm, 5 um. Detection method was diode array (DAD). MS mode was positive electrospray ionization. MS range was 100-1000. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1214] Method 2: 5_95AB_6min-220-254-ELSD

[1215] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 2.60 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was Kinetex C18 2.1*50 mm, 5 um. The detection method was diode array (DAD) and evaporative light scattering detection (ELSD). The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1216] The largest experiment ever run:

[1217] General procedure for the preparation of compound 15 - ET43587-401.

[1218]

[1219] To a solution of compound 14 (200 mg, 299.59 μmol, 1 equivalent), NH4Cl (80.13 mg, 1.50 mmol, 5 equivalents) and DIEA (116.16 mg, 898.78 μmol, 156.55 μL, 3 equivalents) in DMF (4 mL) was added HOBt (60.72 mg, 449.39 μmol, 1.5 equivalents) and the mixture was stirred at 25 ° C for 30 minutes. EDCI (86.15 mg, 449.39 μmol, 1.5 equivalents) was then added at 0 ° C. The mixture was stirred at 25 ° C for 12 hours. LCMS showed that all starting materials were consumed and a new peak with the desired product MS was detected. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3 × 10 mL). The organic layer was separated and the combined organic layers were washed with brine (2×10 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue, which was purified by chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 20 / 1 to 1 / 3) to give compound 15 (170 mg, 76.61% yield) as a white solid.

[1220] 1 H NMR (ET43587-401-P1A, 400MHz, chloroform-d) δ0.10(,9H),0.51-0.59(m,2H),2.85-2.96(m,2H),3.85(s,3H),4.41(s,2H),5.06(s,2H),5.34(br s,1H),6.75(br s,1H),6.85(s,1H),6.91(d,J=8.63Hz,1H),7.17(d,J=8.50Hz,2H),7.28-7.35(m,3H),7.53-7.61(m,2H),7.84(d,J=1.63Hz,1H)

[1221] General procedure for the preparation of compound 16 - ET43587-406.

[1222]

[1223] A mixture of compound 15 (100 mg, 150.02 μmol, 1 equivalent) in TBAF (1 M, 3.00 mL, 20 equivalents) was stirred at 25 ° C for 20 hours. LCMS showed that all starting materials were exhausted and a new peak with the desired product MS was detected. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (3 × 10 mL). The organic layer was separated and the combined organic layer was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue, which was purified by chromatography on silica gel (eluted with petroleum ether / ethyl acetate=20 / 1 to 1 / 2) to produce compound 16 (50 mg, yield 55.93%) as a yellow solid.

[1224] LCMS(ESI+):RT=0.846min,m / z 536.1(M+H) + .

[1225] 5_95AB_2min: LC / MS (the column used for chromatography was Kinetex EVO C18 2.1*30mm, 5um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70 -1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1226] General procedure for the preparation of series 11-3-12 - ET43587-416.

[1227]

[1228] To a solution of compound 16 (50 mg, 93.23 μmol, 1 eq) in DCM (1 mL) was added burgess reagent (33.32 mg, 139.84 μmol, 1.5 eq) at 0°C. The mixture was stirred at 25°C for 3 hours. LCMS indicated that all starting material was consumed and a new peak with the desired product MS was detected. The mixture was purified by preparative HPLC (column: Waters XbridgePrep OBD C18 150*40mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 55%-85%, 8 min) and lyophilized to produce series 11-3-12 (11.2 mg, 23.18% yield) as a white solid.

[1229] 1 H NMR (ET43587-416-P1A, 400MHz, chloroform-d) δ3.85 (s, 3H), 5.06 (s, 2H), 6.70 (s, 1H), 6.73 (d, J = 8.63Hz, 1H), 7.16 (d, J = 8.38Hz, 2 H),7.21(d,J=8.63Hz,1H),7.29-7.34(m,2H),7.53(dd,J=8.13,2.00Hz,1H),7.65(d,J=8.25Hz,1H),7.79(d,J=1.88Hz,1H)

[1230] LCMS(ESI+):RT=3.252min,m / z 518.0(M+H) + .

[1231] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 2.60 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was Kinetex C18 2.1*50 mm, 5 um. The detection method was diode array (DAD) and evaporative light scattering detection (ELSD). The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1232] Synthesis of NUCC-0226659 (ET43587-299-1) and NUCC-0226660 (ET43587-309-1)

[1233]

[1234] Synthesis scheme

[1235] Scheme 36: Preparation of Series 11-3-9 (NUCC-0226659)

[1236]

[1237] Scheme 37: Preparation of Series 11-3-15 (NUCC-0226660)

[1238]

[1239] Chemical synthesis

[1240] LCMS method:

[1241] Method 1:5_95CD_6min-220-254-ELSD

[1242] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O + 10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1243] We use series 11-3-9 (NUCC-0226659) as an example.

[1244] The largest experiment ever run:

[1245] General procedure for the preparation of compound 6 - ET43587-275.

[1246] Reactions were performed in parallel but pooled for purification.

[1247]

[1248] To a solution of series 11-3-core 1 (100 mg, 203.21 μmol, 1 eq) and DIEA (78.79 mg, 609.62 μmol, 106.18 μL, 3 eq) in DCM (1 mL) was added SEMCl (67.76 mg, 406.41 μmol, 71.93 μL, 2 eq) at 0°C. The reaction was stirred at 25°C for 12 hours. TLC (petroleum ether / ethyl acetate = 10 / 1) indicated that the starting material (R f = 0.45) are exhausted and new spots are generated (R f =0.5). Three additional reactions were set up as detailed above, and all four reaction mixtures were combined. The combined reaction mixtures were concentrated under reduced pressure to give a residue, which was purified by preparative TLC (petroleum ether / ethyl acetate=10 / 1) to give compound 6 (340 mg, 60.49% yield) as a white solid.

[1249] 1H NMR (ET43587-275-P2A, 400 MHz, chloroform-d) δ -0.045 (s, 9H), 0.62-0.71 (m, 2H), 3.20-3.28 (m, 2H), 4.83 (s, 2H), 4.98 (s, 2H), 6.73 (d, J = 8.88 Hz, 1H), 7.12 (d, J = 8.50 Hz, 2H), 7.28-7.31 (m, 2H), 7.51-7.56 (m, 3H), 7.81 (s, 1H)

[1250] General procedure for the preparation of compound 7-ET43587-288.

[1251]

[1252] To a solution of compound 6 (70 mg, 112.47 μmol, 1 eq) and compound 11-3-9A (167.42 mg, 449.89 μmol, 4 eq) in dioxane (4 mL) was added Pd(dppf)Cl2 (16.46 mg, 22.49 μmol, 0.2 eq) under nitrogen, and the mixture was stirred at 100° C. for 12 hours. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that the starting material (R f = 0.9) are exhausted and new spots are generated (R f =0.3). The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by preparative TLC (petroleum ether / ethyl acetate=1 / 1) to give Compound 7 (120 mg, yield 76.87%) as a white solid.

[1253] 1 H NMR (ET43587-288-P1A, 400MHz, methanol-d4) δ-0.1(s,9H),0.49-0.57(m,2H),2.87-2.96(m,2H),4.04(s,3H),4.38(s,2H),5.14( s,2H),7.16(d,J=8.63Hz,1H),7.23-7.34(m,4H),7.42(d,J=8.63Hz,1H),7.64-7.72(m,2H),7.76-7.81(m,1H),7.86(s,1H)

[1254] General procedure for the preparation of series 11-3-9 - ET43587-299.

