Amide-containing heterocyclic derivatives and uses thereof

Abstract

A series of amide-containing heterocyclic derivatives and uses thereof are disclosed, specifically compounds of formula (III) and pharmaceutically acceptable salts thereof. TIFF2025529831000240.tif73100

Description

[Technical Field]

[0001] This application claims priority to the following: CN2022109954611, filing date: August 18, 2022; CN2022110677997, filing date: September 1, 2022; CN2022116596067, filing date: December 22, 2022; CN2023101673027, filing date: February 24, 2023. The present invention relates to a series of amide-containing heterocyclic derivatives and uses thereof, and in particular to compounds of formula (III) and pharmaceutically acceptable salts thereof: [Background technology]

[0002] PRMT5 (protein arginine methyltransferase 5) is an important type II protein arginine methyltransferase that controls gene expression by regulating the methylation level of histone arginine. PRMT5 is highly expressed in various tumors (e.g., colorectal cancer, lung cancer, ovarian cancer, prostate cancer, lymphoma, leukemia, and glioblastoma), and its expression level is closely correlated with tumor development, progression, and prognosis. Therefore, inhibition of PRMT5 is expected to be a novel therapeutic approach for these tumors. Currently, many inhibitors have entered clinical studies in this field (e.g., GSK3326595, JNJ64619178, PF06939999, PRT543, PRT811). Among them, GSK-3326595, the first PRMT5 inhibitor to enter clinical studies, is highly effective in inhibiting PRMT5 function. However, due to its lack of selectivity, it inhibits PRMT5 in tumor cells while also inhibiting PRMT5 in normal cells, increasing the risk of clinical hematological side effects such as thrombocytopenia, anemia, and neutropenia. Therefore, selectively inhibiting PRMT5 function in tumor cells without affecting PRMT5 activity in normal cells is expected to improve the therapeutic index of PRMT5 inhibitors and represent a new direction for PRMT5 inhibitor research. MTAP (methylthioadenosine phosphorylase) is the first enzyme in the salvage pathway for methionine and purine synthesis and can catalyze the metabolism of MTA (methylthioadenosine), a by-product of polyamine metabolism. The MTAP gene is often co-deleted with CDKN2A, a common tumor inhibitor gene in the body, and the rate of this co-deletion in tumors can reach 9% to 15%. Deletion of the MTAP gene can lead to intracellular accumulation of MTA, which can compete with SAM to bind to PRMT5, forming a PRMT5·MTA complex and inhibiting the catalytic activity of PRMT5 against SAM. Inhibitors developed for the PRMT5·MTA complex can selectively inhibit PRMT5 in MTAP-deficient tumor cells while minimizing the effect on PRMT5 in normal cells, thereby selectively killing MTAP-deficient tumor cells and achieving superior safety.Currently, only two inhibitors targeting the PRMT5·MTA complex, AMG-193 and MRTX1719, are currently in clinical trials or are about to be developed, and therefore the development of inhibitors targeting the PRMT5·MTA complex has important clinical implications for the treatment of MTAP-deficient tumors. Summary of the Invention

[0003] The present invention provides a compound represented by formula (III) or a pharmaceutically acceptable salt thereof:

[0004] [ka]

[0005] however, E1 is selected from CH2, NH and O; Structural Unit

[0006] [ka]

[0007] is selected from Each R1 is independently H, halogen, or C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R a is optionally replaced by R6 is C 2-4 Alkynyl and C 2-4 alkenyl, wherein C 2-4 Alkynyl and C 2-4 Alkenyl is each independently one, two or three R b is optionally replaced by Alternatively, R6 is halogen, C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3The alkoxy is independently one, two, or three R b is optionally replaced by, in which case the structural unit

[0008] [ka]

[0009] is selected from Ring A is a 5- to 6-membered heteroaryl, C 4-6 cycloalkenyl or 5- to 6-membered heterocycloalkenyl, wherein said 5- to 6-membered heteroaryl, C 4-6 The cycloalkenyl or 5- to 6-membered heterocycloalkenyl each independently has one, two, or three R c is optionally replaced by T1 and T2 are each independently selected from CR7 and N; T3, T4 and T5 are each independently selected from CR4 and N; T6, T7, and T8 are each independently selected from CR5 and N; R3 is H, C 1-3 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, -C 1-3 Alkyl-C 3-6 Cycloalkyl and -C 1-3 alkyl-4 to 6-membered heterocycloalkyl, 1-3 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, -C 1-3 Alkyl-C 3-6 Cycloalkyl and -C 1-3 The alkyl-4 to 6-membered heterocycloalkyl is each independently one, two, or three R e is optionally replaced by R4, R5 and R7 are each independently H, halogen, C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R dis optionally replaced by Each R a , each R c , each R d and each R e are each independently selected from H, D, halogen, OH, NH2, CH3, and CD3; Each R b are independently H, D, halogen, OH, NH2, C 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 alkylamino and 4- to 6-membered heterocycloalkyl, 1-3 Alkyl, C 1-3 Alkoxy and C 1-3 alkylamino is optionally substituted by 1, 2 or 3 R, each independently; each R is independently selected from H, D, halogen, OH, NH2, CH3, CF3, and CD3; n is selected from 0, 1, 2 and 3; m is selected from 0, 1 and 2; p is selected from 0, 1 and 2, and m and p are not simultaneously 0; The "hetero" in the 4- to 6-membered heterocycloalkyl, 5- to 6-membered heteroaryl, or 5- to 6-membered heterocycloalkenyl each independently represents 1, 2, 3, or 4 heteroatoms or heteroatom groups selected from -O-, -NH-, -S-, and N.

[0010] The present invention further provides a compound represented by formula (III) or a pharmaceutically acceptable salt thereof.

[0011] [ka]

[0012] however, Each R1 is independently H, halogen, or C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3The alkoxy is independently one, two, or three R a is optionally replaced by E1 is selected from CH2, NH and O; R6 is

[0013] [ka]

[0014] R2 is selected from H, halogen, OH, NH2 and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R b is replaced by Structural Unit

[0015] [ka]

[0016] is selected from Alternatively, R6 is halogen, C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R b is optionally replaced by, in which case the structural unit

[0017] [ka]

[0018] is selected from T1 and T2 are each independently selected from CR7 and N; Ring A is a 5- to 6-membered heteroaryl, C 4-6 cycloalkenyl and 5- to 6-membered heterocycloalkenyl, wherein said 5- to 6-membered heteroaryl, C 4-6 The cycloalkenyl and 5- to 6-membered heterocycloalkenyl each independently have one, two, or three Rc is optionally replaced by T3, T4 and T5 are each independently selected from CR4 and N; T6, T7, and T8 are each independently selected from CR5 and N; R3 is H and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R e is replaced, R4, R5 and R7 are each independently H, halogen, C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R d is optionally replaced by Each R a , each R b , each R c , each R d and each R e are each independently selected from H, D, halogen, OH, NH2, CH3, and CD3; n is selected from 0, 1, 2 and 3; m is selected from 0, 1 and 2; p is selected from 0, 1 and 2, and m and p are not simultaneously 0; The "hetero" in the 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl represents 1, 2, 3, or 4 heteroatoms or heteroatom groups independently selected from -O-, -NH-, -S-, and -N-.

[0019] The present invention further provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0020] [ka]

[0021] however, Each R1 is independently H, halogen, or C 1-3Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R a is optionally replaced by R6 is

[0022] [ka]

[0023] R2 is selected from H, halogen, OH, NH2 and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R b is replaced by Structural Unit

[0024] [ka]

[0025] is selected from Alternatively, R6 is halogen, C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R b is optionally replaced by, in which case the structural unit

[0026] [ka]

[0027] is selected from T1 and T2 are each independently selected from CR7 and N; Ring A is a 5- to 6-membered heteroaryl, C 4-6 cycloalkenyl and 5- to 6-membered heterocycloalkenyl, wherein said 5- to 6-membered heteroaryl, C 4-6The cycloalkenyl and 5- to 6-membered heterocycloalkenyl each independently have one, two, or three R c is optionally replaced by T3, T4 and T5 are each independently selected from CR4 and N; T6, T7, and T8 are each independently selected from CR5 and N; R3 is H and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R e is replaced by R4, R5 and R7 are each independently H, halogen, C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R d and each R a , each R b , each R c , each R d and each R e are each independently selected from H, D, halogen, OH, NH2, CH3, and CD3; n is selected from 0, 1, 2 and 3; The "hetero" in the 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl represents 1, 2, 3, or 4 heteroatoms or heteroatom groups independently selected from -O-, -NH-, -S-, and -N-.

[0028] The present invention further provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0029] [ka]

[0030] however, Each R1 is independently H, halogen, or C 1-3 Alkyl and C 1-3 alkoxy, wherein C1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R a is optionally replaced by R2 is H, halogen, OH, NH2 and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R b is replaced by E1 is selected from CH2, NH and O; Structural Unit

[0031] [ka]

[0032] is selected from Ring A is a 5- to 6-membered heteroaryl, C 4-6 cycloalkenyl and 5- to 6-membered heterocycloalkenyl, wherein said 5- to 6-membered heteroaryl, C 4-6 The cycloalkenyl and 5- to 6-membered heterocycloalkenyl each independently have one, two, or three R c is optionally replaced by T3, T4 and T5 are each independently selected from CR4 and N; T6, T7, and T8 are each independently selected from CR5 and N; R3 is H and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R e is replaced, R4 and R5 are each independently H, halogen, or C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R d is optionally replaced by Each R a , each R b , each R c , each R d and each Re are each independently selected from H, halogen, OH, NH2, CH3, and CD3; n is selected from 0, 1, 2 and 3; The "hetero" in the 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl represents 1, 2, 3, or 4 heteroatoms or heteroatom groups independently selected from -O-, -NH-, -S-, and -N-.

[0033] The present invention further provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0034] [ka]

[0035] however, Each R1 is independently H, halogen, or C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R a is optionally replaced by R2 is H, halogen, OH, NH2 and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R b is replaced by E1 is selected from CH2, NH and O; Structural Unit

[0036] [ka]

[0037] is selected from Ring A is a 5- to 6-membered heteroaryl, C 4-6 cycloalkenyl and 5- to 6-membered heterocycloalkenyl, wherein said 5- to 6-membered heteroaryl, C 4-6The cycloalkenyl and 5- to 6-membered heterocycloalkenyl each independently have one, two, or three R c is optionally replaced by T3, T4 and T5 are each independently selected from CR4 and N; T6, T7, and T8 are each independently selected from CR5 and N; R3 is H and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R e is replaced by R4 and R5 are each independently H, halogen, or C 1-3 Alkyl and C 1-3 alkoxy, wherein C 1-3 Alkyl and C 1-3 The alkoxy is independently one, two, or three R d is optionally replaced by Each R a , each R b , each R c , each R d and each R e are each independently selected from H, halogen, OH, NH2, CH3, and CD3; n is selected from 0, 1, 2 and 3; The "hetero" in the 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl represents 1, 2, 3, or 4 heteroatoms or heteroatom groups independently selected from -O-, -NH-, -S-, and -N-.

[0038] In some embodiments of the present invention, each of the R a are each independently selected from H, D, F, Cl, OH, NH2, CH3, and CD3, and all other variables are as defined herein.

[0039] In some embodiments of the present invention, each of the R bare each independently selected from H, D, F, Cl, OH, NH, CH, CHCH, OCH, OCHCH, NHCH, NHCHCH, N(CH), pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl, wherein said CH, CHCH, OCH, OCHCH, NHCH, NHCHCH, N(CH), pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl are each independently optionally substituted by 1, 2, or 3 R, and other variables are as defined herein.

[0040] In some embodiments of the present invention, each of the R b are each independently H, D, F, Cl, OH, NH2, CH3, CD3, CF3, -C(CH3)2OH, N(CH3)2,

[0041] [ka]

[0042] and the other variables are as defined in the present invention.

[0043] In some embodiments of the present invention, each of the R b are each independently selected from H, D, F, Cl, OH, NH2, CH3, and CD3, and all other variables are as defined herein.

[0044] In some embodiments of the present invention, each of the R c are each independently selected from H, D, F, Cl, OH, NH2, CH3, and CD3, and all other variables are as defined herein.

[0045] In some embodiments of the present invention, each of the R d are each independently selected from H, D, F, Cl, OH, NH2, CH3, and CD3, and all other variables are as defined herein.

[0046] In some embodiments of the present invention, each of the R eare each independently selected from H, D, F, Cl, OH, NH2, CH3, and CD3, and all other variables are as defined herein.

[0047] In some aspects of the invention, R1 is selected from H, F, Cl, Br, I, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3, and OCH(CH3)2, wherein CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3, and OCH(CH3)2 are each independently selected from 1, 2, or 3 R a and each R a and other variables as defined in the present invention.

[0048] In some aspects of the invention, R1 is selected from F and CH3, and other variables are as defined herein.

[0049] In some aspects of the invention, R2 is selected from H, F, Cl, Br, I, OH, NH2, CH3, CH2CH3, CH2CH2CH3, and CH(CH3)2, wherein CH3, CH2CH3, CH2CH2CH3, and CH(CH3)2 are each independently selected from 1, 2, or 3 R b and each R b and other variables as defined in the present invention.

[0050] In some aspects of the invention, R2 is selected from H and CF3, and other variables are as defined herein.

[0051] In some aspects of the invention, R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, cyclopropyl, cyclobutyl, oxetanyl, azetidinyl, -CH2-cyclopropyl, -CH2CH2-cyclopropyl, and -CH2-oxetanyl, wherein CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, cyclopropyl, cyclobutyl, oxetanyl, azetidinyl, -CH2-cyclopropyl, -CH2CH2-cyclopropyl, and -CH2-oxetanyl are each independently selected from 1, 2, or 3 R e and the other variables are as defined in the present invention.

[0052] In some embodiments of the invention, R3 is selected from the group consisting of H, CH3, CF3, CD3, CH2CH3, CH(CH3)2, cyclopropyl, cyclobutyl,

[0053] [ka]

[0054] and the other variables are as defined in the present invention.

[0055] In some aspects of the invention, R3 is selected from H, CH3, CF3, CD3, CH2CH3, and CH(CH3)2, with other variables as defined herein.

[0056] In some aspects of the invention, R3 above is selected from H and CH3, and other variables are as defined herein.

[0057] In some aspects of the invention, R4 is selected from H, F, Cl, Br, I, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3 and OCH(CH3)2, wherein CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3 and OCH(CH3)2 are each independently selected from 1, 2 or 3 Rd and each R d and other variables as defined in the present invention.

[0058] In some aspects of the invention, R4 is selected from H, F, Cl, Br, and CH3, and other variables are as defined herein.

[0059] In some aspects of the invention, R5 is selected from H, F, Cl, Br, I, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3 and OCH(CH3)2, wherein CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3 and OCH(CH3)2 are each independently selected from 1, 2 or 3 R d and each R d and other variables as defined in the present invention.

[0060] In some aspects of the invention, R5 is selected from H, F, Cl, Br, and CH3, and all other variables are as defined herein.

[0061] In some aspects of the invention, R7 is selected from H, F, Cl, Br, I, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3, and OCH(CH3)2, wherein CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH2CH2CH3, and OCH(CH3)2 are each independently selected from 1, 2, or 3 R d and each R d and other variables are as defined herein. In some aspects of the invention, R7 is selected from H, F, Cl, Br, CH3, and CD3, and other variables are as defined herein.

[0062] In some aspects of the invention, R6 is selected from ethynyl, propynyl, vinyl, and propenyl, wherein the ethynyl, propynyl, vinyl, and propenyl are each independently selected from one, two, or three R b and the other variables are as defined in the present invention.

[0063] In some embodiments of the present invention, R6 is

[0064] [ka]

[0065] and the other variables are as defined in the present invention.

[0066] In some embodiments of the present invention, in the above compound or a pharmaceutically acceptable salt thereof, R6 is

[0067] [ka]

[0068] and the other variables are as defined in the present invention.

[0069] In some embodiments of the present invention, in the above compound or a pharmaceutically acceptable salt thereof, R6 is

[0070] [ka]

[0071] and the other variables are as defined in the present invention.

[0072] In some embodiments of the present invention, R6 is

[0073] [ka]

[0074] and the other variables are as defined in the present invention.

[0075] In some embodiments of the present invention, R6 is

[0076] [ka]

[0077] , CH3 and CF3, with other variables as defined herein.

[0078] In some embodiments of the present invention, R6 is

[0079] [ka]

[0080] and the other variables are as defined in the present invention.

[0081] In some embodiments of the present invention, R6 is

[0082] [ka]

[0083] and the other variables are as defined in the present invention.

[0084] In some embodiments of the present invention, R6 is

[0085] [ka]

[0086] and the other variables are as defined in the present invention.

[0087] In some aspects of the present invention, R6 is selected from CH3 and CF3, and other variables are as defined herein.

[0088] In some embodiments of the present invention, the structural unit

[0089] [ka]

[0090] and the other variables are as defined in the present invention.

[0091] In some embodiments of the present invention, the structural unit

[0092] [ka]

[0093] and the other variables are as defined in the present invention.

[0094] In some embodiments of the present invention, the structural unit

[0095] [ka]

[0096] and the other variables are as defined in the present invention.

[0097] In some embodiments of the present invention, the structural unit

[0098] [ka]

[0099] and the other variables are as defined in the present invention.

[0100] In some embodiments of the present invention, the structural unit

[0101] [ka]

[0102] and the other variables are as defined in the present invention.

[0103] In some embodiments of the present invention, the structural unit

[0104] [ka]

[0105] and the other variables are as defined in the present invention.

[0106] In some embodiments of the present invention, the structural unit

[0107] [ka]

[0108] and the other variables are as defined in the present invention.

[0109] In some embodiments of the present invention, the ring A is selected from a 5- to 6-membered heteroaryl or a 5- to 6-membered heterocycloalkenyl, and the 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl each independently comprises one, two, or three R c and the other variables are as defined in the present invention.

[0110] In some aspects of the invention, the ring A is selected from a 5- to 6-membered heteroaryl, which optionally has one, two, or three R c and the other variables are as defined in the present invention.

[0111] In some aspects of the invention, the ring A is selected from a five-membered heteroaryl, said five-membered heteroaryl optionally having one, two or three R c and the other variables are as defined in the present invention.

[0112] In some aspects of the invention, Ring A is selected from imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl, and oxazolyl, wherein the imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl, and oxazolyl are each independently selected from one, two, or three R c and the other variables are as defined in the present invention.

[0113] In some aspects of the invention, ring A is selected from imidazolyl and pyrazolyl, wherein the imidazolyl and pyrazolyl each independently contain one, two or three R c and the other variables are as defined in the present invention.

[0114] In some aspects of the invention, ring A is selected from pyrazolyl, which optionally contains one, two or three R c and the other variables are as defined in the present invention.

[0115] In some aspects of the invention, ring A is selected from imidazolyl, which optionally contains one, two or three R c and the other variables are as defined in the present invention.

[0116] In some aspects of the invention, Ring A is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl, oxazolyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, oxacyclopentenyl, oxacyclohexenyl, azacyclopentenyl, and azacyclohexenyl, wherein said pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl, oxazolyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, oxacyclopentenyl, oxacyclohexenyl, azacyclopentenyl, and azacyclohexenyl are each independently selected from one, two, or three R c and each R c and other variables as defined in the present invention.