[1255]

[1256] To a solution of compound 7 (120 mg, 192.14 μmol, 1 eq) in THF (1 mL) was added TBAF (1 M, 1.44 mL, 7.5 eq) at 0°C, and the mixture was stirred at 65°C for 12 hours. LCMS indicated that the starting material was consumed and 80% of the product with the desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to produce a residue, which was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 50%-85%, 8 min) to produce series 11-3-9 (34.1 mg, 35.37% yield) as a white solid.

[1257] 1 H NMR (ET43587-299-P1A, 400 MHz, methanol-d4) δ 3.97 (s, 3H), 5.10 (s, 2H), 6.87 (d, J = 8.63 Hz, 1H), 7.19-7.32 (m, 5H), 7.58-7.80 (m, 4H).

[1258] LCMS(ESI+):RT=3.516min,m / z 494.1(M+H) + .

[1259] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1260] Synthesis of NUCC-0226656 (ET43587-277-1) and NUCC-0226657 (ET43587-268-1)

[1261]

[1262] Scheme 38: Preparation of Series 11-3-13 (NUCC-0226656)

[1263]

[1264] Scheme 39: Preparation of Series 11-3-16 (NUCC-0226657)

[1265]

[1266] Chemical synthesis

[1267] LCMS method:

[1268] Method 1: NEG5-95CD_2min

[1269] LC / MS (the column used for chromatography was an Xbridge C18 2.1*50 mm, 5 um. The detection method was diode array (DAD). The MS mode was negative electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 10 mm ammonium bicarbonate in water, and mobile phase B was HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min), with a hold of 0.34 min at 5% B. The flow rate was 1.5 mL / min.

[1270] Method 2:5_95CD_6min-220-254-ELSD

[1271] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O + 10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1272] Method 3: 5_95AB_6min-220-254-ELSD

[1273] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 2.60 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was Kinetex C18 2.1*50 mm, 5 um. The detection method was diode array (DAD) and evaporative light scattering detection (ELSD). The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1274] The largest experiment ever run:

[1275] General procedure for the preparation of Series 11-3-Core 1-ET42365-239.

[1276]

[1277] To a solution of compound 5 (4 g, 9.68 mmol, 1.25 eq) and DIPA (1.57 g, 15.49 mmol, 2.19 mL, 2 eq) in CHCl₃ (80 mL) was added NBS (1.38 g, 7.74 mmol, 1 eq) at -40°C, and the reaction was stirred at -40°C for 4 hours. LCMS indicated that the starting material was consumed and a major new peak was detected. The reaction was diluted with water (100 mL) and extracted with DCM (3×50 mL). The combined organic layers were washed with brine (100 mL), dried over Na₂SO₄, and concentrated under reduced pressure to yield a crude product, which was purified by column chromatography on silica gel (eluting with petroleum ether / ethyl acetate = 30 / 1 to 5 / 1) to yield series 11-3-core 1 (2.5 g, 55.76% yield) as a yellow solid.

[1278] LCMS(ESI+):RT=1.042min,m / z 490.9(MH) - .

[1279] NEG5-95CD_2min: LC / MS (the column used for chromatography was Xbridge C18 2.1*50mm, 5um. The detection method was diode array (DAD). The MS mode was negative electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 10 mm ammonium bicarbonate in water, and mobile phase B was HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min), with a hold of 0.34 min at 5% B. The flow rate was 1.5 mL / min.

[1280] General procedure for the preparation of series 11-3-13 - ET43587-277.

[1281]

[1282] To a solution of series 11-3-core 1 (150 mg, 304.81 μmol, 1 eq), 11-3-13A (164.89 mg, 792.50 μmol, 2.6 eq) and K 3 PO 4 (194.10 mg, 914.42 μmol, 3 eq) in a mixture of THF (5 mL) and H 2 O (1 mL) was added di-tert-butyl(cyclopentyl)phosphane; palladium dichloride; iron (39.73 mg, 60.96 μmol, 0.2 eq) under nitrogen, and the reaction was stirred at 80° C. for 12 hours. LCMS indicated that the starting material was consumed and 67.7% of the product with the desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water(NH4HCO3)-ACN]; B%: 70%-95%, 8 min) to give series 11-3-13 (11.5 mg, yield 7.28%) as a white solid.

[1283] 1 H NMR (ET43587-277-P1A, 400 MHz, methanol-d4) 3.73 (s, 3H), 5.08 (s, 2H), 6.29 (d, J = 1.88 Hz, 1H), 6.83 (d, J = 8.63 Hz, 1H), 7.19-7.23 (m, 3H), 7.27-7.31 (m, 2H), 7.49 (d, J = 2.00 Hz, 1H), 7.58-7.65 (m, 2H), 7.77 (s, 1H)

[1284] LCMS(ESI+):RT=3.650min,m / z 493.1(M+H) + .

[1285] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1286] General procedure for the preparation of series 11-3-16 - ET43587-268.

[1287]

[1288] To a solution of series 11-3-core 1 (200 mg, 406.41 μmol, 1 eq), 11-3-16A (117.28 mg, 528.33 μmol, 1.3 eq) and K 3 PO 4 (172.54 mg, 812.82 μmol, 2 eq) in a mixture of THF (6 mL) and H 2 O (1 mL) was added di-tert-butyl(cyclopentyl)phosphane; palladium dichloride; iron (26.49 mg, 40.64 μmol, 0.1 eq) under nitrogen, and the reaction was stirred at 80° C. for 12 hours. LCMS indicated that the starting material was consumed and 41.9% of the product with the desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water(NH4HCO3)-ACN]; B%: 70%-95%, 8 min) to give series 11-3-16 (17.9 mg, yield 7.21%) as a brown solid.

[1289] 1H NMR (ET43587-268-P1, 400 MHz, methanol-d4) δ 1.42 (d, J = 6.63 Hz, 6H), 4.44 (quin, J = 6.63 Hz, 1H), 5.09 (s, 2H), 6.51 (s, 1H), 6.84 (d, J = 8.50 Hz, 1H), 7.19-7.24 (m, 3H), 7.27-7.32 (m, 2H), 7.57-7.66 (m, 2H), 7.77 (d, J = 1.75 Hz, 1H)

[1290] LCMS(ESI+):RT=3.475min,m / z 589.0(M+H) + .

[1291] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 2.60 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was Kinetex C18 2.1*50 mm, 5 um. The detection method was diode array (DAD) and evaporative light scattering detection (ELSD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Synthesis of NUCC-0226652 (ET42365-279-1)

[1292]

[1293] Synthesis scheme: Preparation of series 11-4-2 (NUCC-0226652)

[1294]

[1295]

[1296] Chemical synthesis

[1297] NEG5-95CD_2min: LC / MS (the column used for chromatography was Xbridge C18 2.1*50mm, 5um. The detection method was diode array (DAD). The MS mode was negative electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 10 mM ammonium bicarbonate in water, and mobile phase B was HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min), with a hold of 0.34 min at 5% B. The flow rate was 1.5 mL / min.

[1298] 50_100AB_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.00 min, held at 100% B for 1.00 min, and then 100-50% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1299] The largest experiment ever run:

[1300] General procedure for the preparation of compound 2 - ET43365-250.

[1301]

[1302] To a solution of compound 1 (0.5 g, 2.89 mmol, 335.57 μL, 1 eq), compound 2A (587.51 mg, 3.76 mmol, 1.3 eq) and K 3 PO 4 (1.53 g, 7.23 mmol, 2.5 eq) in a mixture of THF (10 mL) and H 2 O (2 mL) was added Pd (dppf) Cl 2 .CH 2 Cl 2 (177.01 mg, 216.75 μmol, 0.075 eq) under nitrogen, and the reaction was stirred at 80 ° C for 2 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The crude product was diluted with water (50 mL) and extracted with ethyl acetate (3×50 mL). The organic layer was washed with brine (50 mL), dried over Na 2 SO 4, filtered and concentrated under reduced pressure to provide the crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give Compound 2 (500 mg, yield 80.31%) as a yellow solid.

[1303] 1 H NMR (ET42365-250-P1A, 400 MHz, chloroform-d) δ 4.96 (s, 1H), 6.85-6.96 (m, 1H), 7.12-7.22 (m, 1H), 7.31-7.42 (m, 2H)

[1304] General procedure for the preparation of compound 3 - ET42365-256.

[1305]

[1306] To a solution of compound 2 (500 mg, 2.44 mmol, 1 eq) and DIPA (494.45 mg, 4.89 mmol, 690.57 μL, 2 eq) in CHCl₃ (12.5 mL) was added 1-bromopyrrolidine-2,5-dione (413.10 mg, 2.32 mmol, 0.95 eq) at -40°C and the reaction was stirred at -40°C for 4 hours. LCMS showed that approximately 9.6% of the starting material remained and a new peak (61.8%) was detected. The reactant was diluted with water (50 mL) and extracted with DCM (3×30 mL). The combined organic layers were washed with brine (50 mL), dried over Na₂SO₄, and concentrated to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to produce compound 3 (500 mg, yield 64.96%) as a yellow solid.

[1307] 1H NMR (ET42365-256-P1A, 400 MHz, chloroform-d) δ 5.68 (s, 1H), 6.90 (t, J = 7.84 Hz, 1H), 7.24 (dd, J = 7.65, 1.38 Hz, 1H), 7.39-7.45 (m, 2H), 7.46-7.52 (m, 3H)

[1308] General procedure for the preparation of compound 4-ET42365-257.

[1309]

[1310] To a solution of compound 3 (500 mg, 1.76 mmol, 1 eq) in DMF (12 mL) was added K2CO3 (487.42 mg, 3.53 mmol, 2 eq) and compound 3A (530.47 mg, 1.94 mmol, 1.1 eq) and the reaction was stirred at 25 ° C for 12 hours. LCMS showed that the starting material was exhausted and a new peak was detected. The crude product was diluted with water (50 mL) and extracted with ethyl acetate (3×30 mL). The organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to provide compound 4 (700 mg, yield 75.04%) as a yellow oil. The product was used directly in the next step without further purification.

[1311] 1 H NMR (ET42365-257-P1A, 400 MHz, chloroform-d) δ 4.59 (s, 2H), 7.08-7.15 (m, 1H), 7.25 (dd, J = 8.22, 1.82 Hz, 1H), 7.27-7.30 (m, 1H), 7.35-7.48 (m, 6H), 7.60 (dd, J = 7.97, 1.57 Hz, 1H)

[1312] General procedure for the preparation of compound 5 - ET42365-260.

[1313] Reactions were performed in parallel but pooled for purification.

[1314]

[1315] To a solution of compound 4 (350 mg, 735.12 μmol, 1 eq) and LiOH.HO (61.70 mg, 1.47 mmol, 2 eq) in a mixture of DMSO (6 mL) and HO (1.5 mL) under nitrogen was added CuO (5.26 mg, 36.76 μmol, 3.76 μL, 0.05 eq) and BHMPO (12.06 mg, 36.76 μmol, 0.05 eq). The reaction was stirred at 80°C for 12 hours. LCMS indicated approximately 25% of the starting material remained, and a new peak with the desired product Ms was detected (44%). An additional reaction was set up as described above and combined for purification. The combined reactions were diluted with water (30 mL) and extracted with ethyl acetate (3 x 20 mL). The organic layer was washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield the crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give Compound 5 (500 mg, yield 74.07%) as a yellow solid.

[1316] 1 H NMR (ET42365-260-P1A, 400MHz, chloroform-d) δ4.57 (s, 2H), 5.66 (s, 1H), 6.91 (d, J = 7.63Hz, 1H), 7.02 (d, J = 8.00Hz, 1H), 7.12-7.19 (m, 1H), 7.26 (br d,J=8.13Hz,1H),7.41-7.50(m,4H),7.54(d,J=8.38Hz,2H)

[1317] General procedure for the preparation of compound 6 - ET42365-274.

[1318]

[1319] To a solution of compound 5 (400 mg, 968.02 μmol, 1 equivalent) and DIPA (195.91 mg, 1.94 mmol, 273.61 μL, 2 equivalents) in CHCl (10 mL) was added 1-bromopyrrolidine-2,5-dione (172.29 mg, 968.02 μmol, 1 equivalent) at -40 ° C. The reactants were stirred at -40 ° C for 4 hours. LCMS showed that about 5.6% of the starting material remained and a new peak with the desired product Ms was detected. The reactants were diluted with water (50 mL) and extracted with DCM (3 × 30 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4 and concentrated to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to produce compound 6 (300 mg, 56.68% yield) as a white solid.

[1320] LCMS(ESI+):RT=1.061min,m / z 490.8(MH) + .

[1321] NEG5-95CD_2min: LC / MS (the column used for chromatography was Xbridge C18 2.1*50mm, 5um. The detection method was diode array (DAD). The MS mode was negative electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 10 mM ammonium bicarbonate in water, and mobile phase B was HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min), with a hold of 0.34 min at 5% B. The flow rate was 1.5 mL / min.

[1322] General procedure for the preparation of series 11-4-2 - ET42365-279.

[1323]

[1324] To a solution of compound 6 (200 mg, 406.41 μmol, 1 eq), compound 7 (118.22 mg, 609.62 μmol, 1.5 eq), and K PO (215.67 mg, 1.02 mmol, 2.5 eq) in a mixture of THF (6 mL) and H O (1.5 mL) was added di-tert-butyl(cyclopentyl)phosphane; dichloro-palladium; and iron (52.98 mg, 81.28 μmol, 0.2 eq) under nitrogen. The reaction was stirred at 85° C. for 40 hours. LCMS indicated approximately 12% of the starting material remained, and a new peak with the desired product Ms was detected. The reaction was filtered, and the filtrate was concentrated to yield the crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water(NH4HCO3)-ACN]; B%: 70%-95%, 8 min) to give series 11-4-2 (60.1 mg, yield 26.19%) as an off-white solid.

[1325] 1 H NMR (ET42365-279-P1A, 400 MHz, chloroform-d) δ 3.82 (s, 3H), 4.59 (s, 2H), 6.02 (s, 1H), 6.58 (s, 1H), 6.99 (d, J = 8.03 Hz, 1H), 7.11 (d, J = 8.16 Hz, 1H), 7.25 (s, 1H), 7.41-7.51 (m, 4H), 7.54-7.61 (m, 2H)

[1326] LCMS(ESI+):RT=2.774min,m / z 561.0(M+H) + .

[1327] 50_100AB_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.00 min, held at 100% B for 1.00 min, and then 100-50% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1328] Synthesis of NUCC-0226606 (ET42365-228-P1) and NUCC-0226605 (ET42365-228-P2)

[1329]

[1330] Synthesis scheme

[1331] Scheme 40: Preparation of A02B01C07D01_P1 (NUCC-0226606) and A02B01C07D01 (NUCC-0226605)

[1332]

[1333]

[1334] Scheme 41: Preparation of Compound 13

[1335]

[1336] Chemical synthesis

[1337] LCMS method:

[1338] Method 1:

[1339] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1340] Method 2:

[1341] 5-95 CD_4.5 min: LC / MS (the column used for chromatography was an Xbridge C18 2.1*50 mm, 5 μm). The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 10 mM ammonium bicarbonate in water, and mobile phase B was HPLC-grade acetonitrile. The gradient was 5-95% B in 4.30 min. 5% B in 0.01 min, 5-95% B (0.01-3.00 min), and held at 95% B (in 0.5 min), 95-5% B (3.50-3.51 min), with a 0.79 min hold at 5% B. The flow rate was 1.0 mL / min (0.01-4.30 min).