[0117] In some aspects of the invention, Ring A is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl, oxazolyl, triazolyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, oxacyclopentenyl, oxacyclohexenyl, azacyclopentenyl, and azacyclohexenyl, wherein said pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl, oxazolyl, triazolyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, oxacyclopentenyl, oxacyclohexenyl, azacyclopentenyl, and azacyclohexenyl are each independently selected from one, two, or three R c and each R c and other variables as defined in the present invention.

[0118] In some embodiments of the invention, T1 is CH, and the other variables are as defined herein.

[0119] In some forms of the invention, T2 is CH, and the other variables are as defined herein.

[0120] In some forms of the invention, T3 is CH, and the other variables are as defined herein.

[0121] In some aspects of the invention, T4 is CH, CF, CCl, CCH3, and N, with other variables as defined herein.

[0122] In some forms of the invention, T5 is CH, and the other variables are as defined herein.

[0123] In some embodiments of the invention, T6 is CH and N, with the other variables being as defined herein.

[0124] In some forms of the invention, T7 is CH and N, with the other variables being as defined herein.

[0125] In some forms of the invention, T8 is CH and N, with the other variables being as defined herein.

[0126] In some embodiments of the present invention, the structural unit

[0127] [ka]

[0128] teeth,

[0129] [ka]

[0130] and the other variables are as defined in the present invention.

[0131] In some embodiments of the present invention, the structural unit

[0132] [ka]

[0133] teeth,

[0134] [ka]

[0135] and the other variables are as defined in the present invention.

[0136] In some embodiments of the present invention, the structural unit

[0137] [ka]

[0138] teeth,

[0139] [ka]

[0140] and the other variables are as defined in the present invention.

[0141] In some embodiments of the present invention, the structural unit

[0142] [ka]

[0143] teeth,

[0144] [ka]

[0145] and the other variables are as defined in the present invention.

[0146] In some embodiments of the present invention, the structural unit

[0147] [ka]

[0148] teeth,

[0149] [ka]

[0150] and the other variables are as defined in the present invention.

[0151] In some embodiments of the present invention, the structural unit

[0152] [ka]

[0153] teeth,

[0154] [ka]

[0155] and the other variables are as defined in the present invention.

[0156] In some embodiments of the present invention, the structural unit

[0157] [ka]

[0158] teeth,

[0159] [ka]

[0160] and the other variables are as defined in the present invention.

[0161] In some embodiments of the present invention, the structural unit

[0162] [ka]

[0163] teeth,

[0164] [ka]

[0165] and the other variables are as defined in the present invention.

[0166] In some embodiments of the present invention, E1 is selected from O, with the other variables being as defined herein.

[0167] In some aspects of the invention, the compound or a pharmaceutically acceptable salt thereof is

[0168] [ka]

[0169] is selected from however, Structural Unit

[0170] [ka]

[0171] teeth,

[0172] [ka]

[0173] is selected from Ring A is selected from 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl, and the 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl each independently has one, two, or three R c is optionally replaced by T3, T4 and T5 are each independently selected from CR4; T8 is independently selected from CH and N; Each R1 is independently selected from H, halogen and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R a is replaced by R3 is H and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R e is replaced by R4 is H, halogen and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R d is replaced by R6 is C 2-4 Alkynyl and C 2-4 alkenyl, wherein C 2-4 Alkynyl and C 2-4 Alkenyl is each independently one, two or three R b is optionally replaced by Each R a , each R c , each R d and each R e are each independently selected from H, D, halogen, OH, NH2, CH3, and CD3; Each R b are independently H, D, halogen, C 1-3 Alkyl, C 1-3 alkylamino and 4- to 6-membered heterocycloalkyl, 1-3 Alkyl, C 1-3 Alkoxy and C 1-3alkylamino is optionally substituted by 1, 2 or 3 R, each independently; each R is independently selected from H, D, halogen, OH, NH2, CH3, CF3, and CD3; n is selected from 0 and 1, 2 and 3; m is selected from 0 and 1.

[0174] In some aspects of the invention, the compound or a pharmaceutically acceptable salt thereof is

[0175] [ka]

[0176] is selected from however, Each R1 is independently selected from H, halogen and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R a is replaced by R2 is H, halogen and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R b is replaced by E1 is selected from O; Structural Unit

[0177] [ka]

[0178] teeth,

[0179] [ka]

[0180] is selected from Ring A is selected from 5- to 6-membered heteroaryl and 5- to 6-membered heterocycloalkenyl, and the 5- to 6-membered heteroaryl and 5- to 6-membered heterocycloalkenyl each independently contain 1, 2, or 3 R c is optionally replaced by T3, T4 and T5 are each independently selected from CR4; T6 and T7 are each independently CH; T8 is selected from CH and N; R3 is H and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R e is replaced by R4 is H, halogen and C 1-3 alkyl, wherein C 1-3 The alkyl may optionally have one, two or three R d is replaced by Each R a , each R b , each R c , each R d and each R e are each independently selected from H, halogen, OH, NH2, CH3, and CD3; n is selected from 0, 1, 2 and 3; The "hetero" in the 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl represents 1, 2, 3, or 4 heteroatoms or heteroatom groups independently selected from -O-, -NH-, -S-, and -N-.

[0181] In some aspects of the present invention, in the compounds of formula (P) or formula (I) above or pharmaceutically acceptable salts thereof, ring A is selected from 5- to 6-membered heteroaryl, said 5- to 6-membered heteroaryl optionally containing one, two or three R c and the other variables are as defined in the present invention.

[0182] In some aspects of the invention, in the compounds of formula (P) or formula (I) above or pharmaceutically acceptable salts thereof, ring A is selected from a 5-membered heteroaryl, said 5-membered heteroaryl optionally containing 1, 2 or 3 R c and the other variables are as defined in the present invention.

[0183] In some aspects of the present invention, in the compounds of formula (P) or formula (I) above or pharmaceutically acceptable salts thereof, ring A is selected from imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl and oxazolyl, wherein said imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl and oxazolyl each independently contain one, two or three R c and the other variables are as defined in the present invention.

[0184] In some aspects of the present invention, in the compounds of formula (P) or formula (I) above or pharmaceutically acceptable salts thereof, ring A is selected from imidazolyl and pyrrolyl, wherein said imidazolyl and pyrrolyl each independently contain one, two or three R c and the other variables are as defined in the present invention.

[0185] In some aspects of the present invention, in the compounds of Formula (P) or Formula (I) above or pharmaceutically acceptable salts thereof, T4 and T5 are each independently selected from CH, and T8 is each independently selected from CH and N, and the other variables are as defined herein.

[0186] In some aspects of the present invention, in the compounds of formula (P) or formula (I) above or pharmaceutically acceptable salts thereof, each R is independently selected from F and CH, and other variables are as defined herein.

[0187] In some aspects of the present invention, in the compounds of formula (P) or formula (I) above or pharmaceutically acceptable salts thereof, each R is independently selected from CH, and other variables are as defined herein.

[0188] In some aspects of the present invention, in the compounds of formula (P) or formula (I) above or pharmaceutically acceptable salts thereof, R3 is selected from H, CH3, CF3, CD3, CH2CH3 and CH(CH3)2, and other variables are as defined herein.

[0189] In some aspects of the invention, in the compounds of formula (P) or formula (I) above or pharmaceutically acceptable salts thereof, n is selected from 0 and 1, and the other variables are as defined herein.

[0190] In some aspects of the present invention, in the compounds of formula (P) above or pharmaceutically acceptable salts thereof, R6 is selected from ethynyl and propynyl, and said ethynyl and propynyl are each independently selected from one, two or three R b and the other variables are as defined in the present invention.

[0191] In some aspects of the present invention, in the compounds of formula (P) above or pharmaceutically acceptable salts thereof, R6 is selected from ethynyl and propynyl, and said ethynyl and propynyl are each independently selected from one, two or three R b and the other variables are as defined in the present invention.

[0192] In some aspects of the present invention, in the compound of formula (P) above or a pharmaceutically acceptable salt thereof, R6 is

[0193] [ka]

[0194] and the other variables are as defined in the present invention.

[0195] In some aspects of the present invention, in the compound of formula (P) above or a pharmaceutically acceptable salt thereof, R6 is

[0196] [ka]

[0197] and the other variables are as defined in the present invention.

[0198] In some embodiments of the present invention, in the compound of formula (P) above or a pharmaceutically acceptable salt thereof, the structural unit

[0199] [ka]

[0200] and the other variables are as defined in the present invention.

[0201] In some aspects of the invention, in the compounds of formula (I) above or pharmaceutically acceptable salts thereof, E1 is O, and the other variables are as defined herein.

[0202] Some aspects of the invention are further formed by any combination of the above variables.

[0203] The present invention further provides compounds of the formula: and pharmaceutically acceptable salts thereof.

[0204] [ka]

[0205] [ka]

[0206] [ka]

[0207] [ka]

[0208]

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[0209]

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[0210]

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[0211]

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[0212]

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[0213]

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[0214]

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[0215]

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[0216]

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[0217]

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[0218]

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[0219] [ka]

[0220] [ka]

[0221] [ka]

[0222] [ka]

[0223] [ka]

[0224] [ka]

[0225] [ka]

[0226] In some aspects of the invention, the compound is selected from:

[0227] [ka]

[0228] [ka]

[0229]

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[0230]

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[0231]

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[0232]

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[0233]

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[0234]

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[0235]

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[0236]

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[0237]

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[0238]

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[0239]

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[0240]

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[0241]

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[0242]

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[0243]

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[0244]

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[0246]

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[0247]

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[0248]

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[0249]

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[0250]

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[0251]

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[0252]

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[0253]

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[0254]

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[0255]

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[0256]

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[0257]

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[0258]

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[0259]

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[0260]

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[0261] [ka]

[0262] [ka]

[0263] [ka]

[0264] [ka]

[0265] [ka]

[0266] [ka]

[0267] [ka]

[0268] [ka]

[0269] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralcel OD-3 150×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 1a: 2.214 min.

[0270] In one embodiment of the present invention, the SFC detection conditions are: chromatographic column: Chiralcel OD-3 150×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 1b: 2.715 min.

[0271] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralcel OJ-3 100×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 2a: 1.797 min.

[0272] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralcel OJ-3 100×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 2b: 3.929 min.

[0273] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: ChiralPak AD-3 150 × 4.6 mm ID, 3 μm; mobile phase: phase A: carbon dioxide, phase B: ethanol (0.05% diethylamine); gradient: B%: 5% to 40% (0 to 4.5 min), 40% (4.5 to 6.5 min), 5% (6.5 to 8 min); retention time of compound 3a: 5.283 min; SFC detection conditions were: chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: phase A: carbon dioxide, phase B: methanol (0.05% diethylamine); gradient: 5% to 40% (0 to 2 min), 40% (2 to 4 min), 5% (4 to 6 min); retention time of compound 3a: 5.471 min.

[0274] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: ChiralPak AD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 5% to 40% (0 to 4.5 min), 40% (4.5 to 6.5 min), 5% (6.5 to 8 min); retention time of compound 3b: 5.581 min; SFC detection conditions were: chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: methanol (0.05% diethylamine); gradient: 5% to 40% (0 to 2 min), 40% (2 to 4 min), 5% (4 to 6 min); retention time of compound 3b: 5.458 min.

[0275] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralpak IC-3 100 × 4.6 mm ID, 3 μm; mobile phase: Phase A: carbon dioxide, Phase B: ethanol (0.05% diethylamine); gradient: B%: 60%, retention time of compound 3c: 2.029 min; SFC detection conditions were: chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: Phase A: carbon dioxide, Phase B: methanol (0.05% diethylamine); gradient: 5% to 40% (0 to 2 min), 40% (2 to 4 min), 5% (4 to 6 min), retention time of compound 3c: 5.803 min.

[0276] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralpak IC-3 100 × 4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 60%, retention time of compound 3d: 2.714 min; SFC detection conditions were: chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: methanol (0.05% diethylamine); gradient: 5% to 40% (0 to 2 min), 40% (2 to 4 min), 5% (4 to 6 min), retention time of compound 3d: 5.772 min.

[0277] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralpak IC-3 100×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 60%, retention time of compound 4a: 2.298 min.

[0278] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralpak IC-3 100×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 60%, retention time of compound 4b: 3.083 min.

[0279] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: ChiralPak AD-3 50×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethanolamine); gradient: B%: 40%, retention time of compound 5a: 3.058 min.

[0280] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: ChiralPak AD-3 50×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethanolamine); gradient: B%: 40%, retention time of compound 5b: 3.649 min.

[0281] In one embodiment of the present invention, the SFC detection conditions were as follows: Chromatography column: Chiralpak IG-3 50 × 4.6 mm ID, 3 μm; Mobile phase: Phase A: carbon dioxide, Phase B: isopropanol (0.05% diethylamine); Gradient: B%: 5–40% (0–2 min), 40% (2–3.2 min), 5% (3.2–4 min); Retention time of compound 6a: 2.347 min.

[0282] In one embodiment of the present invention, the SFC detection conditions were as follows: Chromatography column: Chiralpak IG-3 50 × 4.6 mm ID, 3 μm; Mobile phase: Phase A: carbon dioxide, Phase B: isopropanol (0.05% diethylamine); Gradient: B%: 5-40% (0-2 min), 40% (2-3.2 min), 5% (3.2-4 min); Retention time of compound 6b: 2.650 min.

[0283] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralcel OJ-3 150×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: methanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 7a: 2.008 min.

[0284] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralcel OJ-3 150×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: methanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 7b: 2.872 min.

[0285] In one embodiment of the present invention, the SFC detection conditions are: chromatographic column: Chiralcel OD-3 150×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 8a: 1.152 min.

[0286] In one embodiment of the present invention, the SFC detection conditions are: chromatographic column: Chiralcel OD-3 150×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: ethanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 8b: 1.655 min.

[0287] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralpak AS-3 150 × 4.6 mm ID, 3 μm; mobile phase: Phase A: carbon dioxide, Phase B: ethanol (0.05% diethylamine); gradient: B%: 5% to 40% (5 to 4 min), 40% to 5% (4 to 4.2 min), 5% (4.2 to 6 min); retention time of compound 9a: 3.426 min.

[0288] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralpak AS-3 150 × 4.6 mm ID, 3 μm; mobile phase: Phase A: carbon dioxide, Phase B: ethanol (0.05% diethylamine); gradient: B%: 5% to 40% (5 to 4 min), 40% to 5% (4 to 4.2 min), 5% (4.2 to 6 min); retention time of compound 9b: 3.698 min.

[0289] In one embodiment of the present invention, the SFC detection conditions were: chromatographic column: Chiralpak AD-3 150×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: isopropanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 9c: 2.281 min.

[0290] In one embodiment of the present invention, the SFC detection conditions are: chromatographic column: Chiralpak AD-3 150×4.6 mm ID, 3 μm; mobile phase: A phase: carbon dioxide, B phase: isopropanol (0.05% diethylamine); gradient: B%: 40%, retention time of compound 9d: 2.599 min.

[0291] The present invention further provides use of the above compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a PRMT5-associated disease.

[0292] The present invention further provides the following synthesis methods.

[0293] Method 1 (Intermediate):

[0294] [ka]

[0295] wherein R2 is selected from H and CF3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0296] Method 2 (Intermediate):

[0297] [ka]

[0298] wherein R2 is selected from H and CF3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0299] Method 3:

[0300] [ka]

[0301] wherein R2 is selected from H and CF3; R4 is selected from H, F, Cl, Br and CH3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0302] Method 4:

[0303] [ka]

[0304] wherein R2 is selected from H and CF3; R4 is selected from H, F, Cl, Br and CH3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0305] Method 5:

[0306] [ka]

[0307] wherein R2 is selected from H and CF3; R4 is selected from H, F, Cl, Br and CH3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0308] Method 6:

[0309] [ka]

[0310] wherein R2 is selected from H and CF3; R4 is selected from H, F, Cl, Br and CH3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0311] Method 7 (Intermediate):

[0312] [ka]

[0313] wherein R1 is selected from H and CH3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0314] Method 8:

[0315] [ka]

[0316] however, R1 is selected from H and CH3; R6 is CF3 and

[0317] [ka]

[0318] is selected from R4 is selected from H, F, Cl, Br and CH3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0319] Method 9:

[0320] [ka]

[0321] R1 is selected from H and CH3; R6 is CF3 and

[0322] [ka]

[0323] is selected from R4 is selected from H, F, Cl, Br and CH3; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0324] Method 10:

[0325] [ka]

[0326] wherein R1 is selected from H and CH3; R4 is selected from H, F, Cl, Br and CH3; R7 is selected from CH3, CD3 and halogen; Carbon atoms marked with an "*" are chiral carbon atoms and exist in either (R) or (S) single enantiomeric form or in an enantiomerically enriched form.

[0327] The present invention further provides the following test method.

[0328] Test method 1: PRMT5 enzyme inhibitory activity test Experimental objective: To test the inhibitory effects of small molecule compounds on PRMT5·MTA.

[0329] Experimental materials: PRMT5 / MEP50, peptide H4(1-21), LANCE® Ultra Europium anti-methyl histone H4 arginine 3 (H4R3me) antibody, Ulight streptavidin, LANCE detection buffer, SAM (adenosylmethionine), pigskin collagen, MTA (methylthioadenosine).

[0330] Experimental Method: (1) Preparation of buffer solution: Take 10 ml as an example. 10 mM MTA: 10 mg of MTA was added to 3296 μl of DMSO, dissolved, and then aliquoted and stored at -80°C. It can be used immediately on the day of the experiment. The preparation scheme is shown in Tables 1 and 2.

[0331] [Table 1]

[0332] [Table 2]

[0333] (2) Preparation of compounds Compounds were dissolved in DMSO to obtain a 10 mM compound mother solution. A gradient dilution was performed on the compound dilution plate to obtain four compound wells, with sequential concentrations of 1 mM, 37.037 μM, 1.3717 μM, and 0.0508 μM. These four concentrations of compound were transferred to the compound transfer plate in a transfer volume of 8 μL per concentration. DMSO was added to empty wells of the compound transfer plate for later use. The liquid was transferred to the experimental plate using the serial dilution function of the Echo 550 microfluidic device. Once the transfer was complete, the experimental plate was obtained.

[0334] (3) Reaction A mixed solution of the enzyme solution and the substrate was prepared using a buffer solution, and the concentration of PRMT5 was 7.6 nM, the concentration of peptide H4(1-21) was 0.32 μM, and the concentration of SAM was 2.6 μM.

[0335] Using an electronic multichannel pipette, 5 μL of PRMT5 solution was added per well to the compound and negative control wells of the experimental plate, and the same volume of buffer was added to the positive control well. The plates were centrifuged at 1000 rpm for 1 minute, then placed in a constant-temperature incubator and incubated at 25°C for 30 minutes. Next, using the same method, the substrate mixture solution was added to the positive control, negative control, and compound wells of the experimental plate, centrifuged, and incubated at 25°C for 90 minutes.

[0336] (4) Testing and calculation of results Principle: In this experiment, PE's time-resolved fluorescence resonance energy transfer technology (LANCE® Ultra) was used for detection. During the reaction, PRMT methylated the substrate polypeptide H4(1-21), and then two antibodies were added. Here, the LANCE® Ultra europium anti-methyl histone H4 arginine 3 (H4R3me) antibody acts as an energy donor and can specifically bind to the methylation site of polypeptide H4(1-21), while Ulight acts as an energy acceptor and can specifically bind to the biotin tag carried by polypeptide H4(1-21). When a laser of a specific wavelength (340 nm in this experiment) is used for excitation, the energy donor emits light at a wavelength of 615 nm. At the same time, if the spatial distance between the energy donor and the energy acceptor is sufficiently close (i.e., when two antibodies are simultaneously linked to the polypeptide H4(1-21)), energy transfer occurs between the energy donor and the energy acceptor, causing the energy acceptor to emit light at a wavelength of 665 nm. A plate reader was used to detect the two emitted lights, and the ratio of the 665 nm and 615 nm signals was calculated. The relevant parameters of the test sample were then calculated by plotting and calculating.

[0337] The antibody mixture solution was prepared using LANCE buffer. The antibody concentration of LANCE® Ultra Europium anti-methyl histone H4 arginine 3 (H4R3me) was 4 nM, and the Ulight concentration was 53.3 nM. Using an electronic multichannel pipette, the detection solution was added to the positive control, negative control, and compound wells of the experimental plate at a volume of 10 μL per well. The plate was centrifuged, incubated at room temperature for 1 hour, and read using an Envision 2104 plate reader. Compound inhibition rates were calculated using interpolation, and compound inhibition curves were generated using a four-parameter logarithm equation and XLfit software, which allowed for the determination of minimum inhibition rate, maximum inhibition rate, and IC. 50 The relevant parameters were calculated as follows:

[0338]

number

[0339] [Technical Effects] The compounds of the present invention have good binding effects with the PRMT5 MTA complex, exhibit significant inhibitory activity against the PRMT5 MTA enzyme, and exhibit significant anti-proliferative activity against LU99 cells. They also exhibit excellent inhibitory activity against MTAP-deficient HCT116 tumor cells and weak inhibitory activity against wild-type HCT116 tumor cells, demonstrating excellent selectivity. In vivo PK results in mice indicate that the compounds of the present invention have a long half-life, low clearance, high drug exposure, and excellent oral bioavailability, demonstrating excellent in vivo pharmacokinetic properties. The compounds of the present invention exhibit moderate metabolism in human liver microsome tests and good metabolic stability. The compounds of the present invention have a moderate ratio of plasma free drug concentrations in different species, good in vitro permeability through MDCKII-MDR1 monolayer cells, weak inhibition of hERG potassium current, low risk of cardiotoxicity, and good drug formation. Furthermore, the compounds of the present invention exhibit excellent in vivo antitumor effects and good weight maintenance in mice after administration.

[0340] [Definitions and Explanations] Unless otherwise stated, the following terms and phrases used herein have the following meanings. Unless otherwise defined, a particular term or phrase should be understood to have its ordinary definition, rather than being indefinite or unclear. When a trade name appears in this specification, it refers to the corresponding product or its active ingredient.

[0341] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals, without appreciable toxicity, irritation, allergic response or other problem or complication, and consistent with a reasonable benefit / risk ratio.

[0342] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is prepared with a relatively non-toxic acid or base, when the compound has certain substituents found in the present invention. When the compounds of the present invention contain a relatively acidic functional group, a base addition salt can be obtained by contacting these compounds with a sufficient amount of base in a solution of the compound alone or in a suitable inert solvent. When the compounds of the present invention contain a relatively basic functional group, an acid addition salt can be obtained by contacting these compounds with a sufficient amount of acid in a solution of the compound alone or in a suitable inert solvent.

[0343] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing an acidic or basic group in a conventional manner. Typically, such salts are prepared by reacting the compound in its free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both.

[0344] The compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as mixtures enriched in enantiomers or non-enantiomers, and all such mixtures are within the scope of the present invention. Other asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers and mixtures thereof are within the scope of the present invention.

[0345] Unless otherwise specified, the terms "enantiomers" or "optical isomers" are stereoisomers that are mirror images of each other.

[0346] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" refer to the inability to freely rotate about double bonds or single bonds of ring carbon atoms.

[0347] Unless otherwise specified, the term "diastereomer" refers to stereoisomers whose molecules have two or more centers of chirality and whose molecules are not mirror-images of each other.

[0348] Unless otherwise stated, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic.

[0349] [ka]

[0350] Unless otherwise stated, carbon atoms marked with an "*" are chiral carbon atoms and exist in the form of a single enantiomer, either (R) or (S), or enriched in one enantiomer.

[0351] [ka]

[0352] Unless otherwise explained, the terms "enriched in one isomer," "isomer-enriched," "enriched in one enantiomer," or "enantiomer-enriched" mean that the amount of one isomer or enantiomer is less than 100% and that the amount of that isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0353] Unless otherwise stated, the terms "isomeric excess" or "enantiomeric excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is present in an amount of 90% and the other isomer or enantiomer is present in an amount of 10%, the isomeric or enantiomeric excess (ee) is 80%.

[0354] Optically active (R)- and (S)-isomers and D- and L-isomers can be prepared using asymmetric synthesis, chiral reagents, or other conventional techniques. Single enantiomers of certain compounds of the invention can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary is cleaved to provide the desired isolated enantiomer. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxyl) functional group, diastereomeric salts can be formed with an appropriate optically active acid or base, followed by separation of the diastereomers by conventional methods known in the art and subsequent recovery to provide the isolated enantiomers. Separation of enantiomers and diastereomers is also typically accomplished by chromatographic methods using chiral stationary phases and optionally chemical derivatization (e.g., carbamate formation from an amine).

[0355] Unless otherwise defined, when a compound has a double bond structure, such as a carbon-carbon double bond, a carbon-nitrogen double bond, and a nitrogen-nitrogen double bond, and each atom in the double bond has two different substituents bonded to it (in a double bond containing a nitrogen atom, a pair of lone electron pairs on the nitrogen atom is considered to be one substituent bonded to it), the atoms on the double bond of the compound and their substituents are

[0356] [ka]

[0357] When the compound is represented by the formula:

[0358] The compounds of the present invention may contain unnatural atomic isotopes at one or more atoms constituting the compounds. For example, tritium ( 3 H), iodine-125( 125 I) or C-14( 14Compounds can be labeled with radioactive isotopes such as C). For example, deuterium can be substituted for hydrogen to form deuterated drugs, where the bond formed between deuterium and carbon is stronger than the bond formed between normal hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs offer the advantages of reduced toxic side effects, increased drug stability, improved therapeutic efficacy, and a longer biological half-life. Conversion of the isotopic composition of the compounds of the present invention, whether radioactive or not, is within the scope of the present invention.

[0359] The term "substituted" refers to the replacement of any one or more hydrogen atoms at a particular atom with a substituent, which may include variations of deuterium and hydrogen, provided that the particular valence state is correct and the compound is stable after substitution. When the substituent is a keto group (i.e., =0), this means that two hydrogen atoms have been replaced.

[0360] The term "optionally" or "optionally" means that the following item or circumstance may occur, but does not necessarily occur, and that the description includes cases where the described item or circumstance does not occur, even if the item or circumstance occurs.

[0361] The term "optionally substituted" means that the group may be substituted or unsubstituted, and unless otherwise defined, the type and number of substituents are optional as long as they are chemically stable.

[0362] When any variable (e.g., R) occurs more than one time in a compound composition or structure, its definition is independent at each occurrence. So, for example, if a group is substituted with 0 to 2 R, then that group is optionally substituted with up to 2 R, and each occurrence of R is independently optional. Also, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0363] When the number of linking groups is 0, for example, -(CRR)0- means that the linking group is a single bond.

[0364] If one of the variables is a single bond, the two groups connected by it are directly linked; for example, if L in ALZ represents a single bond, the structure actually becomes AZ.

[0365] When there is no substituent, it means that the substituent does not exist. For example, when there is no X in AX, it means that the structure is actually A.

[0366] When the atom through which a substituent is substituted for a given substituent is not specified, such a substituent may be bonded through any atom thereof, for example, pyridinyl as a substituent may be bonded to the substituent through any carbon atom of the pyridine ring. When the given linking group does not specify another direction of attachment, the direction of attachment is arbitrary, for example,

[0367] [ka]

[0368] In the formula, the linking group L is -MW-, and in this case, -MW- constitutes ring A and ring B in the same direction as the reading order from left to right.

[0369] [ka]

[0370] You can also construct rings A and B in the reverse order of reading from left to right.

[0371] [ka]

[0372] Combinations of the above linking groups, substituents and / or variables thereof are permissible only if such combinations result in stable compounds.

[0373] Unless otherwise specified, when a group has one or more bondable sites, any one or more sites of the group can be bonded to other groups by chemical bonds. If the bonding mode of the chemical bond is delocalized and there is an H atom at the bondable site, when the chemical bond is bonded, the number of H atoms at the site will be reduced to the corresponding valence of the group according to the number of bonded chemical bonds. The chemical bond that bonds the site to other groups is:

[0374] [ka]

[0375] For example, the straight solid bond in -OCH3 represents a bond to another group via the oxygen atom in the group,

[0376] [ka]

[0377] The straight-dashed bond represents a bond between the nitrogen atoms in the group and another group,

[0378] [ka]

[0379] The wavy lines indicate that the phenyl is bonded to another group via the 1st and 2nd carbon atoms of the phenyl.

[0380] [ka]

[0381] indicates that any available bonding site of the piperidinyl can be bonded to another group via one chemical bond, and at least

[0382] [ka]

[0383] Even if the H atom is drawn as -N-,

[0384] [ka]

[0385] for

[0386] [ka]

[0387] When one chemical bond is bonded, the H at that site decreases by one to form the corresponding monovalent piperidinyl.

[0388] Unless otherwise defined, the terms "halogen" or "halo," by themselves or as part of another substituent, mean a fluorine, chlorine, bromine, or iodine atom.

[0389] Unless otherwise defined, the term "C 1-3 The term "alkyl," by itself or in combination with other terms, refers to a saturated hydrocarbon group of 1 to 3 carbon atoms, either straight or branched. 1-3 C for alkyl 1-2 and C 2-3 alkyl, which may be monovalent (e.g., methyl), divalent (e.g., methylene), and polyvalent (e.g., methine). 1-3 Examples of alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0390] Unless otherwise defined, "C 2-4 The term "alkenyl," by itself or in combination with other terms, refers to a saturated hydrocarbon group of 2 to 4 carbon atoms, each of which is linear or branched, and contains at least one carbon-carbon double bond, and the carbon-carbon double bond may be located at any position within the group.1-4 Alkenyl has C 2-3 , C4, C3 and C2 alkenyl, etc., 2-4 Alkenyl may be monovalent, divalent or polyvalent. 2-4 Illustrative examples of alkenyl include, but are not limited to, vinyl, propenyl, butenyl, butadienyl, and the like.

[0391] Unless otherwise defined, "C 2-4 The term "alkynyl," by itself or in combination with other terms, refers to a saturated hydrocarbon group of 2 to 4 carbon atoms, either linear or branched, containing at least one carbon-carbon triple bond, and the carbon-carbon triple bond may be located at any position within the group. 2-4 Alkynyl has C 2-3 , C4, C3 and C2 alkynyl, etc. It may be monovalent, divalent or polyvalent. 2-4 Examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, and the like.

[0392] Unless otherwise defined, the term "C 1-3 "Alkoxy," by itself or in combination with other terms, refers to an alkyl group containing 1 to 3 carbon atoms, each linked to the remainder of the molecule via an oxygen atom. 1-3 Alkoxy is C 1-2 , C 2-3 , C3 and C2 alkoxy, etc. It may be monovalent, divalent or polyvalent. C 1-3 Illustrative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy or isopropoxy), and the like.

[0393] Unless otherwise defined, the term "C 1-3 "Alkylamino," by itself or in combination with other terms, refers to an alkyl group containing 1 to 3 carbon atoms, each linked to the remainder of the molecule via a nitrogen atom. 1-3 Alkylamino has C 1-2 , C3 and C2 alkylamino, etc. It may be monovalent, divalent or polyvalent.1-3 Illustrative examples of alkylamino include, but are not limited to, -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH3)CH2CH3, -NHCH2CH2CH3, -NHCH2(CH3)2, and the like.

[0394] Unless otherwise defined, "C 3-6 "Cycloalkyl" by itself or in combination with other terms refers to a saturated cyclic hydrocarbon group of 3 to 6 carbon atoms, each of which is a monocyclic ring system. 3-6 Cycloalkyl is C 3-5 , C 4-5 and C 5-6 cycloalkyl, etc., which may be monovalent, divalent or polyvalent. C 3-6 Illustrative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0395] Unless otherwise defined, "C 4-6 The term "cycloalkenyl," by itself or in combination with other terms, refers to a partially unsaturated monocyclic hydrocarbon group composed of 4 to 6 carbon atoms, each containing at least one carbon-carbon double bond. 4-6 Cycloalkenyl is C 4-5 or C 5-6 cycloalkenyl, etc., which may be monovalent, divalent or polyvalent. C 4-6 Illustrative examples of cycloalkenyl include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.

[0396] Unless otherwise defined, the term "4-6 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group composed of 4 to 6 ring atoms, of which 1, 2, 3, and 4 ring atoms are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms, wherein the carbon atoms are optionally oxidized (i.e., C(O)), the nitrogen atoms are optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O)). p, p is 1 or 2). It is a monocyclic ring system. Also, for the "4- to 6-membered heterocycloalkyl", a heteroatom can occupy the position connecting the heterocycloalkyl to the remainder of the molecule. The 4- to 6-membered heterocyclyl includes 5- to 6-membered, 4-membered, 5-membered, and 6-membered heterocyclyl. It may be monovalent, divalent, or polyvalent. Illustrative examples of 4- to 6-membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl, and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, or hexahydropyridazinyl, etc.

[0397] Unless otherwise defined, "5- to 6-membered heterocycloalkenyl," by itself or in combination with other terms, refers to a partially unsaturated monocyclic group composed of 5 to 6 ring atoms, each containing at least one carbon-carbon double bond, of which 1, 2, 3, and 4 ring atoms are independently selected from O, S, and N heteroatoms, and the remainder are carbon atoms, wherein the carbon atoms are optionally oxidized (i.e., C(O)), the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O)). p , p is 1 or 2.) Furthermore, with respect to "5- to 6-membered heterocycloalkenyl," a heteroatom can occupy the position at which the heterocycloalkenyl is connected to the rest of the molecule. The 5- to 6-membered heterocycloalkenyl includes 5- or 6-membered heterocycloalkenyl. It may be monovalent, divalent, or polyvalent. Illustrative examples of 5- to 6-membered heterocycloalkenyl include:

[0398] [ka]

[0399] Including, but not limited to:

[0400] Unless otherwise specified, the terms "5- to 6-membered heteroaryl ring" and "5- to 6-membered heteroaryl" as used herein may be used interchangeably, and the term "5- to 6-membered heteroaryl" refers to a monocyclic group having a conjugated π-electron system composed of 5 to 6 ring atoms, of which 1, 2, 3, and 4 ring atoms are independently selected from O, S, and N heteroatoms, and the remainder are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., NO and S(O)). p , p is 1 or 2). The 5- to 6-membered heteroaryl can be attached to the remainder of the molecule via a heteroatom or a carbon atom. It may be monovalent, divalent, or polyvalent. The 5- to 6-membered heteroaryl includes a 5- or 6-membered heteroaryl. Illustrative examples of the 5- to 6-membered heteroaryl include pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl, and 5-oxazolyl, etc.), triazolyl (including 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, and 4H-1,2,4-triazolyl, etc.), and the like. thiazolyl (including 2-thiazolyl, 4-thiazolyl, and 5-thiazolyl), tetrazolyl, isoxazolyl (including 3-isoxazolyl, 4-isoxazolyl, and 5-isoxazolyl), thiazolyl (including 2-thiazolyl, 4-thiazolyl, and 5-thiazolyl, etc.), furanyl (including 2-furanyl and 3-furanyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl, and 4-pyridyl, etc.), pyrazinyl, or pyrimidinyl (including 2-pyrimidinyl and 4-pyrimidinyl, etc.).

[0401] The compounds of the present invention can be prepared by various synthetic methods familiar to those skilled in the art, including the specific embodiments listed below, embodiments in combination with other chemical synthetic methods, and equivalent alternative methods familiar to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0402] The structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. When the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional technical means known to those skilled in the art. For example, single crystal X-ray diffraction (SXRD), cultured single crystals are collected by a Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning, and after collecting the relevant data, the absolute configuration can be confirmed by direct crystal structure analysis (Shelxs97).

[0403] All solvents used in this invention are commercially available. Compounds are named according to conventional naming principles in the art or ChemDraw® software, and commercially available compounds use the manufacturer's catalog name. [Brief explanation of the drawings]

[0404] [Figure 1] Schematic diagram of the binding between compound A and the PRMT5·MTA complex. [Figure 2] Schematic diagram of the binding between compound B and the PRMT5·MTA complex. [Figure 3] Schematic diagram of the binding between compound C and the PRMT5·MTA complex. [Figure 4] Schematic diagram of the binding between compound D and the PRMT5·MTA complex. [Figure 5] Schematic diagram of the binding between compound E and the PRMT5·MTA complex. [Figure 6] Schematic diagram of the binding between compound F and the PRMT5·MTA complex. [Figure 7] Schematic diagram of the binding between compound G and the PRMT5·MTA complex. [Figure 8]Schematic diagram of the binding between compound H and the PRMT5·MTA complex. [Figure 9] 1 shows a body weight change curve of mice in a LU99 cell subcutaneous xenograft tumor model (compound 1a). [Figure 10] 1 shows the body weight change curves of mice in a LU99 cell subcutaneous xenograft tumor model (compounds 3d and 4b). [Figure 11] 1 shows the mean tumor volume at different time points in the LU99 cell subcutaneous xenograft tumor model (Compound 1a). [Figure 12] Average tumor volume at different time points in LU99 cell subcutaneous xenograft tumor model (compounds 3d and 4b). DETAILED DESCRIPTION OF THE INVENTION

[0405] The present invention will be specifically described below by way of examples, but is not intended to be an adverse limitation of the present invention. The present invention has been described in detail herein, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0406] Calculation example 1

[0407] [ka]

[0408] The molecular docking process is performed using Maestro (Schrodinger version 2022-1) Induced Fit Docking [1-2] The docking was performed using default settings. The crystal structure of PRMT5 (PDB ID: 7S1S) in the PDB database was used as a docking template. Maestro was used to generate the protein. [2] Hydrogen atoms were added using the Protein Preparation Wizard module of the LigPrep library, and energy minimization was performed using the OPLS4 force field.[3] The three-dimensional molecular structure was generated using the OPLS4 force field, and energy minimization was performed using the OPLS4 force field. A box was automatically generated centered on Trp579 of the 7S1S, and the example compounds were placed in the molecular docking process. The interaction between the protein and the example compounds was analyzed, and reasonable docking conformations were selected and saved according to the calculated IFD scores and binding schemes. The binding schemes for compounds A to H are shown in Figures 1 to 8.