[1342] Method 3:

[1343] 50_100CD_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 0.40-3.40 min, held at 100% B for 0.45 min, and then 100-50% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1344] Method 4:

[1345] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1346] The largest experiment ever run:

[1347] General procedure for the preparation of compound 13 - ET42365-161.

[1348]

[1349] To a solution of compound 11 (300 mg, 2.33 mmol, 1.2 eq) in THF (6 mL) was added t-BuOK (1 M, 2.33 mL, 1.2 eq) at 0 ° C. The reaction was stirred at 0 ° C. for 1 hour, then compound 12 (390.56 mg, 1.94 mmol, 1 eq) was added and the reaction was stirred at 50 ° C. for 12 hours. LCMS showed that all the starting material was consumed and two new peaks were detected. The reaction was filtered and the solid was collected and dried to produce compound 13 (300 mg, 62.08% yield) as an off-white solid.

[1350] 1 H NMR (ET42365-161-P1B, 400MHz, DMSO-d6) δ2.94 (q, J=5.09Hz, 2H), 6.82-6.89 (m, 2H), 7.17-7.24 (m, 2H)

[1351] General procedure for the preparation of compound 7 - ET42365-170.

[1352]

[1353] To a solution of core A3_1 (400 mg, 835.47 μmol, 1 eq) and pyridine (264.34 mg, 3.34 mmol, 269.74 μL, 4 eq) in DCM (12 mL) was added Tf2O (353.58 mg, 1.25 mmol, 206.77 μL, 1.5 eq) dropwise at 0 ° C. The reaction was stirred at 20 ° C for 3 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reactant was diluted with water (50 mL) and extracted with DCM (3 × 25 mL). The organic layer was separated and the combined organic layer was washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give Compound 7 (0.4 g, yield 78.38%) as a white solid.

[1354] LCMS(ESI+):RT=0.915min,m / z 611.1(M+H) + .

[1355] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1356] General procedure for the preparation of compound 8 - ET42365-200.

[1357]

[1358] To a solution of compound 7 (200 mg, 327.42 μmol, 1 eq), compound 13 (188.32 mg, 757.88 μmol, 2.31 eq) and K 3 PO 4 (208.50 mg, 982.26 μmol, 3 eq) in a mixture solution of H 2 O (2 mL) and toluene (10 mL) was added RuPhos Pd G 3 (54.77 mg, 65.48 μmol, 0.2 eq) under nitrogen and the reactants were stirred at 110 ° C for 20 hours. LCMS showed that the starting material was consumed and a new peak with the desired product Ms was detected. The reactants were filtered and the filtrate was concentrated to produce a crude product. The crude product was purified by preparative TLC (petroleum ether / ethyl acetate=2 / 1) to produce compound 8 (50 mg, yield 20.25%) as a yellow solid.

[1359] LCMS(ESI+):RT=0.988min,m / z 603.2(M+H) + .

[1360] 5-95AB_2min:LC / MS (the column used for chromatography was Agilent Poroshell SB-C18 3.0*30mm, 2.7um. The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was a solution of 0.04% trifluoroacetic acid in water, and mobile phase B was a solution of 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 1.50 min. 5% B in 0.01 min, 5-95% B (0.01-0.70 min), 95% B (0.70-1.16 min), 95-5% B (1.16-1.50 min). The flow rate was 1.5 mL / min.

[1361] General procedure for the preparation of compound 8 - ET42365-201.

[1362]

[1363] To a solution of compound 8 (80 mg, 106.08 μmol, 80% purity, 1 eq) in MeOH (3 mL) was added KCO (29.32 mg, 212.15 μmol, 2 eq), and the reaction was stirred at 20°C for 12 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was diluted with water (10 mL) and extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine (10 mL), dried over NaSO, and concentrated to yield a crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 um; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 70%-95%, 8 min) to yield compound 9 (40 mg, 67.18% yield) as a white solid.

[1364] LCMS(ESI+):RT=3.108min,m / z 561.0(M+H) + .

[1365] 5-95 CD_4.5 min: LC / MS (the column used for chromatography was an Xbridge C18 2.1*50 mm, 5 um). The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 10 mM ammonium bicarbonate in water, and mobile phase B was HPLC grade acetonitrile. The gradient was 5-95% B in 4.30 min. 5% B in 0.01 min, 5-95% B (0.01-3.00 min), and held at 95% B (in 0.5 min), 95-5% B (3.50-3.51 min), with a 0.79 min hold at 5% B. The flow rate was 1.0 mL / min (0.01-4.30 min).

[1366] General procedure for the preparation of A02B01C07D01_P1 and A02B01C07D01-ET42365-228.

[1367]

[1368] To a solution of compound 9 (50 mg, 89.08 μmol, 1 eq) in AcOH (2 mL) was added NCS (11.89 mg, 89.08 μmol, 1 eq) at 0°C, and the reaction was stirred at 60°C for 12 hours. LCMS indicated approximately 33% of the starting material remained and two new peaks with the desired Ms were detected. The reaction was concentrated to yield the crude product. The crude product was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 70%-90%, 8min) to give a crude product, which was further purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 70%-90%, 8min) to give A02B01C07D01_P1 (NUCC-0226606) (15.2mg, yield 27.9%) and A02B01C07D01 (NUCC-0226605) (22.3mg, yield 41.05%) as white solids.

[1369] A02B01C07D01 (NUCC-0226606):

[1370] 1H NMR (ET42365-228-P1B, 400MHz, chloroform-d) δ3.84(s,3H),4.65-4.78(m,2H),4.93(br s,1H),6.75(d,J=8.88Hz,2H),7.23(d,J=8.88Hz,2H),7.33-7.43(m,2H),7.54(brd,J=8.25Hz,1H),7.67(d,J=8.13Hz,1H),7.76(d,J=1.38Hz,1H)

[1371] LCMS(ESI+):RT=2.467min,m / z 596.9(M+H) + .

[1372] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1373] A02B01C07D01(NUCC-0226605):

[1374] 1 H NMR (ET42365-228-P1C, 400 MHz, chloroform-d) δ 3.90 (s, 3H), 4.71-4.78 (m, 1H), 4.79-4.86 (m, 1H), 5.02 (br s, 1H), 6.65 (s, 1H), 6.77 (d, J = 9.01 Hz, 2H), 7.18-7.26 (m, 2H), 7.45 (s, 1H), 7.52 (dd, J = 8.19, 1.81 Hz, 1H), 7.62 (d, J = 8.13 Hz, 1H), 7.76 (d, J = 1.63 Hz, 1H)

[1375] LCMS(ESI+):RT=2.513min,m / z 596.9(M+H) + .

[1376] 5_95CD_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.40 min, held at 95% B for 0.45 min, and then 95-5% B in 0.01 min, with a flow rate of 0.8 ml / min. Mobile phase A was H2O+10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge C18 2.1*50 mm column (5 um particles). The detection method was diode array (DAD) detection. The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1377] Synthesis of A02B01C07D01_P1 (NUCC-0226606) and A02B01C07D01_P2 (NUCC-0227064)

[1378]

[1379] Scheme 42: Preparation of A02B01C07D01_P1 (NUCC-0226606)

[1380]

[1381]

[1382]

[1383] Scheme 43: Preparation of A02B01C07D01_P2 (NUCC-0227064)

[1384]

[1385] Chemical synthesis

[1386] LCMS method:

[1387] 5_95AB_6min-220-254-ELSD

[1388] LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C1850*2.1 mm column (5 μm particles). The detection methods were diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1389] The largest experiment ever run:

[1390] General procedure for the preparation of compound 2-ET52382-5.