[0409] [1]Glide, Schrodinger, LLC, New York, NY, 2021. [2] Prime, Schrodinger, LLC, New York, NY, 2021. [3] Maestro, Schrodinger, LLC, New York, NY, 2022. [4] LigPrep, Schrodinger, LLC, New York, NY, 2022.

[0410] Conclusion: The compounds of this invention have good binding effect with the PRMT5·MTA complex.

[0411] Reference Example 1 Synthesis of Intermediate A

[0412] [ka]

[0413] Step 1 N-Bromosuccinimide (21.04 g, 118.24 mmol) was slowly added to a solution of compound A-1 (20 g, 118.24 mmol) in acetonitrile (200 mL) at 0°C, and the mixture was stirred at room temperature and 25°C for 16 hours. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated aqueous sodium chloride (50 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to column chromatography (silica gel, petroleum ether:ethyl acetate = 3:1) to obtain compound A-2. MS: m / z 248.0 / 250.0 [M+H] + .

[0414] Step 2 To a solution of compound A-2 (5.00 g, 20.16 mmol) and bis(pinacolato)diboron (6.14 g, 24.19 mmol) in dioxane (50 mL) at 0 °C, potassium acetate (5.93 g, 60.47 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (737.47 mg, 1.01 mmol) were added, and the mixture was stirred at 100 °C for 20 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to column chromatography (silica gel, petroleum ether:ethyl acetate=20:1) to obtain compound A-3. MS: m / z 296.2 [M+H] + .

[0415] Step 3 To a solution of compound A-4 (1 g, 8.19 mmol) in water (15 mL) was slowly added a solution of hydrogen bromide (24.84 g, 122.82 mmol, 40% aqueous solution) and sodium nitrite (847.47 mg, 12.28 mmol) in water (15 mL) at 0 to 5°C. Next, cuprous bromide (1.88 g, 13.10 mmol) was added to the mixture. After the addition was complete, the reaction mixture was continuously stirred at 0 to 5°C for 3 hours, allowed to warm to 25°C, and continuously stirred for 18 hours. The reaction mixture was cooled to 0-5°C, ice water (60 mL) was added, and the mixture was stirred at 0-5°C for 1 hour. The reaction mixture was filtered, the cake was washed with ice water (50 mL), and the filtrate was extracted with dichloromethane (50 mL x 3). The organic phases were combined and washed with saturated aqueous sodium chloride (30 mL x 3). The organic phase and cake were combined and concentrated under reduced pressure to give compound A-5, which was used directly in the next step. MS: m / z 186.0 / 187.9 [M+H] + .

[0416] Step 4 To a solution of compound A-5 (1 g, 5.38 mmol) and compound A-3 (1.59 g, 5.38 mmol) in dioxane (16 mL) and water (4 mL) was added potassium phosphate (667.55 mg, 4.83 mmol) and bis[di-tert-butyl-(4-dimethylaminophenyl)phosphine]dichloropalladium(II) (761.33 mg, 1.08 mmol), and the mixture was stirred in an oil bath at 90 °C for 6 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, dichloromethane:methanol = 10:1) to obtain compound A-6. MS: m / z 275.0 [M+H] + .

[0417] Step 5 To a solution of compound A-6 (510 mg, 1.86 mmol) in water (5 mL), methanol (5 mL), and tetrahydrofuran (5 mL) was added lithium hydroxide monohydrate (156.06 mg, 3.72 mmol), and the mixture was stirred at room temperature and 25°C for 19 hours. The pH of the reaction mixture was adjusted to 5-6 with 2 mol / L dilute hydrochloric acid, and the mixture was stirred for 5 minutes, filtered, and the cake was washed with water (10 mL x 3). The cake was dried under reduced pressure to obtain compound A. MS: m / z 261.0 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ = 8.76-8.69 (m, 1H), 8.27 (s, 1H), 7.50 (br s, 2H), 7.28-7.23 (m, 1H), 4.38 (s, 3H).

[0418] Example 1

[0419] [ka]

[0420] Step 1 Compound 1-1 (4.5 g, 22.39 mmol) was dissolved in dimethyl sulfoxide (60 mL), trimethylsulfoxonium iodide (5.91 g, 26.86 mmol) was added, and the mixture was cooled to 0 °C. Potassium tert-butoxide (3.01 g, 26.86 mmol) was added in batches. The reaction mixture was slowly warmed to 25 °C under nitrogen gas protection and stirred for 2 h. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (ethyl acetate / petroleum ether = 0-25%) to give compound 1-2. 1H NMR (400 MHz, CDCl3) δ ppm 7.28 (d, J=5.77 Hz, 1H), 7.09 (dd, J=7.91, 1.63 Hz, 1H), 7.06 (d, J=1.25 Hz, 1H), 5.33 (dd, J=6.53, 2.51 Hz, 1H), 4.55-4.61 (m, 1H), 4.44-4.49 (m, 1H).

[0421] Step 2 Compound 1-2 (1.7 g, 7.91 mmol) was dissolved in toluene (27 mL) and, under nitrogen gas protection, diphenylphosphoryl azide (2.39 g, 8.70 mmol) was slowly added dropwise at 0 °C. Next, a solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (1.32 g, 8.70 mmol) in toluene (1 mL) was slowly added dropwise. The reaction mixture was slowly warmed to 25 °C under nitrogen gas protection and stirred for 3.5 h. The reaction mixture was diluted with ethyl acetate (80 mL). The organic phase was washed with saturated brine (40 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column (ethyl acetate / petroleum ether = 0-2%) to give compound 1-3. 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.46 (d, J=8.03 Hz, 1H), 7.22 (d, J=1.51 Hz, 1H), 7.18 (dd, J=7.91, 1.63 Hz, 1H), 5.34 (dd, J=7.03, 2.26 Hz, 1H), 4.57-4.66 (m, 1H), 4.46-4.55 (m, 1H).

[0422] Step 3 Compound 1-3 (1.03 g, 4.29 mmol) was dissolved in tetrahydrofuran (26 mL), triphenylphosphine (1.69 g, 6.44 mmol) was added, and the reaction mixture was stirred at 25 °C for 1 h under nitrogen gas protection. A solution of potassium hydroxide (601.82 mg, 10.73 mmol) in water (6.5 mL) was added dropwise, and the reaction mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with ethyl acetate (60 mL), washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 1-4. The crude product was purified using a flash silica gel column (methanol / ethyl acetate = 0 to 3%) to give crude product 1-4, which was used directly in the next step.

[0423] Step 4 The crude product 1-4 (918 mg, 4.29 mmol) was dissolved in dichloromethane (15 mL) and cooled to 0 °C. Under nitrogen gas protection, triethylamine (867.91 mg, 8.58 mmol) was added, and a solution of di-tert-butyl dicarbonate (935.96 mg, 4.29 mmol) in dichloromethane (5 mL) was added dropwise. The reaction mixture was warmed to 25 °C and stirred for 12 h. The reaction mixture was diluted with dichloromethane (30 mL). The organic phase was washed with water (20 mL × 2), then with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. This was purified by flash silica gel column chromatography (ethyl acetate / petroleum ether = 0-15%) to give compound 1-5. 1 H NMR (400 MHz, CDCl3) δ ppm 7.20 (d, J=8.03 Hz, 1H), 7.06 (dd, J=7.91, 1.63 Hz, 1H), 7.01 (d, J=1.51 Hz, 1H), 5.31 (br s, 1H), 4.84 (br s, 1H), 4.65-4.73 (m, 1H), 4.36 (dd, J=10.04, 4.27 Hz, 1H), 1.47 (s, 9H).

[0424] Step 5 Compound 1-5 (500 mg, 1.59 mmol) was dissolved in tetrahydrofuran (10 mL) and cooled to 0 °C. Sodium hydride (127.31 mg, 3.18 mmol, 60% purity) was added under nitrogen gas protection and stirred at 0 °C for 2 h. Iodomethane (451.78 mg, 3.18 mmol) was added dropwise, and the reaction mixture was slowly warmed to 25 °C and stirred under nitrogen gas protection for 12 h. The reaction mixture was quenched by the dropwise addition of water (10 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column (ethyl acetate / petroleum ether = 0-10%) to give compound 1-6. 1 H NMR (400 MHz, CDCl3) δ ppm 7.04-7.14 (m, 2H), 7.01 (s, 1H), 5.56-6.16 (m, 1H), 4.63 (br t, J=9.54 Hz, 1H), 4.39 (br d, J=7.28 Hz, 1H), 2.55 (br s, 3H), 1.50 (s, 9H).

[0425] Step 6 Compound 1-6 (500 mg, 1.52 mmol) and trimethylsilylacetylene (1.50 g, 15.23 mmol) were dissolved in triethylamine (5 mL). Dichlorobis(triphenylphosphine)palladium(II) (213.86 mg, 304.69 μmol) and copper(I) iodide (58.03 mg, 304.69 μmol) were added, and the reaction mixture was stirred at 75 °C for 3 h under nitrogen gas protection. The reaction mixture was diluted with ethyl acetate (20 mL), filtered, and the cake was washed with ethyl acetate (10 mL × 2). The filtrate was collected, washed with water (10 mL × 2), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (ethyl acetate / petroleum ether = 0-10%) to give compound 1-7. 1H NMR (400 MHz, CDCl3) δ ppm 7.16 (br d, J=7.03 Hz, 1H), 7.06 (dd, J=7.65, 1.13 Hz, 1H), 6.93 (s, 1H), 5.70-6.17 (m, 1H), 4.62 (br t, J=9.29 Hz, 1H), 4.38 (BR S, 1H), 2.51 (BR S, 3H), 1.50 (S, 9H), 0.25 (S, 9H).

[0426] Step 7 Compound 1-7 (100 mg, 289.43 μmol) was dissolved in dichloromethane (1 mL), trifluoroacetic acid (383.75 mg, 3.37 mmol) was added dropwise, and the reaction mixture was stirred at 25° C. for 1 hour. The reaction mixture was concentrated under reduced pressure, diluted with dichloromethane (2 mL), and concentrated under reduced pressure to give the crude trifluoroacetate salt of 1-8, which was used directly in the next step.

[0427] Step 8 Crude product 1-8 (70 mg, trifluoroacetate salt crude product) was dissolved in N,N-dimethylacetamide (1 mL), and N,N-diisopropylethylamine (73.74 mg, 570.52 μmol) was added dropwise. Intermediate A (74.23 mg, 285.26 μmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (130.16 mg, 342.31 μmol) were then added, and the reaction mixture was stirred at 25°C for 12 hours. Water (5 mL) was added dropwise to the reaction mixture, followed by extraction with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude product 1-9. MS: m / z 488.1 [M+H] + .

[0428] Step 9 The crude product 1-9 (130 mg, 266.61 μmol) was dissolved in methanol (2 mL), potassium carbonate (73.69 mg, 533.22 μmol) was added, and the reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was diluted with N,N-dimethylformamide (1 mL), filtered, and the filtrate was collected. The crude product was separated and purified by preparative high-performance liquid chromatography (chromatography column: Welch Xtimate C18 150 × 30 mm × 5 μm; mobile phase: [A phase: water (0.225% formic acid); B phase: acetonitrile]; gradient: B%: 8% to 48%) to give compound 1. MS: m / z 416.0 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.41 (d, J=6.88 Hz, 1H), 8.32 (s, 1H), 7.42-7.55 (m, 1H), 7.26-7.41 (m, 1H), 7.04-7.18 (m, 1H), 6.88-6.99 (m, 1H), 5.59-6.55 (m, 1H), 4.76-4.84 (m, 1H), 4.66 (br dd, J=10.38, 3.50 Hz, 1H), 4.43-4.52 (m, 3H), 3.52 (d, J=3.25 Hz, 1H), 2.67-2.85 (m, 3H).

[0429] Step 10 Compound 1 was separated and purified by preparative SFC (chromatographic column: DAICEL CHIRALCEL OD-H (250 mm × 30 mm, 5 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: ethanol (0.1% aqueous ammonia)]; gradient: B%: 40%) to obtain compounds 1a and 1b.

[0430] Compound 1a: SFC analytical detection conditions: Chromatography column: Chiralcel OD-3 150 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 2.214 min, ee=94.56%. MS: m / z 416.3 [M+H] + .1 H NMR (400 MHz, CD3OD) δ ppm 8.24 (br d, J=7.28 Hz, 1H), 8.11 (d, J=2.26 Hz, 1H), 7.14-7.37 (m, 2H), 6.94-7.06 (m, 1H), 6.77-6.89 (m, 1H), 5.51-6.42 (m, 1H), 4.65-4.72 (m, 1H), 4.52-4.58 (m, 1H), 4.30-4.41 (m, 3H), 3.41 (d, J=3.51 Hz, 1H), 2.57-2.74 (m, 3H).

[0431] Compound 1b: SFC analysis, extraction conditions: クロマトグラフィーカラム: Chiralcel OD-3 150×4.6mm ID, 3 μm; mobile phase: A phase: supercritical carbonic acid carbonate, B phase: エタノール (0.05% のジエチルアミン); blending: B%: 40%, retention time: 2.715min, ee=88.50%. MS: m / z 416.3 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.34 (d, J=7.28 Hz, 1H), 8.21 (d, J=2.76 Hz, 1H), 7.26-7.47 (m, 2H), 7.04-7.16 (m, 1H), 6.88-6.98 (m, 1H), 5.61-6.53 (m, 1H), 4.76-4.82 (m, 1H), 4.65 (dd, J=10.67, 3.64 Hz, 1H), 4.42-4.50 (m, 3H), 3.51 (d, J=4.02 Hz, 1H), 2.68-2.83 (m, 3H).

[0432] Example 2

[0433]

change

[0434] ステップ1 Sodium hydride (10.19 g, 254.82 mmol, 60% purity) was dissolved in tetrahydrofuran (200 mL). Ethyl glycolate (12.73 g, 122.31 mmol) was added at 0 °C and stirred for 30 minutes. Compound 2-1 (methyl 2,6-dichloronicotinate, 21 g, 101.93 mmol) in tetrahydrofuran (200 mL) was added and the mixture was heated to 75 °C and stirred for 2.5 hours. After the reaction was completed, ethanol was slowly added with stirring until hydrogen gas no longer evolved. The mixture was then neutralized with dilute hydrochloric acid or acetic acid and diluted with ethyl acetate (500 mL). The organic phase was washed with water (500 mL × 2), saturated brine (500 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 2-2, which was directly used in the next step.

[0435] Step 2 Compound 2-2 (22 g, 91.42 mmol) was dissolved in dioxane (220 mL) and hydrochloric acid (58 mL), and the reaction mixture was stirred at 95 °C for 12 hours. The reaction mixture was diluted with ethyl acetate (200 mL), and the pH was adjusted to 8 with sodium bicarbonate. The organic phase was washed with water (200 mL x 2), then with saturated brine (200 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified using a flash silica gel column (0-25% ethyl acetate / petroleum ether) to give compound 2-3. MS: m / z 169.8 [M+H] + .

[0436] Step 3 Compound 2-3 (3 g, 17.69 mmol) was dissolved in ethanol (30 mL) and sodium borohydride (1.69 g, 44.67 mmol) was added in batches at 0 °C. The reaction mixture was warmed to 25 °C and stirred for 2 h. The reaction mixture was quenched with saturated aqueous ammonium chloride (100 mL), diluted with ethyl acetate (100 mL), and the pH was adjusted to less than 3 with dilute hydrochloric acid. The organic phase was washed with water (100 mL x 2), saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-30% ethyl acetate / petroleum ether) to give compound 2-4. MS: m / z 171.8 [M+H] + .

[0437] Step 4 Compound 2-4 (1 g, 5.83 mmol) was dissolved in toluene (10 mL). Diphenylphosphoryl azide (2.41 g, 8.74 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (1.33 g, 8.74 mmol) were added at 0 °C. The mixture was stirred under nitrogen gas for 2 h, then warmed to 25 °C and continued stirring for 10 h. The reaction mixture was diluted with ethyl acetate (100 mL). The organic phase was washed with water (100 mL x 2), saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-20% ethyl acetate / petroleum ether) to give compound 2-5. MS: m / z 196.8 [M+H] + .

[0438] Step 5 Compound 2-5 (1.7 g, 8.65 mmol) was dissolved in tetrahydrofuran (20 mL), triphenylphosphine (3.40 g, 12.97 mmol) was added, and the mixture was stirred at 25 °C for 1 h under nitrogen gas protection. A solution of potassium hydroxide (1.21 g, 21.62 mmol) in water (5 mL) was added, and the mixture was heated to 50 °C and continued stirring for 11 h. The reaction mixture was diluted with ethyl acetate (100 mL). The organic phase was washed successively with water (100 mL × 2) and saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give compound 2-6. The crude product was used directly in the next step.

[0439] Step 6 Compound 2-6 (400 mg, 703.42 μmol) was dissolved in dichloromethane (4 mL), triethylamine (106.77 mg, 1.06 mmol, 146.86 μL) and di-tert-butyl dicarbonate (161.20 mg, 738.59 μmol) were added, and the reaction mixture was stirred at 25 °C for 12 h. Ethyl acetate (100 mL) was added for dilution, and the organic phase was washed sequentially with water (100 mL × 2) and saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-20% ethyl acetate / petroleum ether) to give compound 2-7. MS: m / z 214.8 [M- t Bu+H] + .

[0440] Step 7 Compound 2-7 (1.2 g, 4.43 mmol) was dissolved in tetrahydrofuran (12 mL). Sodium hydride (354.59 mg, 8.87 mmol, 60% purity) was added at 0 °C and stirred for 1 h. Iodomethane (1.26 g, 8.87 mmol) was added dropwise to the reaction mixture. The mixture was stirred at 0 °C for 30 min, then warmed to 25 °C and stirred for 10.5 h. Ethanol was slowly added to the reaction mixture and stirred until hydrogen gas evolution ceased. Next, dilute hydrochloric acid was added to adjust the solution to pH 7, and ethyl acetate (100 mL) was added to dilute the mixture. The organic phase was washed sequentially with water (100 mL × 2), saturated brine (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by flash silica gel column chromatography (0-20% ethyl acetate / petroleum ether) to obtain compound 2-8. MS: m / z 228.8 [M- t Bu+H] + .

[0441] Step 8 Compound 2-8 (150 mg, 526.80 μmol) and trimethylsilylacetylene (517.41 mg, 5.27 mmol) were dissolved in triethylamine (3 mL). Dichlorobis(triphenylphosphine)palladium(II) (147.90 mg, 210.72 μmol) and copper(I) iodide (50.16 mg, 263.40 μmol) were added, and the reaction mixture was stirred at 75 °C for 12 h under nitrogen gas protection. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-20% ethyl acetate / petroleum ether) to give compound 2-9. MS: m / z 290.9 [M- t Bu+H] + .