[1391]

[1392] To a solution of compound 1 (25 g, 108.20 mmol) in MeOH (250 mL) was added sulfuric acid (9.20 g, 93.80 mmol, 5 mL) dropwise at 20 ° C. The mixture was heated to 70 ° C and stirred for 12 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, Rf = 0.25) showed that the starting material was consumed and a new spot was generated. Two more reactions were set up as detailed above and the three reaction mixtures were combined. The reaction mixture was concentrated to produce a crude product, which was diluted with water (500 mL), adjusted to pH = 6-7 with K2CO3, and extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with brine (100 mL) and dried over Na2SO4 and concentrated to produce compound 2 (76 g, 90.76% yield) as a white solid, which was used directly in the next step without further purification.

[1393] 1 H NMR (400 MHz, chloroform-d) δ 3.93 (s, 3H), 3.94 (s, 3H), 7.02 (dd, J = 8.00, 1.38 Hz, 1H), 7.26-7.28 (m, 1H), 7.30-7.36 (m, 1H)

[1394] General procedure for the preparation of compound 3 - ET52382-10.

[1395]

[1396] At 20 ℃ under nitrogen, to 2-bromo-3-methoxy-benzoic acid methyl ester (25g, 102.01mmol), [4-chloro-3-(trifluoromethyl)phenyl] boronic acid (27.47g, 122.41mmol) in dioxane (200mL) and H O (100mL) solution, add Na CO (21.62g, 204.02mmol) and Pd (dppf) Cl .CH Cl (4.17g, 5.10mmol). The mixture was heated to 85 ℃ and stirred for 12 hours. TLC (petroleum ether / ethyl acetate=10 / 1) showed that the starting material (Rf=0.4) was consumed and a new spot (Rf=0.45) was produced. As described in detail above, two other reactions were set up and the three reaction mixtures were merged. The reaction mixture was poured into water (800mL) and extracted with ethyl acetate (200mL×3). The organic layers were combined, washed with brine (2×20 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate=0 / 1 to 20 / 1) to give compound 3 (85 g, 72.52% yield) as a white solid.

[1397] 1 H NMR (400 MHz, chloroform-d) δ 3.62 (s, 3H), 3.77 (s, 3H), 7.14 (d, J = 8.00 Hz, 1H), 7.36 (dd, J = 8.19, 1.44 Hz, 1H), 7.41-7.47 (m, 1H), 7.48-7.54 (m, 2H), 7.57 (d, J = 1.50 Hz, 1H)

[1398] General procedure for the preparation of compound 4-ET52382-14.

[1399]

[1400] A mixture of methyl 2-[4-chloro-3-(trifluoromethyl)phenyl]-3-methoxybenzoate (35 g, 101.53 mmol) and pyridine; hydrochloride (117.33 g, 1.02 mol) was heated to 190°C under nitrogen and stirred for 10 hours. TLC (petroleum ether / ethyl acetate = 5 / 1) showed that the starting material (Rf = 0.6) was consumed and a new spot (Rf = 0.2) was produced. Two more reactions were set up as detailed above and the three reaction mixtures were combined. The reaction mixture was diluted with water (1000 mL) and extracted with ethyl acetate (300 mL x 3). The combined organic layers were washed with brine (100 mL x 3) and dried over Na2SO4 and concentrated to produce compound 4 (90 g, 88.64% yield) as a white solid, which was used directly in the next step without further purification.

[1401] 1 H NMR(400MHz,DMSO-d6)δ7.12(d,J=7.75Hz,1H),7.25-7.35(m,2H),7.51(br d,J=8.25Hz,1H),7.60(s,1H),7.70(d,J=8.25Hz,1H),9.88(s,1H),12.70(br s,1H)

[1402] General procedure for the preparation of compound 5-ET52382-16.

[1403]

[1404] To a solution of 2-[4-chloro-3-(trifluoromethyl)phenyl]-3-hydroxy-benzoic acid (40 g, 126.32 mmol) in THF (1000 mL) was added BH3-Me2S (10 M, 63.16 mL) dropwise at 0 ° C. The mixture was heated to 40 ° C and stirred for 16 hours. LCMS showed that the starting material was consumed. The reaction mixture was cooled to 0 ° C and slowly quenched with methanol (200 mL) at 0 ° C. Another reaction was set up as described in detail above and the two reaction mixtures were combined. The mixture was concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) to produce compound 5 (72 g, 89.45% yield) as a white solid.

[1405] 1 H NMR (400MHz, DMSO-d6) δ4.15(d,J=5.25Hz,2H),5.00(t,J=5.32Hz,1H),6.86(d,J=8.00Hz,1H),7.01(d,J=7.50Hz, 1H),7.17-7.25(m,1H),7.56(dd,J=8.25,1.75Hz,1H),7.68(d,J=1.75Hz,1H),7.74(d,J=8.25Hz,1H),9.43(s,1H)

[1406] General procedure for the preparation of compound 6 - ET52382-20.

[1407]

[1408] To a solution of 2-[4-chloro-3-(trifluoromethyl)phenyl]-3-(hydroxymethyl)phenol (30 g, 99.12 mmol) and DIPA (30.09 g, 297.35 mmol, 42.02 mL) in CHCl₃ (700 mL) was added 1-bromopyrrolidine-2,5-dione (17.64 g, 99.12 mmol) portionwise at 0°C. The mixture was warmed to 20°C and stirred for 12 hours. LCMS indicated 16.1% of the starting material remained and 55% of the product with the desired Ms was detected (Rt = 0.854 min). An additional reaction was set up as detailed above and the two reaction mixtures were combined. The mixture was poured into water (1000 mL) and extracted with DCM (3 x 300 mL). The organic layers were combined, washed with brine (2 x 100 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to yield a residue. The residue was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give compound 6 (40 g, 50.24% yield) as a white solid.

[1409] 1 H NMR (400MHz, DMSO-d6) δ4.08 (br d, J=3.26Hz, 2H), 5.11 (br t,J=4.64Hz,1H),7.03(d,J=8.28Hz,1H),7.51-7.60(m,2H),7.69(d,J=1.88Hz,1H),7.78(d,J=8.16Hz,1H),8.93(br s,1H)

[1410] General procedure for the preparation of compound 7-ET52382-23.

[1411]

[1412] To a solution of 6-bromo-2-[4-chloro-3-(trifluoromethyl)phenyl]-3-(hydroxymethyl)phenol (20 g, 52.41 mmol) in ACN (200 mL) was added PBr3 (5.68 g, 20.97 mmol) dropwise at 0 ° C. The mixture was warmed to 20 ° C and stirred for 12 hours. LCMS showed that the starting material was exhausted and 69.7% of the product with the desired Ms was detected (Rt = 0.975 min). The reaction mixture was poured into water (300 mL) and extracted with ethyl acetate (150 mL × 3). The organic layers were combined, washed with brine (2 × 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 1 / 0 to 50 / 1) to produce compound 7 (20 g, 77.26% yield) as a white solid.

[1413] 1 H NMR(400MHz,DMSO-d6)δ4.32(br d,J=5.50Hz,2H),7.07(d,J=8.38Hz,1H),7.57-7.65(m,2H),7.73(d,J=1.38Hz,1H),7.84(d,J=8.25Hz,1H),9.19(s,1H)

[1414] General procedure for the preparation of compound 8 - ET52382-25.

[1415]

[1416] To a solution of 4-chlorophenol (28.92 g, 224.99 mmol, 22.08 mL) in a mixture of THF (100 mL) and DMF (100 mL) was added t-BuONa (21.62 g, 224.99 mmol). The reactants were stirred at 25 ° C for 30 minutes and compound 7 (20 g, 45.00 mmol) in THF (50 mL) was added dropwise at 0 ° C. The reactants were stirred at 20 ° C for 12 hours. LCMS showed that the starting material was exhausted and 51.9% of the peak with the expected Ms was detected (Rt = 1.019 min). TLC (petroleum ether / ethyl acetate = 20 / 1, Rf = 0.4) showed that a new spot was generated. The reaction mixture was poured into water (300 mL) and extracted with ethyl acetate (3 × 100 mL). The organic layers were combined, washed with brine (2 × 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 1 / 0 to 100 / 1) to give Compound 8 (20 g, 85.80% yield) as a white solid.

[1417] 1 H NMR(400MHz,DMSO-d6)δ4.69(br s,2H),6.75-6.85(m,2H),7.06(d,J=8.25Hz,1H),7.21-7.30(m,2H),7.55-7.65(m,2H),7.67-7.76(m,2H),9.16(s,1H)

[1418] General procedure for the preparation of compound 9-ET52382-33.

[1419]

[1420] To a solution of compound 8 (10 g, 20.32 mmol), [2-methyl-5-(trifluoromethyl)pyrazol-3-yl]-boronic acid (7.88 g, 40.64 mmol) in dioxane (80 mL), H2O (40 mL) and ACN (80 mL) was added 4-di-tert-butylphosphanyl-N,N-dimethyl-aniline; palladium chloride (1.44 g, 2.03 mmol) and K3PO4 (12.94 g, 60.96 mmol) at 20 ° C under nitrogen. The mixture was heated to 80 ° C and stirred for 12 hours. LCMS showed that the starting material was exhausted and 73.3% of the peak with the desired Ms was detected (Rt = 1.041 min). TLC (petroleum ether / ethyl acetate = 5 / 1, Rf = 0.5) showed that a new spot was generated. The reaction mixture was poured into water (200 mL) and extracted with ethyl acetate (60 mL × 3). The organic layers were combined, washed with brine (2×50 mL), dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate=1 / 0 to 10 / 1) to give compound 9 (9 g, 71.02% yield) as a white solid.

[1421] 1 H NMR (400MHz, DMSO-d6) δ3.78(s,3H),4.61-4.91(m,2H),6.80(s,1H),6.82-6.91(m,2H),7.21-7. 31(m,3H),7.37(d,J=7.88Hz,1H),7.65(dd,J=8.19,1.69Hz,1H),7.71-7.80(m,2H),9.01(s,1H)

[1422] General procedure for the preparation of compound 10-ET52382-58.

[1423]

[1424] To a solution of compound 9 (10 g, 17.82 mmol) and TEA (2.70 g, 26.72 mmol, 3.72 mL) in DCM (150 mL) was added acetyl chloride (1.68 g, 21.38 mmol, 1.53 mL) dropwise at ℃. The mixture was warmed to 20 ℃ and stirred for 2 hours. LCMS showed that the starting material was exhausted and 93.4% of the peak with the desired Ms was detected (Rt = 0.916 min). TLC (petroleum ether / ethyl acetate = 5 / 1, Rf = 0.4) showed that a new spot was produced. The reaction mixture was poured into water (200 mL) and extracted with DCM (60 mL × 3). The organic layers were combined, washed with brine (2 × 20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue. The residue was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give compound 10 (10.2 g, 85.40% yield) as a white solid.

[1425] 1 H NMR (400MHz, DMSO-d6) δ1.71(s,3H),3.82(s,3H),4.80-4.99(m,2H),6.78(s,1H),6.84-6.91(m,2H),7.24-7.32(m,2H),7.61(br d,J=7.88Hz,1H),7.72(br s,1H),7.75(s,2H),7.79(d,J=8.25Hz,1H)

[1426] General procedure for the preparation of compound 11 - ET52382-61.

[1427]

[1428] To a solution of compound 10 (10 g, 16.57 mmol) in DMF (100 mL) was added 1-chloropyrrolidine-2,5-dione (8.85 g, 66.30 mmol) at 20 ° C. The mixture was heated to 100 ° C and stirred for 12 hours. LCMS and HPLC showed that 2.5% of the starting material remained and 67.3% of the product with the desired Ms was detected. The reaction mixture was poured into water (600 mL) and extracted with ethyl acetate (3 × 200 mL). The organic layers were combined, washed with brine (2 × 50 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue. The residue was purified by preparative HPLC to produce compound 11 (4.5 g, 42.36% yield) and compound 11_ by-product (0.50 g, 4.44% yield) as a white solid.

[1429] Compound 11:1 H NMR (400MHz, DMSO-d6) δ1.65(s,3H),3.73(s,3H),4.72-4.97(m,2H),6.82(br d,J=8.82Hz,2H),7.22(br d,J=8.82Hz,2H),7.47-7.70(m,2H),7.70-7.80(m,3H)

[1430] Compound 11_by-product: 1 H NMR(400MHz,DMSO-d6)δ1.72(s,3H),3.80(s,3H),4.94-5.15(m,2H),7.09(br d,J=8.88Hz,1H),7.33(dd,J=8.88,2.50Hz,1H),7.56(d,J=2.38Hz,1H),7.59-7.77(m,2H),7.78-7.90(m,3H)

[1431] Preparative HPLC separation method:

[1432] Instrument: Shimadzu LC-8A preparative HPLC

[1433] Column: Phenomenex luna C18 (250*70mm, 15um)

[1434] Mobile phase: A represents H2O (0.09% TFA) and B represents CAN

[1435] Gradient: 73% to 90% B in 20 min

[1436] Flow rate: 130mL / min

[1437] Wavelength: 220 and 254 nm

[1438] General procedure for the preparation of A02B01C07D01_P1 - ET52382-63.

[1439]

[1440] To a solution of compound 11 (6.5 g, 10.19 mmol) in MeOH (100 mL) and ACN (100 mL) was added K2CO3 (4.23 g, 30.57 mmol) at 20 ° C. The mixture was stirred for 12 hours. LCMS showed that the starting material was exhausted and 96.6% of the peak with the desired Ms was detected (Rt = 0.941 min). The reaction mixture was concentrated under reduced pressure to remove methanol and acetonitrile. Water (200 mL) was then added and extracted with ethyl acetate (3 × 60 mL). The organic layers were combined, washed with brine (2 × 20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue. The residue was co-evaporated 3 times with DCM (60 mL) to produce A02B01C07D01_P1 (5.2 g, 84.70% yield) as a white solid.

[1441] 1 H NMR(400MHz,DMSO-d6)δ3.77(s,3H),4.78(br s,2H),6.82-6.91(m,2H),7.24-7.32(m,3H),7.40(d,J=8.00Hz,1H),7.64-7.70(m,1H),7.72-7.76(m,1H),7.78(s,1H),9.24(s,1H)

[1442] LCMS(ESI+):m / z 596.9(M+H)+,RT:3.260min

[1443] 30_90AB_6min-220-254-ELSD:

[1444] 30-90AB_6min-220-254-ELSD: LC / MS (gradient was 30% B in 0.40 min and 30-90% B in 2.60 min, held at 90% B for 1.00 min, and then 90-30% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was Kinetex C18 2.1*50 mm, 5 um. The detection methods were diode array (DAD) and evaporative light scattering detection (ELSD). The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1445] General procedure for the preparation of A02B01C07D01_P2-ET52382-64.