[0442] Step 9 Compound 2-9 (117 mg, 337.67 μmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1.20 g, 10.50 mmol) was added, and the reaction mixture was stirred for 2 hours at 25° C. The reaction mixture was directly concentrated to give the crude trifluoroacetate salt of compound 2-10, which was directly carried on to the next step.

[0443] Step 10 Compound 2-10 (65 mg, crude trifluoroacetate salt) was dissolved in N,N-dimethylacetamide (1.5 mL). Intermediate A (72.08 mg, 277.01 μmol), N,N-diisopropylethylamine (102.29 mg, 791.46 μmol), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (130.41 mg, 342.97 μmol) were added sequentially and stirred at 25 °C for 12 h. The reaction mixture was diluted with ethyl acetate (10 mL). The organic phase was washed with water (10 mL x 2), saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 2-11, which was directly carried on to the next step. MS: m / z 489.1 [M+H] + .

[0444] Step 11 Compound 2-11 (110 mg, 225.14 μmol) was dissolved in methanol (3 mL), potassium carbonate (62.23 mg, 450.28 μmol) was added, and the mixture was stirred at 25 °C for 3 h. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (20 mL). The organic phase was washed with water (20 mL × 2), saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was isolated and purified by preparative high-performance liquid chromatography (chromatography column: Welch Xtimate C18 150 × 30 mm × 5 μm; mobile phase: [A phase: water (0.225% formic acid); B phase: acetonitrile]; gradient: B%: 8% to 48%) to obtain compound 2.

[0445] Step 12 Compound 2 was separated and purified by SFC (chromatographic column: DAICEL CHIRALCEL OJ (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: ethanol (0.1% aqueous ammonia)]; gradient: B%: 55%) to give compounds 2a and 2b.

[0446] Compound 2a: SFC detection conditions: Chromatography column: Chiralcel OJ-3 100 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 1.797 min, ee=100%. MS: m / z 439.2 [M+Na] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.20-8.40 (m, 1H), 8.10 (s, 1H), 7.65-7.80 (m, 1H), 7.23-7.34 (m, 1H), 7.06-7.20 (m, 1H), 5.55-6.45 (m, 1H), 4.50-4.70 (m, 2H), 4.27-4.40 (m, 3H), 4.03-4.08 (m, 1H), 2.63-2.80 (m, 3H).

[0447] Compound 2b: SFC detection conditions: Chromatography column: Chiralcel OJ-3 100 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 3.929 min, ee=99.30%. MS: m / z 439.4 [M+Na] + . 1H NMR (400 MHz, CD3OD) δ ppm 8.28-8.45 (m, 1H) ,8.20 (s, 1 H), 7.75-7.90 (m, 1H), 7.30-7.45 (m, 1H), 7.17-7.23 (m, 1H), 5.67-6.53 (m, 1H), 4.89-4.97 (m, 1H), 4.69-4.78 (m, 1H), 4.38-4.50 (m, 3H), 3.33-3.37 (m, 1H), 2.66-2.91 (m, 3H).

[0448] Example 3

[0449] [ka]

[0450] [ka]

[0451] Step 1 Compound 3-1 (9 g, 29.13 mmol) was dissolved in tetrahydrofuran (135 mL) and, under nitrogen gas protection, a 1 M solution of diisobutylaluminum hydride in toluene (1 M, 43.70 mL) was added dropwise at −78°C. The reaction mixture was stirred slowly at −78°C for 3 h under N2 protection. At 0°C, the reaction mixture was slowly poured into a mixture of glacial acetic acid (22.5 mL) and water (112.5 mL). The mixture was warmed to 25°C and stirred for 1.5 h. The mixture was then cooled to 0°C. Saturated aqueous sodium bicarbonate solution was added dropwise to the reaction mixture to adjust the pH to 8. The mixture was extracted with ethyl acetate (200 mL × 2). The combined organic phases were washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-10% ethyl acetate / dichloromethane) to give compound 3-2. 1 H NMR (400 MHz, CDCl3) δ ppm 10.10 (s, 1H), 7.88 (d, J=8.28 Hz, 1H), 7.55 (d, J=8.53 Hz, 1H).

[0452] Step 2 Compound 3-2 (3 g, 11.32 mmol) was dissolved in tetrahydrofuran (45 mL) and methylmagnesium bromide (3 M, 7.55 mL) was added dropwise at −78°C under nitrogen gas protection. The reaction mixture was stirred at −78°C for 0.5 h, then warmed to 25°C and stirred for 0.5 h. Under nitrogen gas, the reaction mixture was quenched by the slow dropwise addition of saturated aqueous ammonium chloride solution (30 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0–10% ethyl acetate / petroleum ether) to give compound 3-3. 1 H NMR (400 MHz, CDCl3) δ ppm 7.69 (d, J=8.28 Hz, 1H), 7.30 (d, J=8.28 Hz, 1H), 5.10 (q, J=6.44 Hz, 1H), 1.47 (d, J=6.27 Hz, 3H).

[0453] Step 3 Compound 3-3 (1.8 g, 6.41 mmol) and compound 3-4 (2.94 g, 19.22 mmol) were dissolved in tetrahydrofuran (72 mL) and cooled to 0 °C. Sodium hydride (512.56 mg, 12.81 mmol, 60% purity) was added in batches. The reaction mixture was stirred at 0 °C for 4 h under a nitrogen atmosphere, then slowly warmed to 25 °C and continued stirring for 12 h. The reaction mixture was cooled to 0 °C and quenched by the slow dropwise addition of saturated aqueous ammonium chloride (50 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-15% ethyl acetate / petroleum ether) to give compound 3-5. MS: m / z 351.7, 353.7, 355.7 [M+H] + .

[0454] Step 4 Compound 3-5 (1.5 g, 4.25 mmol) was dissolved in tetrahydrofuran (8 mL), methanol (8 mL) and water (4 mL) were added, followed by lithium hydroxide monohydrate (356.62 mg, 8.50 mmol). The reaction mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran and methanol. 1 M diluted hydrochloric acid was added dropwise to the aqueous phase to adjust the pH to 3-4, followed by extraction with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 3-6. MS: m / z 337.7, 339.7, 341.7 [M+H] + .

[0455] Step 5 Compound 3-6 (1.55 g, 4.57 mmol) and compound 3-7 (535.23 mg, 5.49 mmol) were dissolved in N,N-dimethylformamide (15 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (2.09 g, 5.49 mmol) and N,N-diisopropylethylamine (1.77 g, 13.72 mmol) were added, and the reaction mixture was stirred at 25 °C for 12 h. Water (20 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (30 mL × 2). The combined organic phases were washed with water (20 mL × 2), saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-50% ethyl acetate / petroleum ether) to give compound 3-8. MS: m / z 380.8, 382.8, 384.8 [M+H] + .

[0456] Step 6 Compound 3-8 (700 mg, 1.83 mmol) was dissolved in tetrahydrofuran (23 mL) and cooled to -78 °C. n-Butyllithium (2.5 M, 879.47 μL) was added dropwise, and the reaction mixture was stirred at -78 °C for 4 h under nitrogen gas protection. The reaction mixture was slowly warmed to 0 °C and quenched by the slow dropwise addition of saturated aqueous ammonium chloride (10 mL). The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-15% ethyl acetate / petroleum ether) to give compound 3-9. MS: m / z 241.7, 243.7 [M+H] + .

[0457] Step 7 Compound 3-9 (250 mg, 1.03 mmol) was dissolved in methanol (2.5 mL), and methylamine tetrahydrofuran solution (1 M, 2.52 mL) and zinc chloride (211.15 mg, 1.55 mmol) were added. The mixture was stirred at 25 °C for 2 h. Sodium cyanoborohydride (97.35 mg, 1.55 mmol) was added, and the reaction mixture was stirred at 40 °C for 40 h under nitrogen gas protection. The reaction mixture was quenched with 10% aqueous sodium bicarbonate (20 mL) and then extracted with dichloromethane (20 mL × 4). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-10% methanol / dichloromethane) to give compound 3-10. MS: m / z 256.9, 258.9 [M+H] + .

[0458] Step 8 Compound 3-10 (170 mg, 661.15 μmol) and trimethylsilylacetylene (649.37 mg, 6.61 mmol) were dissolved in triethylamine (3.4 mL). Dichlorobis(triphenylphosphine)palladium(II) (92.81 mg, 132.23 μmol) and copper(I) iodide (25.18 mg, 132.23 μmol) were added, and the reaction mixture was stirred at 75 °C for 3 h under nitrogen gas protection. The reaction mixture was diluted with ethyl acetate (10 mL), filtered, and the cake was washed with ethyl acetate (10 mL × 2). The filtrate was collected and washed with water (10 mL × 2), saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-10% methanol / dichloromethane) to give compound 3-11. MS: m / z 274.9 [M+H] + .

[0459] Step 9 Compound 3-11 (140 mg, 510.14 μmol) and Compound A (119.48 mg, 459.13 μmol) were dissolved in N,N-dimethylacetamide (2.8 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (232.77 mg, 612.17 μmol) and N,N-diisopropylethylamine (131.86 mg, 1.02 mmol) were added, and the reaction mixture was stirred at 25 °C for 12 h. Water (10 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified using a flash silica gel column (0-10% methanol / dichloromethane) to give Compound 3-12. MS: m / z 517.1 [M+H] + .

[0460] Step 10 Compound 3-12 (280 mg, 541.96 μmol) was dissolved in methanol (3 mL), potassium carbonate (149.80 mg, 1.08 mmol) was added, and the reaction mixture was stirred at 25°C for 2 hours. The reaction mixture was filtered, the cake was washed with methanol (1 mL), and the filtrate was collected. The filtrate was separated and purified by preparative high-performance liquid chromatography (chromatography column: Welch Xtimate C18 150 × 30 mm × 5 μm; mobile phase: [A phase: water (0.225% formic acid); B phase: acetonitrile]; gradient: B%: 7% to 47%) to give compound 3. MS: m / z 445.0 [M+H] + .

[0461] Step 11 Compound 3 was separated and purified by SFC (chromatographic column: DAICEL CHIRALCEL OD-H (250 mm × 30 mm, 5 μm); mobile phase: [A phase: supercritical carbon dioxide, B phase: methanol (0.1% aqueous ammonia)]; gradient: B%: 40%) to give two crude compounds. SFC detection (chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A phase: supercritical carbon dioxide, B phase: methanol (0.05% diethylamine); gradient: B%: 5%–40% (0–2 min), 40% (2–4 min), 5% (4–6 min)) gave the first crude compound with a retention time of 5.483 min, and the second crude compound with a retention time of 5.820 min.

[0462] The first crude product compound was further separated and purified by SFC (separation conditions: chromatography column: DAICEL CHIRALPAK AD (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: ethanol (0.1% aqueous ammonia)]; gradient: B%: 30%) to obtain compounds 3a and 3b.

[0463] Compound 3a: Under SFC detection conditions 1 (chromatographic column: ChiralPak AD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% diethylamine); gradient: B: 5%–40% (0–4.5 min), 40% (4.5–6.5 min), 5% (6.5–8 min)), the retention time was 5.283 min. Under SFC detection conditions 2 (chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A: supercritical carbon dioxide, B: methanol (0.05% diethylamine); gradient: 5%–40% (0–2 min), 40% (2–4 min), 5% (4–6 min)), the retention time was 5.471 min and the chiral purity was 91.55%. MS: m / z 445.0 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.47 (d, J=6.75 Hz, 1H), 8.34-8.37 (m, 1H), 7.84 (d, J=7.88 Hz, 1H), 7.54 (d, J=8.13 Hz, 1H), 7.49 (d, J=10.88 Hz, 1H), 5.89 (br s, 1H), 4.76 (br d, J=6.13 Hz, 1H), 4.50 (s, 3H), 4.38 (br d, J=12.38 Hz, 1H), 4.17 (dd, J=12.82, 4.07 Hz, 1H), 3.75-3.79 (m, 1H), 2.87 (s, 3H), 1.63 (d, J=6.63 Hz, 3H).

[0464] Compound 3b: Under SFC detection condition 1 (chromatographic column: ChiralPak AD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% diethylamine); gradient: B: 5%–40% (0–4.5 min), 40% (4.5–6.5 min), 5% (6.5–8 min)), the retention time was 5.581 min. Under SFC detection condition 2 (chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A: supercritical carbon dioxide, B: methanol (0.05% diethylamine); gradient: 5%–40% (0–2 min), 40% (2–4 min), 5% (4–6 min)), the retention time was 5.458 min and the chiral purity was 100%. MS: m / z 445.1 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.39 (d, J=7.50 Hz, 1H), 8.20-8.24 (m, 1H), 7.79 (d, J=7.63 Hz, 1H), 7.51-7.56 (m, 1H), 7.37-7.43 (m, 1H), 5.92-6.06 (m, 1H), 4.81 (br s, 1H), 4.43-4.47 (m, 3H), 4.36 (dd, J=11.76, 5.63 Hz, 1H), 4.06 (dd, J=11.57, 7.07 Hz, 1H), 3.74-3.78 (m, 1H), 2.80-2.89 (m, 3H), 1.59 (d, J=6.75 Hz, 3H).

[0465] The second crude product compound was further separated and purified by SFC (separation conditions: chromatographic column: DAICEL CHIRALPAK IC (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: ethanol (0.1% aqueous ammonia)]; gradient: B%: 60%) to give compounds 3c and 3d.

[0466] Compound 3c: Under SFC detection condition 3 (chromatographic column: Chiralpak IC-3 100 × 4.6 mm ID, 3 μm; mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% diethylamine); gradient: B: 60%), the retention time was 2.029 min. Furthermore, under SFC detection condition 4 (chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A: supercritical carbon dioxide, B: methanol (0.05% diethylamine); gradient: 5% to 40% (0 to 2 min), 40% (2 to 4 min), 5% (4 to 6 min)), the retention time was 5.803 min and the chiral purity was 93.45%. MS: m / z 445.0 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.39 (d, J=7.38 Hz, 1H), 8.22 (s, 1H), 7.76-7.85 (m, 1H), 7.54 (br d, J=7.88 Hz, 1H), 7.35-7.44 (m, 1H), 6.00 (s, 1H), 4.81 (br s, 1H), 4.44-4.47 (m, 3H), 4.36 (dd, J=11.63, 5.88 Hz, 1H), 4.06 (br dd, J=11.69, 7.07 Hz, 1H), 3.74-3.78 (m, 1H), 2.79-2.89 (m, 3H), 1.53-1.69 (m, 3 H).

[0467] Compound 3d: Under SFC detection condition 3 (chromatographic column: Chiralpak IC-3 100 × 4.6 mm ID, 3 μm; mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% diethylamine); gradient: B: 60%), the retention time was 2.714 min. Furthermore, under SFC detection condition 4 (chromatographic column: Chiralcel OD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A: supercritical carbon dioxide, B: methanol (0.05% diethylamine); gradient: 5% to 40% (0 to 2 min), 40% (2 to 4 min), 5% (4 to 6 min), the retention time was 5.772 min and the chiral purity was 98.56%. MS: m / z 445.0 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.38 (br d, J=7.25 Hz, 1H), 8.22 (s, 1H), 7.84 (br d, J=7.63 Hz, 1H), 7.54 (br d, J=8.13 Hz, 1H), 7.37 (br d, J=11.88 Hz, 1H), 5.89 (br s, 1H), 4.76 (br d, J=6.00 Hz, 1H), 4.46 (s, 3H), 4.37 (br d, J=13.13 Hz, 1H), 4.17 (dd, J=12.94, 3.94 Hz, 1H), 3.74-3.78 (m, 1H), 2.88 (s, 3H), 1.63 (br d, J=6.50 Hz, 3H).

[0468] Example 4

[0469] [ka]

[0470] Step 1 Compound 4-1 (6.7 g, 22.72 mmol) was dissolved in ethanol (67 mL) and, under nitrogen gas protection, sodium borohydride (2.51 g, 66.35 mmol) was added in batches at 0 °C. The reaction mixture was slowly warmed to 25 °C under N2 protection and stirred for 12 h. Under N2, the reaction mixture was quenched by the slow dropwise addition of saturated aqueous ammonium chloride (30 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-10% ethyl acetate / petroleum ether) to give compound 4-2. MS: m / z 265.7, 267.7, 269.7 [M+H] + .

[0471] Step 2 Compound 4-2 (3 g, 11.24 mmol) and compound 4-3 (5.16 g, 33.72 mmol) were dissolved in tetrahydrofuran (90 mL) and cooled to 0 °C. Sodium hydride (899.15 mg, 22.48 mmol, 60% purity) was added in a batchwise manner. The reaction mixture was heated to 40 °C under a nitrogen atmosphere and stirred for 36 h. The reaction mixture was cooled to 0 °C and quenched by the slow dropwise addition of saturated aqueous ammonium chloride (50 mL). The mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-20% ethyl acetate / petroleum ether) to give compound 4-4. MS: m / z 337.8, 339.8, 341.8 [M+H]. + .

[0472] Step 3 Compound 4-4 (3.15 g, 9.29 mmol) was dissolved in tetrahydrofuran (16 mL), methanol (16 mL) and water (8 mL) were added, followed by lithium hydroxide monohydrate (779.83 mg, 18.59 mmol). The reaction mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran and methanol. 1M diluted hydrochloric acid was added dropwise to the aqueous phase to adjust the pH to 3-4, followed by extraction with ethyl acetate (50 mL x 3). The combined organic phases were washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 4-5. MS: m / z 323.7, 325.7, 327.7 [M+H] + .

[0473] Step 4 Compound 4-5 (3.02 g, 9.29 mmol) and compound 4-6 (1.09 mg, 11.15 mmol) were dissolved in N,N-dimethylformamide (30 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (4.24 g, 11.15 mmol) and N,N-diisopropylethylamine (3.60 g, 27.88 mmol) were added, and the reaction mixture was stirred at 25 °C for 12 h. Water (30 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (50 mL × 3). The combined organic phases were washed with water (30 mL × 2), saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-50% ethyl acetate / petroleum ether) to give compound 4-7. MS: m / z 3660.8, 368.8, 370.8 [M+H] + .

[0474] Step 5 Compound 4-7 (1.8 g, 4.89 mmol) was dissolved in tetrahydrofuran (54 mL) and cooled to -78 °C. n-Butyllithium (2.5 M, 2.35 mL) was added dropwise, and the reaction mixture was stirred at -78 °C for 4 h under nitrogen gas protection. The reaction mixture was slowly warmed to 0 °C and quenched by the slow dropwise addition of saturated aqueous ammonium chloride (20 mL). The mixture was extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-10% ethyl acetate / petroleum ether) to give compound 4-8. MS: m / z 228.0, 230.0 [M+H] + .

[0475] Step 6 Compound 4-8 (450 mg, 1.97 mmol) was dissolved in methanol (4.5 mL), and methylamine tetrahydrofuran solution (1 M, 4.25 mL) and zinc chloride (403.4 mg, 2.96 mmol) were added. The mixture was stirred at 25 °C for 2 h. Sodium cyanoborohydride (186.0 mg, 2.96 mmol) was added, and the reaction mixture was stirred at 40 °C for 12 h under nitrogen gas protection. The reaction mixture was quenched with water (10 mL) and then extracted with dichloromethane (10 mL × 4). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-10% methanol / dichloromethane) to give compound 4-9. MS: m / z 242.8, 244.8 [M+H] + .

[0476] Step 7 Compound 4-9 (100 mg, 411.35 μmol) and trimethylsilylacetylene (404.0 mg, 4.11 mmol) were dissolved in triethylamine (2 mL). Dichlorobis(triphenylphosphine)palladium(II) (57.8 mg, 82.27 μmol) and copper(I) iodide (15.7 mg, 82.27 μmol) were added, and the reaction mixture was stirred at 75 °C for 3 h under nitrogen gas protection. The reaction mixture was diluted with ethyl acetate (20 mL), filtered, and the cake was washed with ethyl acetate (10 mL × 2). The filtrate was collected and washed with water (10 mL × 2), saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-10% methanol / dichloromethane) to give compound 4-10. MS: m / z 260.9 [M+H] + .

[0477] Step 8 Compound 4-10 (90 mg, 345.61 μmol) and Compound A (89.9 mg, 345.61 μmol) were dissolved in N,N-dimethylacetamide (1 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (157.7 mg, 414.74 μmol) and N,N-diisopropylethylamine (89.3 mg, 691.23 μmol) were added, and the reaction mixture was stirred at 25 °C for 12 h. Water (5 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (10 mL × 4). The combined organic phases were washed with water (10 mL × 2), saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified using a flash silica gel column (0-3% methanol / dichloromethane) to give Compound 4-11. MS: m / z 503.1 [M+H] + .

[0478] Step 9 Compound 4-11 (85 mg, 169.12 μmol) was dissolved in methanol (1.7 mL), potassium carbonate (46.8 mg, 338.23 μmol) was added, and the reaction mixture was stirred at 25° C. for 1 hour. Water (5 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL×3). The organic phases were combined, washed with water (10 mL×2), washed with saturated brine (10 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound 4. MS: m / z 431.2 [M+H] + .

[0479] Step 10 Compound 4 was separated and purified by SFC (separation conditions: chromatographic column: DAICEL CHIRALPAK IC (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: ethanol (0.1% aqueous ammonia)]; gradient: B%: 60%) to obtain compounds 4a and 4b.

[0480] Compound 4a: SFC detection conditions: Chromatography column: Chiralpak IC-3 100 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 60%, Retention time: 2.298 min, ee=98.98%. MS: m / z 431.0 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.28 - 8.39 (m, 2 H) 7.78 (br d, J=7.78 Hz, 1 H) 7.57 (br d, J=7.28 Hz, 3 H) 7.34 - 7.44 (m, 1 H) 5.84 (br s, 1 H) 4.64 - 4.87 (m, 2 H) 4.40 (br d, J=6.53 Hz, 3 H) 4.16 (br s, 2 H) 4.04 (br s, 1 H) 2.67 - 2.81 (m, 3 H).

[0481] Compound 4b: SFC detection conditions: Chromatography column: Chiralpak IC-3 100 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 60%, Retention time: 3.083 min, ee=98.78%. MS: m / z 431.0 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.24 - 8.38 (m, 2 H) 7.77 (br d, J=7.78 Hz, 1 H) 7.57 (br d, J=7.53 Hz, 1 H) 7.27 - 7.38 (m, 3 H) 5.84 (br s, 1 H) 4.63 - 4.90 (m, 2 H) 4.39 (br d, J=5.27 Hz, 3 H) 4.16 (br s, 2 H) 4.04 (br s, 1 H) 2.67 - 2.81 (m, 3 H).

[0482] Example 5

[0483] [ka]

[0484] Step 1 Compound 5-1 (5 g, 23.47 mmol) was dissolved in tetrahydrofuran (40 mL), methanol (20 mL), and water (20 mL). Lithium hydroxide monohydrate (2.95 g, 70.41 mmol) was added at 25° C., and the reaction mixture was stirred for 12 hours at 25° C. The reaction mixture was concentrated and diluted with water (30 mL). 1N diluted hydrochloric acid was added to adjust the pH to 3-4. The precipitated solid was filtered and the cake was dried to obtain compound 5-2. The crude product was used directly in the next step.

[0485] Step 2 Compound 5-2 (5 g, 21.64 mmol) and bromoacetic acid (3.31 g, 23.81 mmol) were dissolved in tetrahydrofuran (200 mL). Sodium hydride (3.46 g, 86.56 mmol, 60% purity) was added in batches at 25 °C. Sodium iodide (324.38 mg, 2.16 mmol) was then added, and the reaction mixture was heated to 65 °C and stirred continuously for 12 h. The reaction mixture was cooled to room temperature and quenched with water (150 mL). The mixture was then washed with methyl tert-butyl ether (90 mL × 3). The aqueous phase was adjusted to pH 3-4 with 1N dilute hydrochloric acid and extracted with ethyl acetate (180 mL × 3). The combined organic phase was washed with saturated brine (180 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give compound 5-3, and the crude product was used directly in the next step.

[0486] Step 3 Compound 5-3 (2 g, 6.92 mmol) was dissolved in acetic anhydride (40 mL), potassium acetate (2.92 g, 29.75 mmol) was added, and the reaction mixture was stirred at 140 ° C. for 2 hours. The reaction mixture was concentrated, diluted with water (20 mL), and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. After removing the solvent, the mixture was dissolved in ethanol (20 mL). Sodium hydroxide (1.11 g, 27.67 mmol) was added, and the reaction mixture was stirred at 40 ° C. for 2 hours. The reaction mixture was directly concentrated, diluted with water (20 mL), and then extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude product, which was purified by silica gel column chromatography (0-10% ethyl acetate / petroleum ether) to give compound 5-4. MS: m / z 226.8, 228.8 [M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.87 (d, J=8.28 Hz, 1H), 7.62 (dd, J=1.76, 8.28 Hz, 1H), 7.58 (s, 1H), 4.89 (s, 2H), 4.34 (s, 2H).

[0487] Step 4 Compound 5-4 (500 mg, 2.20 mmol) was dissolved in methanol (5 mL), methylamine (2.51 mL) and zinc chloride (450.21 mg, 3.30 mmol) were added, and the mixture was stirred for 0.5 h. Sodium cyanoborohydride (207.58 mg, 3.30 mmol) was added, and the reaction mixture was stirred at 20 °C for 48 h. The reaction mixture was quenched with 10% aqueous sodium bicarbonate (10 mL) and filtered. The filtrate was extracted with dichloromethane (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude product. The crude product was purified by silica gel column chromatography (0-10% methanol / dichloromethane) to give compound 5-5. MS: m / z 241.8, 243.8 [M+H] + .

[0488] Step 5 Compound 5-5 (220 mg, 908.67 μmol) and trimethylsilylacetylene (89.25 mg, 908.67 μmol) were dissolved in triethylamine (2 mL). The mixture was purged with nitrogen gas three times. Copper(I) iodide (34.61 mg, 181.73 μmol) and dichlorobis(triphenylphosphine)palladium (127.56 mg, 181.73 μmol) were added, and the reaction mixture was heated to 75°C under a nitrogen atmosphere and stirred continuously for 2 hours. After completion of the reaction, the reaction mixture was directly concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (0-100% ethyl acetate / petroleum ether) to obtain compound 5-6.

[0489] Step 6 Compound 5-6 (50 mg, 192.74 μmol) and Compound A (50.16 mg, 192.74 μmol) were dissolved in N,N-dimethylacetamide (1 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (87.94 mg, 231.29 μmol) and N,N-diisopropylethylamine (99.64 mg, 770.96 μmol) were added. The reaction mixture was stirred at 20° C. for 12 hours. Water (10 mL) was added to the reaction mixture, followed by extraction with dichloromethane (10 mL × 3). The organic phases were combined, washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give Compound 5-7. MS: m / z 502.1 [M+H] + .

[0490] Step 7 Compound 5-7 (50 mg, 99.68 μmol) was dissolved in methanol (1 mL), potassium carbonate (41.33 mg, 299.03 μmol) was added, and the reaction mixture was stirred at 20 °C for 1 h. Water (10 mL) was added to the reaction mixture, followed by extraction with dichloromethane (10 mL × 3). The organic phases were combined, washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was separated and purified by preparative high-performance liquid chromatography (chromatography column: Welch Xtimate C18 150 × 30 mm × 5 μm; mobile phase: [A phase: water (0.225% formic acid); B phase: acetonitrile]; gradient: B%: 0% to 40%) to obtain compound 5. MS: m / z 430.0 [M+H] + .

[0491] Step 8 Compound 5 was separated and purified by SFC (separation conditions: chromatography column: DAICEL CHIRALPAK AD (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: ethanol (0.1% aqueous ammonia)]; B%: 30%) to obtain compounds 5a and 5b.

[0492] Compound 5a: SFC detection conditions: Chromatography column: ChiralPak AD-3 50 × 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 3.058 min, ee=100%. MS: m / z 430.1 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm8.36 (d, J=7.28 Hz, 1H), 8.20-8.21 (m, 1H), 7.20-7.46 (m, 4H), 5.86 (br s, 1H), 4.79-4.83 (m, 1H), 4.67-4.78 (m, 1H), 4.42-4.47 (m, 3H), 4.23-4.29 (m, 1H), 4.11-4.17 (m, 1H), 3.50-3.54 (m, 1H), 2.76-2.94 (m, 3H).

[0493] Compound 5b: SFC detection conditions: Chromatography column: ChiralPak AD-3 50 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 3.649 min, ee=95.82%. MS: m / z 430.1 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.36 (d, J=7.03 Hz, 1H), 8.20-8.25 (m, 1H), 7.21-7.45 (m, 4H), 5.86 (br s, 1H), 4.76-4.81 (m, 1H), 4.67-4.73 (m, 1H), 4.41-4.47 (m, 3H), 4.23-4.29 (m, 1H), 4.11-4.17 (m, 1H), 3.51-3.54 (m, 1H), 2.78-2.95 (m, 3H).

[0494] Example 6

[0495] [ka]

[0496] Step 1 Compound 1-5 (100 mg, 318.29 μmol) was dissolved in tetrahydrofuran (2 mL) and cooled to 0 °C. Sodium hydride (25.46 mg, 636.59 μmol, 60% purity) was added under nitrogen gas protection and stirred at 0 °C for 2 h. Deuterated iodomethane (92.28 mg, 636.59 μmol) was added dropwise, and the reaction mixture was slowly warmed to 25 °C and stirred under nitrogen gas protection for 12 h. Water (10 mL) was added dropwise to the reaction mixture, and the mixture was extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-10% ethyl acetate / petroleum ether) to give compound 6-1.

[0497] Step 2 Compound 6-1 was used as the starting material, and the crude product of compound 6 was obtained by following the procedures of steps 6 to 9 in Example 1. This crude product was then separated and purified by preparative high-performance liquid chromatography (chromatography column: Welch Xtimate C18 150 x 30 mm x 5 μm; mobile phase: [phase A: water (0.225% formic acid); phase B: acetonitrile]; gradient: B%: 8% to 48%) to obtain compound 6. MS: m / z 419.0 [M+H] + .

[0498] Step 3 Compound 6 was separated and purified by SFC (separation conditions: chromatography column: DAICEL CHIRALPAK IG (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: isopropanol (0.1% aqueous ammonia)]; B%: 50%) to obtain compounds 6a and 6b.

[0499] Compound 6a: SFC detection conditions: Chromatography column: Chiralpak IG-3 50 × 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: isopropanol (0.05% diethylamine); Gradient: B%: 5–40% (0–2 min), 40% (2–3.2 min), 5% (3.2–4 min); Retention time: 2.347 min, ee=99.82%. MS: m / z 419.0 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.35 (d, J=7.25 Hz, 1 H) 8.21 (d, J=2.63 Hz, 1 H) 7.27 - 7.45 (m, 2 H) 7.05 - 7.15 (m, 1 H) 6.87 - 6.99 (m, 1 H) 5.61 - 6.52 (m, 1 H) 4.76 - 4.83 (m, 1 H) 4.65 (dd, J=10.44, 3.69 Hz, 1 H) 4.42 - 4.49 (m, 3 H) 3.51 (d, J=3.75 Hz, 1 H).

[0500] Compound 6b: SFC detection conditions: Chromatography column: Chiralpak IG-3 50 × 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: isopropanol (0.05% diethylamine); Gradient: B%: 5–40% (0–2 min), 40% (2–3.2 min), 5% (3.2–4 min); Retention time: 2.650 min, ee=97.90%. MS: m / z 419.3 [M+H] + . 1H NMR (400 MHz, CD3OD) δ ppm 8.34 (br d, J=7.25 Hz, 1 H) 8.21 (d, J=1.75 Hz, 1 H) 7.26 - 7.45 (m, 2 H) 7.04 - 7.16 (m, 1 H) 6.88 - 6.98 (m, 1 H) 5.60 - 6.52(m, 1 H) 4.75 - 4.84 (m, 1 H) 4.64 (br dd, J=10.32, 3.56 Hz, 1 H) 4.42 - 4.50 (m, 3 H) 3.51 (d, J=4.00 Hz, 1 H).

[0501] Example 7

[0502] [ka]

[0503] Step 1 Compound 1-6 (500 mg, 1.52 mmol) and 1-dimethylamino-2-propyne (1.27 g, 15.23 mmol) were dissolved in triethylamine (10 mL). Dichlorobis(triphenylphosphine)palladium(II) (213.9 mg, 304.69 μmol) and copper(I) iodide (58.0 mg, 304.69 μmol) were added, and the reaction mixture was stirred at 75 °C for 12 h under nitrogen gas protection. The reaction mixture was diluted with ethyl acetate (20 mL), filtered, and the cake was washed with ethyl acetate (20 mL × 2). The filtrate was collected and washed with water (20 mL × 2), saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-70% ethyl acetate / petroleum ether) to give compound 7-1. MS: m / z 331.0 [M+H] + .

[0504] Step 2 Compound 7-1 (200 mg, 605.29 μmol) was dissolved in a 4 M solution of hydrochloric acid and ethyl acetate (2 mL), and the reaction mixture was stirred at 25° C. for 3 hours. The reaction mixture was directly concentrated under reduced pressure, diluted with ethyl acetate (1 mL), and concentrated under reduced pressure to give the crude hydrochloride product of compound 7-2. MS: m / z 230.9 [M+H] + .

[0505] Step 3 Compound 7-2 (139 mg, crude hydrochloride salt) and compound A (172.8 mg, 663.90 μmol) were dissolved in N,N-dimethylacetamide (2.8 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (275.4 mg, 724.26 μmol) and N,N-diisopropylethylamine (195.0 mg, 1.51 mmol) were added, and the reaction mixture was stirred at 25 °C for 12 h. The reaction mixture was directly concentrated under reduced pressure to give the crude product. The crude product was separated and purified by preparative high-performance liquid chromatography (chromatography column: Phenomenex C18 80 × 40 mm × 3 μm; mobile phase: [A phase: water (0.0125% aqueous ammonia + 10 mm ammonium bicarbonate); B phase: acetonitrile]; gradient: B%: 41% to 71%) to give compound 7. MS: m / z 473.0 [M+H] + .

[0506] Step 4 Compound 7 was separated and purified by SFC (separation conditions: chromatography column: DAICEL CHIRALCEL OJ (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: methanol (0.1% aqueous ammonia)]; B%: 40%) to obtain compounds 7a and 7b.

[0507] Compound 7a: SFC detection conditions: Chromatography column: Chiralcel OJ-3 150 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: methanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 2.008 min, ee=99.36%. MS: m / z 473.0 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.34 (br d, J=7.38 Hz, 1 H) 8.21 (s, 1 H) 7.23 - 7.46 (m, 2 H) 7.00 - 7.15 (m, 1 H) 6.83 - 6.96 (m, 1 H) 6.48 - 6.52 (m, 0.5 H) 5.58-5.62 (m, 0.5 H) 4.64 (br d, J=7.00 Hz, 2 H) 4.40 - 4.50 (m, 3 H) 3.48 (br d, J=5.75 Hz, 2 H) 2.67 - 2.85 (m, 3 H) 2.37 (br d, J=5.50 Hz, 6 H).

[0508] Compound 7b: SFC detection conditions: Chromatography column: Chiralcel OJ-3 150 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: methanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 2.872 min, ee=97.94%. MS: m / z 473.1 [M+H] + . 1H NMR (400 MHz, CD3OD) δ ppm 8.34 (br d, J=7.00 Hz, 1 H) 8.21 (s, 1 H) 7.25 - 7.44 (m, 2 H) 7.01 - 7.14 (m, 1 H) 6.84 - 6.96 (m, 1 H) 6.48 - 6.52 (m, 0.5 H) 5.58-5.62 (m, 0.5 H) 4.64 (br d, J=6.75 Hz, 2 H) 4.37 - 4.50 (m, 3 H) 3.50 (br d, J=6.50 Hz, 2 H) 2.67 - 2.84 (m, 3 H) 2.38 (br d, J=5.88 Hz, 6 H).

[0509] Example 8

[0510] [ka]

[0511] Step 1 Compound 1-6 (1 g, 3.05 mmol) and compound 8-1 (2.53 g, 30.47 mmol) were dissolved in triethylamine (10 mL). Dichlorobis(triphenylphosphine)palladium(II) (320.79 mg, 457.04 μmol) and copper(I) iodide (87.04 mg, 457.04 μmol) were added, and the reaction mixture was stirred at 75 °C for 3 h under nitrogen gas protection. The reaction mixture was diluted with ethyl acetate (20 mL), filtered, and the cake was washed with ethyl acetate (10 mL x 2). The filtrate was collected, washed with water (10 mL x 2), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (0-20% ethyl acetate / petroleum ether) to give compound 8-2. MS: m / z 315.1 [M-NH2+H] + .

[0512] Step 2 Under a nitrogen atmosphere, compound 8-2 (300 mg, 907.93 μmol) was dissolved in trifluoroethanol (6 mL), paraformaldehyde (40.86 mg, 1.36 mmol) was added, and the mixture was stirred at 25 °C for 1 hour. Sodium borohydride (51.52 mg, 1.36 mmol) was added, and the mixture was stirred at 80 °C for 6 hours. The reaction mixture was cooled to room temperature, quenched by adding water (5 mL), extracted with ethyl acetate (10 mL × 3), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product. The crude product was purified by preparative TLC (petroleum ether:ethyl acetate = 5:1) to give compound 8-3. MS: m / z 359.2 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.13 (br s, 1H), 7.00 (br dd, J = 0.9, 7.7 Hz, 1H), 6.91 - 6.84 (m, 1H), 6.13 - 5.70 (m, 1 H) 4.64 - 4.54 (m, 1H), 4.40 - 4.30 (m, 1H), 2.50 (br s, 3H), 2.34 (s, 6H), 1.48 (s, 9H), 1.44 (s, 6H).

[0513] Step 3 Compound 8 was obtained from compound 8-3 by following the steps 2 and 3 of Example 7. MS: m / z 501.2 [M+H] + .

[0514] Step 4 Compound 8 was separated and purified by SFC (separation conditions: chromatography column: DAICEL CHIRALCEL OJ (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: methanol (0.1% aqueous ammonia)]; B%: 35%) to obtain compounds 8a and 8b.