[1446]

[1447] To a solution of compound 11_ by-product (0.5 g, 743.79 μmol) in MeOH (20 mL) and ACN (20 mL) was added K2CO3 (308.39 mg, 2.23 mmol) at 20 ° C. The mixture was stirred for 12 hours. LCMS showed that the starting material was exhausted and 98.9% of the peak with the desired Ms was detected (Rt = 0.957 min). The reaction mixture was concentrated under reduced pressure to remove methanol and acetonitrile. Water (60 mL) was then added and extracted with ethyl acetate (20 mL × 3). The organic layers were combined, washed with brine (2 × 10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to produce a residue. The residue was co-evaporated 3 times with DCM (30 mL) to produce A02B01C07D01_P2 (424 mg, 89.46% yield) as a white solid.

[1448] 1 H NMR(400MHz,DMSO-d6)δ3.78(s,3H),4.90(br s,2H),7.05(d,J=8.88Hz,1H),7.26-7.37(m,2H),7.39-7.47(m,1H),7. 55(d,J=2.25Hz,1H),7.64-7.71(m,1H),7.72-7.82(m,2H),9.27(s,1H)

[1449] LCMS(ESI+):m / z 630.9(M+H)+,RT:2.670min

[1450] 50-100AB_6min-220-254-ELSD: LC / MS (gradient was 50% B in 0.40 min and 50-100% B in 2.60 min, held at 100% B for 1.00 min, and then 100-50% B in 0.01 min, with a flow rate of 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in acetonitrile. The column used for chromatography was Kinetex C18 2.1*50 mm, 5 um. The detection methods were diode array (DAD) and evaporative light scattering detection (ELSD). The MS mode was positive electrospray ionization. The MS range was 100-1000.

[1451] Synthesis of NUCC-0226605 (ET42365-477-1 and ET42365-496-1)

[1452]

[1453] Synthesis scheme:

[1454]

[1455]

[1456] Chemical synthesis

[1457] 5_95AB_6min-220-254-ELSD: LC / MS (gradient was 5% B in 0.40 min and 5-95% B in 0.40-3.00 min, held at 95% B for 1.00 min, and then 95-5% B in 0.01 min, with a flow rate of 1.0 mL / min. Mobile phase A was a solution of 0.037% trifluoroacetic acid in water, and mobile phase B was a solution of 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was a Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and evaporative light scattering (ELSD) detection with positive electrospray ionization. The MS range was 100-1000.

[1458] The largest experiment ever run:

[1459] General procedure for the preparation of compound 2 - ET42365-393.

[1460] Reactions were performed in parallel but pooled for purification.

[1461]

[1462] To a solution of compound 1 (10 g, 43.28 mmol, 1 eq) in MeOH (100 mL) was added H2SO4 (3.68 g, 37.52 mmol, 2 mL), and the reaction was stirred at 70°C for 12 hours. The reaction was cooled to room temperature, and LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. An additional reaction was set up as detailed above, and the two reaction mixtures were combined. The combined reaction mixture was concentrated to produce a residue, which was diluted with water (100 mL), adjusted to pH 6-7 with aqueous K2CO3, and extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, and concentrated to produce compound 2 (21 g, 94.04% yield) as a yellow solid, which was used directly in the next step without further purification.

[1463] 1H NMR (ET42365-393-P1A, 400 MHz, chloroform-d) δ 3.94 (s, 3H), 3.95 (s, 3H), 7.03 (dd, J = 8.03, 1.51 Hz, 1H), 7.26-7.30 (m, 1H), 7.31-7.37 (m, 1H)

[1464] General procedure for the preparation of compound 3 - ET42365-403.

[1465]

[1466] To a solution of compound 2 (14 g, 57.13 mmol, 1 eq), [4-chloro-3-(trifluoromethyl)phenyl]-boronic acid (15.38 g, 68.55 mmol, 1.2 eq) and NaCO (12.11 g, 114.25 mmol, 2 eq) in a mixture of toluene (500 mL), EtOH (100 mL) and HO (25 mL) was added Pd(dppf)Cl.CHCl (2.33 g, 2.86 mmol, 0.05 eq) under nitrogen and the reaction was stirred at 85° C. for 12 hours. LCMS indicated that the starting material was consumed and a new peak with the desired product Ms was detected. The reaction was filtered and the filtrate was concentrated to yield the crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 30 / 1 to 4 / 1) to give Compound 3 (13 g, yield 62.72%) as a yellow solid.

[1467] 1 H NMR (ET42365-403-P1A, 400 MHz, chloroform-d) δ 3.62 (s, 3H), 3.77 (s, 3H), 7.14 (dd, J = 8.07, 1.06 Hz, 1H), 7.36 (dd, J = 8.19, 1.81 Hz, 1H), 7.41-7.47 (m, 1H), 7.47-7.54 (m, 2H), 7.57 (d, J = 2.00 Hz, 1H)

[1468] General procedure for the preparation of compound 4A - ET42365-431.

[1469]

[1470] A mixture of compound 3 (10 g, 29.01 mmol, 1 eq) in pyridine hydrochloride (50.29 g, 435.15 mmol, 15 eq) was stirred at 190 ° C for 6 hours. The reactants were cooled to room temperature, and LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with brine (150 mL), dried over Na2SO4 and concentrated to produce compound 4A (8 g, yield 87.09%) as a yellow solid, which was used directly in the next step without further purification.

[1471] 1 H NMR (ET42365-431-P1A, 400 MHz, chloroform-d) δ 7.21 (dd, J = 8.19, 1.06 Hz, 1H), 7.38-7.45 (m, 2H), 7.58-7.65 (m, 2H), 7.69 (dd, J = 7.82, 1.06 Hz, 1H)

[1472] General procedure for the preparation of compound 5 - ET42365-438.

[1473]

[1474] To a solution of compound 4A (8 g, 25.26 mmol, 1 equivalent) in THF (300 mL) was added dropwise BH3-Me2S (10 M, 12.63 mL, 5 equivalents) at 0 ° C. The reactant was stirred at 25 ° C. for 12 hours. LCMS showed that the starting material was exhausted and a new peak with the desired product Ms was detected. The reaction mixture was cooled to 0 ° C., slowly quenched with methanol (100 mL) and concentrated in vacuo to produce a crude product. The crude product was purified by column chromatography on silica gel (eluted with petroleum ether / ethyl acetate = 10 / 1 to 2 / 1) to produce compound 5 (6.6 g, yield 86.31%) as a white solid.

[1475] 1 H NMR (ET42365-438-P1A, 400MHz, chloroform-d) δ4.39 (s, 2H), 4.72 (s, 1H), 6.93 (dd, J = 8.13, 0.75Hz, 1H), 7.15 (d, J = 7 .50Hz,1H),7.30-7.37(m,1H),7.49(dd,J=8.13,1.88Hz,1H),7.64(d,J=8.25Hz,1H),7.70(d,J=1.88Hz,1H)

[1476] General procedure for the preparation of compound 9 - ET42365-452.

[1477]

[1478] To a solution of compound 5 (5 g, 16.52 mmol, 1 eq) and DIPA (5.01 g, 49.56 mmol, 7.00 mL, 3 eq) in CHCl (125 mL) was added portionwise NBS (2.79 g, 15.69 mmol, 0.95 eq) at -50 ° C. The reaction was slowly warmed to 25 ° C and stirred for 12 hours. LCMS showed that approximately 3% of the starting material remained and a new peak with the desired product Ms was detected. Another reaction was set up as detailed above and the two reaction mixtures were combined. The combined reaction mixture was diluted with water (300 mL) and extracted with DCM (3×150 mL). The combined organic layer was washed with brine (250 mL) and dried over Na SO and concentrated to produce the crude product. The crude product was purified by preparative HPLC (column: Agela DuraShell C18 250*70mm*10um; mobile phase: [water (TFA)-ACN]; B%: 38%-68%, 20 min) to give compound 9 (8 g, yield 63.46%) as a white solid.

[1479] 1 H NMR (ET42365-452-P1A, 400 MHz, chloroform-d) δ 4.38 (s, 2H), 5.54 (br s, 1H), 7.09 (d, J = 8.38 Hz, 1H), 7.44 (dd, J = 8.13, 2.00 Hz, 1H), 7.56 (d, J = 8.38 Hz, 1H), 7.60 (d, J = 8.13 Hz, 1H), 7.66 (d, J = 2.00 Hz, 1H)

[1480] General procedure for the preparation of compound 11 - ET42365-464.

[1481]

[1482] To a solution of compound 9 (8 g, 20.97 mmol, 1 equivalent...

Claims

1. A compound having formula I: in W is CR 8 or N; Y is CH or N; Q is for CR 2 or N; Z is C(Alk 2 ) q (X) p (Alk 1 ) n R 1 or N; R 1 is hydrogen, halogen, alkyl, aryl, benzyl, heteroaryl, cycloalkyl, alkoxy or cycloheteroalkyl, wherein R 1 Optionally, at one or more positions, alkyl, alkoxy, cycloalkyl, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, carboxyl, -(CH2)-NH-R 9 、-(O(CH2)2) m -R 15 OR 11 One or more substitutions in; Alk 1 is a straight chain or branched chain alkylene or cycloalkylene; n is 0, 1, or 2; p is 0 or 1; Alk 2 is a straight chain or branched chain alkylene group; q is 0 or 1; r is 0 or 1; m is an integer selected from 1 to 20; X is O or NR 13 ; R 2 is hydrogen or a halogen; R 3 is alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, -OC(O)-alkyl or halogen; R 4 is hydrogen, halogen, amino, alkyl or haloalkyl; or R 4 is aryl or benzyl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, carboxyl, aryloxy, and alkylaryloxy; or R 4 is an alkyl group optionally substituted with a haloaryloxy group; or R 4 With R 1 together form a cycloheteroalkyl group fused to ring A, wherein the cycloheteroalkyl group fused to ring A is optionally substituted with aryl or alkylaryl, and the aryl or alkylaryl group is optionally substituted with halogen; R 5 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 5 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy; R 6 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 6 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy; R 7 is an alkyl group; R 8 is hydrogen, cyano, amino, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, benzyl, hydroxy, halogen, amido and carboxyl; R 9 is optionally substituted with one or more -P(O)(OR 10 )2 substituted alkyl; R 10 is hydrogen or alkyl; R 11 is optionally substituted with one or more -P(O)(OR 12 )2 substituted alkyl; R 12 is hydrogen or alkyl; R 13 is hydrogen, alkyl or -C(O)R 14 ; R 14 is aryl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido and carboxy; R 15 Yes OR 16 、-OS(O)2-R 16 or NR 17 R 18 ; R 16 is an alkyl or aryl group, wherein R 16 is optionally substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido, and carboxyl; and R 17 and R 18 are independently hydrogen or alkyl, Provided that the compound is not 4'-chloro-6-((4-chlorobenzyl)oxy)-3-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-3'-(trifluoromethyl)-[1,1'-biphenyl]-2-ol.

2. The compound of claim 1, which has formula I(a):

3. The compound of claim 2, wherein R 1 is hydrogen, alkyl, aryl, benzyl, where R 1 Optionally, at one or more positions, alkyl, alkoxy, aryl, halogen, -(CH2)-NH-R 9 OR 11 One or more substitutions in; R 2 is hydrogen or chlorine; R 3 is hydroxy or -OC(O)Me; R 4 is hydrogen or a halogen; R 5 is hydrogen or a halogen; R 6 is hydrogen, aryl, benzyl, optionally R 6 substituted at one or more positions with one or more of haloalkyl, halogen, and cyano; R 8 is cyano, amino, haloalkyl or amido; and R 14 is aryl optionally substituted at one or more ring positions with one or more of haloalkyl or halogen.

4. The compound of claim 3, wherein Y is N and W is CCF3.

5. The compound of claim 4, wherein R 3 It's a hydroxyl group.

6. The compound of claim 5, wherein r is 0 and R 6 It is a phenyl group substituted by chlorine and trifluoromethyl.

7. The compound of claim 6, wherein R 2 It's hydrogen.

8. The compound of claim 2, wherein the compound is selected from:

9. Compounds having formula II: in R 1 is hydrogen, cyano, amino, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, benzyl, hydroxy, halogen, amido and carboxyl; R 2 is an alkyl group; R 3 is alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, -OC(O)-alkyl or halogen; R 4 is hydrogen, halogen, alkyl, aryl, alkylaryl, heteroaryl, cycloalkyl or cycloheteroalkyl, optionally R 4 substituted at one or more positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, hydroxy, halogen, cyano, carboxyamido, carboxyl, aryloxy, and heteroaryloxy; X is O or NR 5 ; R 5 is hydrogen, alkyl or -C(O)R 6 ; R 6 is aryl optionally substituted at one or more ring positions with one or more of alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, hydroxy, halogen, cyano, amido and carboxy; Y is alkylene, arylene, benzylidene, heteroarylene, cycloalkylene or cycloheteroalkylene; L is a bond or a linker selected from: -(O(CH2)2) a -、-(O(CH2)2) a -NH-C(O)-Alk 3 -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -、-(O(CH2)2) a -C(O)-NH-Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-Alk 3 -NH-C(O)-CH2- and -(O(CH2)2) a -C(O)-NH-Alk 3 -; a is an integer selected from 1-20; b is an integer selected from 1-20; Alk 3 is a straight chain or branched chain alkylene group; M E3 Selected from and R 11 is hydrogen or an alkyl group.

10. The compound of claim 9, wherein the compound has the structure of Formula II(a): in R 3 is hydroxy or -OC(O)-alkyl; R 4 is hydrogen or phenyl substituted at one or more positions with one or more of haloalkyl or halogen; X is O or -NC(O)R 6 ; R 6 is aryl substituted at one or more ring positions with one or more of haloalkyl or halogen; Y is an alkylene group or a benzylidene group; L is a bond or a linker selected from: -(O(CH2)2) a -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -、-(O(CH2)2) a -C(O)-NH-Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -、-(O(CH2)2) a -C(O)-NH-((CH2)2-O) b -Alk 3 -C(O)-, -(O(CH2)2) a -C(O)-NH-Alk 3 -NH-C(O)-CH2- and -(O(CH2)2) a -C(O)-NH-Alk 3 -;and R 8 is an alkyl or aryl group, wherein R 8 Optionally substituted at one or more positions with one or more alkyl groups.

11. The compound of claim 10, wherein R 3 is a hydroxyl group, and R 4 It is a phenyl group substituted with trifluoromethyl and chlorine.

12. The compound of claim 11, wherein X is -NC(O)R 6 , and R 6 It is a phenyl group substituted with trifluoromethyl and chlorine.

13. The compound of claim 11, wherein X is O, and Y is propylene or benzylidene.

14. The compound of claim 9, wherein the compound is selected from:

15. A pharmaceutical composition comprising the molecule of claim 1 and a suitable pharmaceutical carrier, excipient or diluent.

16. A method of treating cancer, comprising administering the composition of claim 15 to a subject suffering from the cancer.

17. The method of claim 16, wherein the cancer is selected from the group consisting of multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and breast cancer.

18. A pharmaceutical composition comprising the molecule of claim 9 and a suitable pharmaceutical carrier, excipient or diluent.

19. A method of treating cancer, comprising administering the composition of claim 18 to a subject suffering from the cancer.

20. The method of claim 19, wherein the cancer is selected from the group consisting of multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, and breast cancer.

Images