[0515] Compound 8a: SFC detection conditions: Chromatography column: Chiralcel OD-3 150 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 1.152 min, ee=100%. MS: m / z 501.2 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) 8.25 (s, 1H), 7.39 - 7.32 (m, 1H), 7.30 (br s, 2H), 7.07 - 6.96 (m, 1H), 6.88 (br d, J = 19.3 Hz, 1H), 6.40 - 6.44 (m, 0.5H), 5.58 - 5.50 (m, 0.5H), 4.62 (br s, 1H), 4.39 (br d, J = 10.3 Hz, 3H), 2.67 (s, 2H), 2.55 (s, 2H), 2.22 (br s, 6H), 1.35 (br s, 6H).

[0516] Compound 8b: SFC detection conditions: Chromatography column: Chiralcel OD-3 150 x 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: ethanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 1.655 min, ee=99.04%. MS: m / z 501.2 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 8.26 (br s, 1H), 7.28-7.32 (m, 3H), 7.14 - 6.96 (m, 1H), 6.88 (br d, J = 19.3 Hz, 1H), 6.41 - 6.45 (m, 0.5H), 5.52 - 5.46 (m, 0.5H), 4.63 (br s, 1H), 4.39 (br d, J = 9.3 Hz, 3H), 2.67 (br s, 2H), 2.55 (br s, 2H), 2.22 (br s, 6H), 1.35 (br s, 6H).

[0517] Example 9

[0518] [ka]

[0519] Step 1 Using compound 9-1 as the starting material, the crude product of compound 9 was obtained by following the procedures of steps 2 to 10 of Example 3. The crude product was separated and purified by preparative high-performance liquid chromatography (chromatography column: Welch Xtimate C18 150 x 25 mm x 5 μm; mobile phase: [phase A: water (0.0125% aqueous ammonia + 10 mm ammonium bicarbonate); phase B: acetonitrile]; gradient: B%: 39% to 59%) to obtain compound 9. MS: m / z 444.1 [M+H] + .

[0520] Step 2 Compound 9 was separated and purified by SFC (chromatographic column: DAICEL CHIRALPAK AD (250 mm × 30 mm, 10 μm); mobile phase: [A phase: supercritical carbon dioxide, B phase: isopropanol (0.1% aqueous ammonia)]; gradient: B%: 35%) to give three crude compounds. SFC detection (chromatographic column: Chiralpak AD-3 150 × 4.6 mm ID, 3 μm; mobile phase: A phase: supercritical carbon dioxide, B phase: isopropanol (0.05% diethylamine); gradient: B%: 40%) gave retention times of 1.934 min for the first crude compound, 2.278 min for the second crude compound, and 2.596 min for the third crude compound.

[0521] The first crude product compound was further separated and purified by SFC (separation conditions: chromatography column: ChiralPak IH (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: ethanol (0.1% aqueous ammonia)]; gradient: B%: 30%) to obtain compounds 9a and 9b.

[0522] The second crude product compound was further separated and purified by SFC (separation conditions: chromatographic column: DAICEL CHIRALCEL OD (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: methanol (0.1% aqueous ammonia)]; gradient: B%: 40%) to obtain compound 9c.

[0523] The third crude product compound was further separated and purified by SFC (separation conditions: chromatographic column: DAICEL CHIRALPAK AD (250 mm × 30 mm, 10 μm); mobile phase: [phase A: supercritical carbon dioxide, phase B: isopropanol (0.1% aqueous ammonia)]; gradient: B%: 35%) to obtain compound 9d.

[0524] Compound 9a: SFC detection conditions: Chromatography column: Chiralpak AS-3 150 × 4.6 mm ID, 3 μm; Mobile phase: Phase A: supercritical carbon dioxide, Phase B: ethanol (0.05% diethylamine); Gradient: B%: 5% to 40% (5 to 4 min), 40% to 5% (4 to 4.2 min), 5% (4.2 to 6 min); Retention time: 3.426 min; Chiral purity: 100%. MS: m / z 444.1 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.35 (d, J = 7.3 Hz, 1H), 8.22 - 8.16 (m, 1H), 7.47 - 7.27 (m, 4H), 5.95 (s, 1H), 5.03 - 4.95 (m, 1H), 4.85 - 4.79 (m, 2H), 4.60 (br s, 1H), 4.47 - 4.38 (m, 3H), 3.99 (dd, J = 7.0, 11.5 Hz, 1H), 3.52 (s, 1H), 2.87 (s, 1H), 1.57 - 1.37 (m, 3H).

[0525] Compound 9b: SFC detection conditions: Chromatography column: Chiralpak AS-3 150 × 4.6 mm ID, 3 μm; Mobile phase: Phase A: supercritical carbon dioxide, Phase B: ethanol (0.05% diethylamine); Gradient: B%: 5% to 40% (5 to 4 min), 40% to 5% (4 to 4.2 min), 5% (4.2 to 6 min); Retention time: 3.698 min; Chiral purity: 99.27%. MS: m / z 444.1 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.37 (d, J = 7.3 Hz, 1H), 8.23 ​​(s, 1H), 7.51 - 7.34 (m, 4H), 5.80 (d, J = 3.3 Hz, 1H), 4.82 - 4.74 (m, 1H), 4.61 (br s, 2H), 4.47 (s, 3H), 4.41 - 4.35 (m, 1H), 4.13 (dd, J = 4.0, 12.5 Hz, 1H), 3.58 - 3.52 (m, 1H), 3.00 (s, 1H), 1.60 (d, J = 6.5Hz, 3H).

[0526] Compound 9c: SFC detection conditions: Chromatography column: Chiralpak AD-3 150 × 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: isopropanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 2.281 min, Chiral purity: 99.83%. MS: m / z 444.1 [M+H] + . 1H NMR (400 MHz, CD3OD) δ ppm 8.35 (d, J = 7.3 Hz, 1H), 8.21 - 8.14 (m, 1H), 7.45 - 7.27 (m, 4H), 5.94 (t, J = 6.1 Hz, 1H), 5.00 - 4.92 (m, 1H), 4.44 (s, 2H), 4.40 (s, 1H), 4.32 (dd, J = 5.5, 11.8 Hz, 1H), 3.98 (br dd, J = 6.9, 11.7 Hz, 1H), 3.52 (s, 1H), 2.89 - 2.71 (m, 3H), 1.57 - 1.37 (m, 3H).

[0527] Compound 9d: SFC detection conditions: Chromatography column: Chiralpak AD-3 150 × 4.6 mm ID, 3 μm; Mobile phase: A phase: supercritical carbon dioxide, B phase: isopropanol (0.05% diethylamine); Gradient: B%: 40%, Retention time: 2.599 min, Chiral purity: 99.29%. MS: m / z 444.1 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ ppm 8.37 (d, J = 7.0 Hz, 1H), 8.22 (s, 1H), 7.48 - 7.33 (m, 4H), 5.78 (d, J = 2.5 Hz, 1H), 4.59 (s, 3H), 4.46 (s, 3H), 4.36 (d, J = 12.3 Hz, 1H), 4.12 (dd, J = 4.1, 12.7 Hz, 1H), 3.56 - 3.51 (m, 1H), 2.99 (s, 1H), 1.58 (d, J = 6.3 Hz, 3H).

[0528] Biological Test Data Test Example 1: LU99 cell proliferation inhibition test Experimental objective: To determine the antiproliferative activity of compounds on LU99 cells. Materials: 1640 medium and penicillin / streptomycin antibiotics were purchased from Gibco, fetal bovine serum was purchased from Biosera, CellTiter-Glo (a chemiluminescent detection reagent for cell viability) reagent was purchased from Promega, and LU99 cell line was purchased from JCRB, Envision multilabel analyzer (PerkinElmer).

[0529] Experimental Procedure: 1) Cell culture: LU99 cells were seeded into an ultra-low-adsorption 96-well black-wall plate, with 80 μL of cell suspension per well, containing 1,000 LU99 cells. The cell plate was cultured overnight in a carbon dioxide incubator. 2) Administration: Test compounds were diluted 5-fold with DMSO using a pipette to eight concentrations, i.e., from 2 μM to 25.6 nM, and two wells were set up under the same conditions. 78 μL of medium was added to the middle plate, and 2 μL / well of the gradient diluted compound was transferred to the middle plate according to the corresponding positions. After uniform mixing, 20 μL / well was transferred to the cell plate. The compound concentrations transferred to the cell plate ranged from 10 μM to 0.128 nM. The cell plate was placed in a carbon dioxide incubator and cultured for 6 days. A separate cell plate was prepared, and the signal value was read as the maximum value (Max value in the formula below) on the day of drug addition and used for data analysis. 3) Cell proliferation detection: 100 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate and incubated at room temperature for 30 minutes to stabilize the luminescence signal. The data were read using a multi-label analyzer. 4) Data analysis: Convert raw data into percent inhibition using the equation (Sample-Min) / (Max-Min) × 100% to IC 50 Values ​​were obtained by curve fitting using four parameters (log(inhibitor) vs. response - obtained with the Variable slope motor in GraphPad Prism).

[0530] Experimental Results: The experimental results are shown in Table 3.

[0531] [Table 3]

[0532] Conclusion: The compounds of the present invention have significant antiproliferative activity against LU99 cells.

[0533] Test Example 2: Pharmacokinetic evaluation in mice Experimental Method: Test compounds were mixed with 5% DMSO / 10% polyethylene glycol-15 hydroxystearate / 85% water, vortexed, and sonicated to produce a clear solution at 0.2 mg / mL, which was then filtered through a microporous filter membrane for later use. Male Balb / c mice weighing 18-20 g were selected and administered the candidate compound solution intravenously at a dose of 1 mg / kg. Oral administration of the candidate compound solution at a dose of 2 mg / kg was also performed. Whole blood samples were collected at designated time points, and plasma samples were prepared. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, Inc., USA).

[0534] Definition of each parameter: IV: intravenous injection, PO: oral administration, C0: concentration required immediately after intravenous injection, C max : Maximum blood drug concentration after administration, T max : The time required to reach the maximum drug concentration after administration, T 1 / 2 : Time required for blood drug concentration to decrease by half, V dss : Apparent volume of distribution refers to the proportionality constant between the amount of drug in the body and the blood drug concentration when the drug reaches dynamic equilibrium in the body. Cl: Clearance refers to the apparent volume of distribution of a drug excreted from the body per unit time. T last : Time of final detection point, AUC 0-last : Area under the drug-time curve, which refers to the area enclosed by the blood drug concentration curve on the time axis, F: A measure of the rate and extent of drug absorption into the blood circulation, and is an important index for evaluating the degree of drug absorption. The experimental results are shown in Table 4.

[0535] [Table 4]

[0536] "--" indicates not tested or data unavailable.

[0537] Conclusion: The compounds of the present invention exhibit longer half-life, lower clearance, higher drug exposure and oral bioavailability, and have relatively good in-vivo pharmacokinetic properties.

[0538] Test Example 3: Enzyme inhibitory activity test Experimental objective: To test the inhibitory effect of the compounds of the present invention on PRMT5·MTA.

[0539] Experimental materials: PRMT5 / MEP50, peptide H4(1-21), LANCE® Ultra Europium anti-methyl histone H4 arginine 3 (H4R3me) antibody, Ulight streptavidin, LANCE detection buffer, SAM (adenosylmethionine), pigskin collagen, MTA (methylthioadenosine).

[0540] Experimental Method: (1) Preparation of buffer solution: Take 10 ml as an example. 10 mM MTA: Add 10 mg of MTA to 3296 μl of DMSO, dissolve, then aliquot and store at -80°C. It can be used immediately on the day of the experiment. The preparation scheme is shown in Table 5.

[0541] [Table 5]

[0542] (1) Preparation of compounds Compounds were dissolved in DMSO to obtain a 10 mM compound mother solution. A gradient dilution was performed on the compound dilution plate to obtain four compound wells, with sequential concentrations of 1 mM, 37.037 μM, 1.3717 μM, and 0.0508 μM. These four concentrations of compound were transferred to the compound transfer plate in a transfer volume of 8 μL per concentration. DMSO was added to empty wells of the compound transfer plate for later use. The liquid was transferred to the experimental plate using the serial dilution function of the Echo 550 microfluidic device. Once the transfer was complete, the experimental plate was obtained.

[0543] (2) Reaction A mixed solution of the enzyme solution and the substrate was prepared using a buffer solution, and the concentration of PRMT5 was 7.6 nM, the concentration of peptide H4(1-21) was 0.32 μM, and the concentration of SAM was 1.3 μM. Using an electronic multichannel pipette, 5 μL of PRMT5 solution was added per well to the compound and negative control wells of the experimental plate, and the same volume of buffer was added to the positive control well. After centrifugation at 1000 rpm for 30 seconds and gentle mixing on an orbital shaker for 30 seconds, the experimental plate was placed in a constant-temperature incubator and incubated at 25°C for 30 minutes. Next, using the same method, the substrate mixture solution was added to the positive control, negative control, and compound wells of the experimental plate, centrifuged, and incubated at 25°C for 90 minutes.

[0544] (4) Testing and calculation of results Principle: In this experiment, PE's time-resolved fluorescence resonance energy transfer technology (LANCE® Ultra) was used for detection. During the reaction, PRMT methylated the substrate polypeptide H4(1-21), and then two antibodies were added. Here, the LANCE® Ultra europium anti-methyl histone H4 arginine 3 (H4R3me) antibody acts as an energy donor and can specifically bind to the methylation site of polypeptide H4(1-21), while Ulight acts as an energy acceptor and can specifically bind to the biotin tag carried by polypeptide H4(1-21). When a laser of a specific wavelength (340 nm in this experiment) is used for excitation, the energy donor emits light at a wavelength of 615 nm. At the same time, if the spatial distance between the energy donor and the energy acceptor is sufficiently close (i.e., when two antibodies are simultaneously linked to the polypeptide H4(1-21)), energy transfer occurs between the energy donor and the energy acceptor, causing the energy acceptor to emit light at a wavelength of 665 nm. A plate reader was used to detect the two emitted lights, and the ratio of the 665 nm and 615 nm signals was calculated. The relevant parameters of the test sample were then calculated by plotting and calculating.

[0545] The antibody mixture solution was prepared using LANCE buffer. The antibody concentration of LANCE® Ultra Europium anti-methyl histone H4 arginine 3 (H4R3me) was 4 nM, and the Ulight concentration was 53.3 nM. Using an electronic multichannel pipette, the detection solution was added to the positive control, negative control, and compound wells of the experimental plate at a volume of 10 μL per well. The plate was centrifuged, incubated at room temperature for 1 hour, and read using an Envision 2104 plate reader. Compound inhibition rates were calculated using interpolation, and compound inhibition curves were generated using a four-parameter logarithm equation and XLfit software, which allowed for the determination of minimum inhibition rate, maximum inhibition rate, and IC. 50 The relevant parameters were calculated as follows:

[0546]

number

[0547] Experimental Results: The experimental results are shown in Table 6.

[0548] [Table 6]

[0549] Conclusion: The compounds of the present invention showed significant inhibitory activity against PRMT5·MTA.

[0550] Test Example 4: In vitro detection of SDMA levels in HCT116 WT / MTAP KO cells Test materials: RPMI-1640 medium was purchased from GIbco, penicillin / streptomycin antibiotics from Vicente, and fetal bovine serum from Biosera. Protein primary antibodies and fluorescent secondary antibodies were purchased from Cell Signaling Technology. HCT116 WT cell line was purchased from Nanjing Cobioer Biosciences Co., Ltd. HCT116 MTAP KO cells were purchased from Wuhan Heyan Biomedical Technology Co., Ltd. 4% paraformaldehyde fixative (PFA), Triton-100, and other fixatives were purchased from Beyotime Biotechnology. Envision Multilabel Analyzer (PerkinElmer) was used.

[0551] Experimental Method: Cells were seeded into a 96-well black-wall plate, with 80 μL of cell suspension per well, containing 2000 cells, and the cell plate was incubated overnight in a carbon dioxide incubator. Test compounds were diluted 5-fold at eight concentrations, from 2 μM to 25.6 nM, using a pipette. Two wells were placed under the same conditions. 78 μL of medium was added to the middle plate, and 2 μL / well of the gradient diluted compound was transferred to the corresponding positions. After uniform mixing, 20 μL / well was transferred to the cell plate. The compound concentrations transferred to the cell plate ranged from 10 μM to 0.128 nM. After culturing the cell plates in a carbon dioxide incubator for 4 days, the cells were removed, the medium discarded, and fixed with 4% paraformaldehyde fixative for 30 minutes. Then, the cells were washed three times with PBS (phosphate buffered saline), permeabilized with 0.1% Triton for 20 minutes, washed three times with PBS, blocked with 0.1% BSA (bovine serum albumin) for 1 hour at room temperature, incubated overnight at 4°C with a protein primary antibody (anti-SDMA or anti-β-actin, 1:1000 dilution), washed three times with PBST (phosphate buffered saline + 0.1% Tween 20), incubated with a fluorescent secondary antibody (FITC or Cy5, 2:1000 dilution) for 2 hours at room temperature, washed three times with PBST, and read on an Envision (FITC and Cy5) microscope.

[0552] Data Analysis: The inhibition rate (%) of the sample wells treated with FITC / Cy5 and compounds was calculated based on the measured fluorescence intensity. FITC / Cy5=(FITCsample-FITCblank) / (FITCsample-FITCblank)

[0553] inhibition%=[1-(FITC / Cy5comp.) / (FITC / Cy5DMSO)]×100

[0554] Furthermore, four-parameter curve fitting was performed to obtain IC 50 The values ​​(obtained using GraphPad Prism's log(inhibitor) vs. response - Variable slope mode) were obtained. The experimental results are shown in Table 7.

[0555] [Table 7]

[0556] Conclusion: The compounds of the present invention can significantly inhibit SDMA levels in MTAP-deficient tumor cells, but have weak inhibitory activity against SDMA levels in non-MTAP-deficient cells, demonstrating excellent selectivity.

[0557] Test Example 5: In vitro liver microsome metabolic stability test of compounds Test materials: Liver microsomes: purchased from Corning or Xenotech and stored in a -80°C freezer; reduced nicotinamide adenine dinucleotide phosphate (NADPH); supplier: Chem-impex international; catalog number: 00616; control compounds: testosterone, diclofenac, propafenone.

[0558] Experimental steps: (1) Preparation of working solution Stock solution: 10 mM in DMSO. Working concentration preparation: Stock solution was diluted to 100 μM with 100% acetonitrile (organic phase content: 99% acetonitrile, 1% DMSO). (2) Experimental steps Two 96-well incubation plates, named T60 incubation plate and NCF60 incubation plate, respectively, were prepared. 445 μL of microsome working solution (liver microsome protein concentration: 0.56 mg / mL) was added to each of the T60 and NCF60 incubation plates, and then the incubation plates were placed in a 37°C water bath and incubated for approximately 10 minutes. After preincubation, 5 μL of sample or control compound working solution was added to each well of the T60 and NCF60 incubation plates and mixed thoroughly. 50 μL of potassium phosphate buffer was added to each well of the NCF60 incubation plate to initiate the reaction. 180 μL of stop solution (acetonitrile containing 200 ng / mL tolbutamide and 200 ng / mL labetalol) and 6 μL of NADPH regenerating system working solution were added to the TO stop plate. 54 μL of sample from the T60 incubation plate was transferred to the TO stop plate (TO sample production). 44 μL of NADPH regenerating system working solution was added to each well of the T60 incubation plate to initiate the reaction. 54 μL of microsome working solution, 6 μL of NADPH regenerating system working solution, and 180 μL of stop solution alone were added to a blank plate. Therefore, in the test compound or control compound samples, the final concentrations of the compounds, testosterone, diclofenac, and propafenone were 1 μM, the concentration of liver microsomes was 0.5 mg / mL, and the final concentrations of DMSO and acetonitrile in the reaction system were 0.01% and 0.99%, respectively.

[0559] After incubation for an appropriate time (e.g., 5, 15, 30, 45, and 60 minutes), 180 μL of stop solution (a solution in acetonitrile containing 200 ng / mL tolbutamide and 200 ng / mL labetalol) was added to each sample well of the stop plate, and then 60 μL of the sample was removed from the T60 incubation plate to stop the reaction.

[0560] All sample plates were shaken uniformly and centrifuged at 3220 × g for 20 minutes. 80 μL of the supernatant was then taken from each well and diluted with 240 μL of pure water for liquid chromatography-tandem mass spectrometry. The experimental results are shown in Table 8.

[0561] [Table 8]

[0562] Conclusion: The compounds of the present invention showed moderate metabolism in human liver microsome tests and good metabolic stability.

[0563] Test Example 6: Plasma protein binding rate test of compounds Experimental objective: To evaluate the protein binding rate of the compound of the present invention in the plasma of CD-1 mice, SD rats, beagles and humans by equilibrium dialysis.

[0564] Experimental scheme: 796 μL of blank plasma was collected from humans, beagles, SD rats, and CD-1 mice (plasma purchased from Bioreclamation IVT), and the test compound working solution or warfarin working solution was added to achieve final concentrations of 2 μM for both the test compound and warfarin in the plasma sample. The samples were mixed thoroughly. The final concentration of DMSO in the organic phase was controlled at 0.5%, and 50 μL of the test compound and warfarin plasma sample was transferred to a sample receiving plate. The corresponding volume of blank plasma or buffer was immediately added to each sample well so that the final volume of each well was 100 μL and the plasma:dialysis buffer volume ratio was 1:1. A stop solution was then added to each sample, and this sample was used as the TO sample for recovery and stability measurements. The test compound and warfarin plasma sample were added to the dosing end of each dialysis well, and blank dialysis buffer was added to the corresponding receiving end of the dialysis well. The dialysis plate was then sealed with a gas-permeable membrane and placed in a humidified 5% CO2 incubator, where it was incubated at 37°C with shaking at 100 rpm for 4 hours. After dialysis was completed, 50 μL of the dialysis buffer sample and the dialyzed plasma sample were pipetted into a new sample receiving plate. The final volume of each sample well was 100 μL, and the corresponding volume of plasma or buffer was added to the sample so that the plasma:dialysis buffer volume ratio was 1:1. All samples were analyzed by LC / MS / MS after protein precipitation, and the compound release rate was calculated using the following formula: PPB_Unbound(%)=100×F C / T C where F C is the concentration of the compound at the buffer end of the dialysis plate, and T Cis the concentration of the compound at the plasma end of the dialysis plate, and T0 is the concentration of the compound in the plasma sample at time zero. The experimental results are shown in Table 9. Here, H stands for human, R for rat, M for mouse, and D for dog.

[0565] [Table 9]

[0566] Conclusion: The compounds of the present invention have a reasonable ratio of free drug concentration in the plasma of different species and good drug formation.

[0567] Test Example 7: Antitumor activity test of compounds in BALB / c nude mouse subcutaneous xenograft tumor model Experimental Objective: To evaluate the tumor inhibitory effects of compounds of the present disclosure in a subcutaneous xenograft tumor model of human large cell lung carcinoma LU99.

[0568] Experimental Method: Female BALB / c nude mice were subcutaneously inoculated with the human large cell lung cancer LU99 cell line, and then randomly divided into groups of 6 mice each based on body weight and tumor volume. Administration plan 1: After inoculation, tumor volume is 150-190 mm 3 Administration began when the rats reached 100 mg / kg, and the vehicle was 5% DMSO / 10% Solutol / 85% double distilled water. Control group: The vehicle was administered intragastrically (po) at a dose of 0.1 mL / 10 g once daily (QD). Treatment group: The test compound was dissolved in the vehicle and administered intragastrically once daily at a dose of 7.5 mg / kg or 15 mg / kg. Administration regimen 2: After inoculation, tumor volume is 110-150 mm 3 Administration began when the rats reached 100 mg / kg, and the vehicle was 5% DMSO / 10% Solutol / 85% double distilled water. Control group: The vehicle was administered intragastrically (po) at a dose of 0.1 mL / 10 g once daily (QD). Treatment group: The test compound was dissolved in the vehicle and administered intragastrically once daily at a dose of 5 mg / kg, 15 mg / kg, or 50 mg / kg. After dividing the experimental groups, the mice were weighed twice a week, and tumor diameters were measured using calipers to calculate tumor volumes. Calculation formula: tumor volume = major diameter × minor diameter. 2 / 2. Next, the tumor growth inhibition rate (TGI) was calculated. Calculation formula: TGI (%) = [1-(T i -T0) / (C i -C0)] × 100, where T i is the mean tumor volume of a particular treatment group on a particular day, T0 is the mean tumor volume of the treatment group at the start of treatment, and C is the mean tumor volume of the treatment group at the start of treatment on a particular day (T i C0 is the average tumor volume of the control group at the start of administration (the same day as the start of administration), and C0 is the average tumor volume of the control group at the start of administration. On day 19 after experimental administration, the mice were euthanized and samples were collected.

[0569] Test Results: The weight change curves of mice in each group are shown in Figures 9 and 10, and the average tumor volumes of each group at each time point are shown in Figures 11 and 12. TGI was calculated based on the average tumor volumes 19 days after administration, and the specific experimental results are shown in Table 10.

[0570] [Table 10]

[0571] Experimental conclusion: The compound of the present invention exhibits excellent antitumor effects in vivo, and the body weight of mice after administration is well maintained.

[0572] Test Example 8: In vitro MDCKII-MDR1 monolayer cell permeability test Experimental Objective: To evaluate the permeability and efflux ratio of the compounds of the present invention using the MDCKII-MDR1 monolayer cell test system, and to determine the compound's ability to cross the blood-brain barrier and its efflux potential through the P-GP transporter.

[0573] Experimental Method: MDR1-MDCKII cells (obtained from the Netherlands Cancer Institute) were plated in 96-well plates (obtained from Corning) at a cell density of 2.5 × 10 5Cells were seeded at 1000 cells / mL and cultured for 4 to 7 days to form a copolymerized cell monolayer. Hank's balanced salt buffer (pH 7.40 ± 0.05) containing 10 mM 4-hydroxyethylpiperazineethanesulfonic acid was used as the transport buffer. Bidirectional transport of test compounds was evaluated at a concentration of 50 μM while maintaining the DMSO concentration in the culture reaction system at less than 1%. After adding the sample, the cell plate was incubated at 37 ± 1°C, 5% CO2, and saturated humidity for 150 minutes. All samples were quantitatively analyzed using LC-MS / MS. The apparent permeability coefficient (P app , cm / s), and the discharge ratio was calculated using the following formula:

[0574] Apparent permeability coefficient (P app , cm / s) was calculated using the following formula: app =(dC r / d t )×V r / (A×C0), where dC r / d t is the cumulative concentration of the compound at the receiving end in unit time (μM / s), and V r is the volume of the solution at the receiving end (the volumes of the solutions at the upper and lower ends are 0.075 mL and 0.250 mL, respectively), and A is the relative surface area of ​​the cell monolayer (0.0804 cm 2 ), where C0 is the starting concentration (nM) of the test substance or the peak area ratio of the control substance. The efflux ratio was calculated using the following formula: efflux ratio = P app (BA) / P app (AB). The experimental results are shown in Table 11.

[0575] [Table 11]

[0576] Conclusion: In the permeability experiment of MDCKII-MDR1 monolayer cells (highly expressing human p-gp), the compounds of the present invention showed good permeability.

[0577] Test Example 9: hERG potassium channel inhibition test Experimental Objective: To detect the effects of the compounds of the present invention on the hERG potassium ion channel using electrophysiological manual whole-cell voltage clamp technique.

[0578] Test materials: The extracellular solution (mM): 140 sodium chloride, 5 potassium chloride, 1 calcium chloride, 1.25 magnesium chloride, 10 HEPES, 10 glucose, pH adjusted to 7.4 with sodium hydroxide. The intracellular solution (mM): 140 potassium chloride, 2 magnesium chloride, 1 calcium chloride, 10 EGTA, 10 HEPES, pH adjusted to 7.2 with potassium hydroxide. HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid); EGTA: ethylenebis(oxyethylenenitrilo)tetraacetic acid.

[0579] Experimental Method: (1)Cell culture CHO cells stably expressing hERG were cultured in 35 mm cell dishes at 300,000 cells per dish and placed in a 37°C, 5% CO2 incubator. They were subcultured at a 1:5 ratio every 48 hours in a medium formulation of 90% F12, 10% fetal bovine serum, 100 μg / mL G418, and 100 μg / mL hygromycin B. On the day of the experiment, the cell medium was aspirated, the cells were washed once with extracellular solution, and then 0.25% trypsin-EDTA solution was added and digested for 3-5 minutes at room temperature. The digestion solution was aspirated, and the cells were resuspended in extracellular solution and transferred to a dish for electrophysiological recording.

[0580] (2) Sample preparation Test compounds were prepared as 15.00 mM stock solutions in DMSO. On the day of testing, the test compound stock solutions were serially diluted 3-fold with DMSO. 10 μL of test compound stock solution was added to 20 μL of DMSO to obtain intermediate concentrations of test compounds serially diluted with DMSO: 5.00, 1.67, 0.56, 0.19, 0.062, and 0.021 mM. Next, 10 μL of the intermediate test compound concentration was added to 4990 μL of extracellular solution and diluted 500-fold to obtain the final concentrations required for testing. The highest test concentration was 10.00 μM, followed by 10.00, 3.33, 1.11, 0.37, 0.12, and 0.041 μM, respectively.

[0581] Preparation of the positive control substance, cisapride: 150.00 μM cisapride stock solution was serially diluted 3-fold using 100% DMSO. 10 μL of the 150.00 μM cisapride stock solution was added to 20 μL of DMSO to obtain five intermediate concentrations of cisapride serially diluted with DMSO: 150.00, 50.00, 16.67, 5.56, and 1.85 μM. 10 μL of the intermediate concentrations of cisapride were then added to 4990 μL of extracellular solution and diluted 500-fold to obtain the final concentrations required for testing. The highest test concentration was 300.00 μM, followed by five concentrations of 300.00, 100.00, 33.33, 11.11, and 3.70 nM, respectively. The DMSO content of the final test concentrations did not exceed 0.2%, indicating that DMSO at these concentrations did not affect hERG potassium channel currents.

[0582] (3) Electrophysiological recording process hERG potassium channel currents were recorded from CHO cells stably expressing the hERG potassium channel using the whole-cell patch clamp technique at room temperature. Glass microelectrodes were prepared by stretching glass electrode blanks (BF150-86-10, Sutter) in a stretching machine. After filling the electrode with the internal solution, the tip resistance was approximately 2-5 MΩ. The glass microelectrode was inserted into the amplifier probe and connected to an Axopatch 200B (Molecular Devices) patch clamp amplifier. The clamp voltage and data recording were computer-controlled and recorded using pClamp 10 software, with a sampling frequency of 10 kHz and a filtering frequency of 2 kHz. After whole-cell recordings were obtained, the cells were clamped at -100 mV. The hERG potassium current (I hERG) was induced by a voltage step: a 2-second depolarization from -100 mV to +20 mV, followed by a repolarization to -50 mV and a 1-second return to -100 mV. The voltage stimulus was applied every 5 seconds, and the peak value of the hERG potassium current was detected when repolarization reached -50 mV. The administration process was initiated after confirming that the hERG potassium current was stable (1 minute). Sequential administration of the sample was initiated from the lowest test concentration, and each test concentration was administered for at least 1 minute. Test compound concentrations were administered sequentially by gravity, starting with the lowest test concentration, and each test concentration was administered for at least 1 minute or up to 3 minutes until an inhibitory steady state was reached. Each concentration of the test compound was tested in at least three cells (n≧3), and each concentration of the positive control substance was tested in at least two cells (n≧2).

[0583] (4) Data processing Data analysis and processing were performed using pClamp 10, GraphPad Prism 8 and Excel software. The inhibition of hERG potassium current (peak hERG tail current induced at -50 mV) at different test compound concentrations was calculated using the following formula: Inhibition%=[1-(I / Io)]×100% Here, Inhibition % represents the rate of inhibition of the hERG potassium current by the test compound, and I and Io represent the amplitude of the hERG potassium current after and before drug administration, respectively.

[0584] IC of test compound 50 was calculated using GraphPad Prism 8 software by fitting the following equation: Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X))×HillSlope)) Here, X is the Log value of the detected concentration of the test compound, Y is the inhibition rate at the corresponding concentration, Bottom and Top are the minimum and maximum values ​​of the inhibition rate, respectively, and HillSlope is the slope of the curve.

[0585] Compound of the present invention hERG IC 50 The values ​​are as shown in Table 12.

[0586] [Table 12]

[0587] Conclusion: The compounds of the present invention have weak inhibition of hERG potassium current and low risk of cardiotoxicity.

Claims

1. A compound represented by formula (III) or a pharmaceutically acceptable salt thereof: 【Chemical 1】 (however, E １ is CH ２ , NH and O; Structural Unit 【Chemistry 2】 teeth, 【Chemistry 3】 is selected from Each R １ are each independently H, halogen, or C １－３ Alkyl and C １－３ alkoxy, wherein C １－３ Alkyl and C １－３ Alkoxy is each independently one, two or three R ａ is optionally replaced by R ６ is C ２－４ Alkynyl and C ２－４ alkenyl, wherein C ２－４ Alkynyl and C ２－４ Alkenyl is each independently one, two or three R ｂ is optionally replaced by Or, R ６ is a halogen, C １－３ Alkyl and C １－３ alkoxy, wherein C １－３ Alkyl and C １－３ Alkoxy is each independently one, two or three R ｂ is optionally replaced by, in which case the structural unit 【Chemistry 4】 is selected from Ring A is a 5- to 6-membered heteroaryl, C ４－６ cycloalkenyl or 5- to 6-membered heterocycloalkenyl, wherein said 5- to 6-membered heteroaryl, C ４－６ The cycloalkenyl or 5- to 6-membered heterocycloalkenyl each independently has one, two, or three R ｃ is optionally replaced by T １ and T ２ are each independently CR ７ and N; T ３ , T ４ and T ５ are each independently CR ４ and N; T ６ , T ７ and T ８ are each independently CR ５ and N; R ３ is H, C １－３ Alkyl, C ３－６ cycloalkyl, 4- to 6-membered heterocycloalkyl, —C １－３ Alkyl-C ３－６ Cycloalkyl and -C １－３ alkyl-4 to 6 membered heterocycloalkyl, １－３ Alkyl, C ３－６ cycloalkyl, 4- to 6-membered heterocycloalkyl, —C １－３ Alkyl-C ３－６ Cycloalkyl and -C １－３ The alkyl-4 to 6-membered heterocycloalkyl is each independently one, two, or three R ｅ is optionally replaced by R ４ , R ５ and R ７ are each independently H, halogen, or C １－３ Alkyl and C １－３ alkoxy, wherein C １－３ Alkyl and C １－３ Alkoxy is each independently one, two or three R ｄ is optionally replaced by Each R ａ , each R ｃ , each R ｄ and each R ｅ are each independently H, D, halogen, OH, or NH ２ , C.H. ３ and CDs ３ is selected from Each R ｂ are each independently H, D, halogen, OH, or NH ２ , C １－３ Alkyl, C １－３ Alkoxy, C １－３ alkylamino and 4- to 6-membered heterocycloalkyl; １－３ Alkyl, C １－３ Alkoxy and C １－３ alkylamino is optionally substituted by 1, 2 or 3 R, each independently; Each R is independently H, D, halogen, OH, or NH ２ , C.H. ３ , C.F. ３ and CDs ３ is selected from n is selected from 0, 1, 2 and 3; m is selected from 0, 1 and 2; p is selected from 0, 1 and 2, and m and p are not simultaneously 0; The "hetero" in the 4- to 6-membered heterocycloalkyl, 5- to 6-membered heteroaryl, or 5- to 6-membered heterocycloalkenyl each independently represents 1, 2, 3, or 4 heteroatoms or heteroatom groups selected from -O-, -NH-, -S-, and N.

2. Each R ｂ are each independently H, D, F, Cl, OH, or NH ２ , C.H. ３ , C.H. ２ CH ３ , OCH ３ , OCH ２ CH ３ , NHCH ３ , NHCH ２ CH ３ , N(CH ３ ) ２ , pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl, wherein said CH ３ , C.H. ２ CH ３ , OCH ３ , OCH ２ CH ３ , NHCH ３ , NHCH ２ CH ３ , N(CH ３ ) ２ , pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl are each independently optionally substituted by 1, 2, or 3 R, or each R ｂ are each independently H, D, F, Cl, OH, or NH ２ , C.H. ３ , CDs ３ , C.F. ３ , -C(CH ３ ) ２ OH, N(CH ３ ) ２ , 【Chemistry 5】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

3. R １ are H, F, Cl, Br, I, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , OCH ３ , OCH ２ CH ３ , OCH ２ CH ２ CH ３ and OCH(CH ３ ) ２ wherein said CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , OCH ３ , OCH ２ CH ３ , OCH ２ CH ２ CH ３ and OCH(CH ３ ) ２ each independently represents one, two or three R ａ or optionally substituted by R １ is F and CH ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

4. R ３ is H, CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , cyclopropyl, cyclobutyl, oxetanyl, azetidinyl, -CH ２ -cyclopropyl, -CH ２ CH ２ -cyclopropyl and -CH ２ -oxetanyl, wherein said CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , cyclopropyl, cyclobutyl, oxetanyl, azetidinyl, -CH ２ -cyclopropyl, -CH ２ CH ２ -cyclopropyl and -CH ２ -oxetanyl is each independently one, two or three R ｅ or optionally substituted by R ３ is H, CH ３ , C.F. ３ , CDs ３ , C.H. ２ CH ３ , CH(CH ３ ) ２ , cyclopropyl, cyclobutyl, 【Chemistry 6】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

5. Each R ４ are each independently H, F, Cl, Br, I, or CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , OCH ３ , OCH ２ CH ３ , OCH ２ CH ２ CH ３ and OCH(CH ３ ) ２ wherein said CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , OCH ３ , OCH ２ CH ３ , OCH ２ CH ２ CH ３ and OCH(CH ３ ) ２ each independently represents one, two or three R ｄ or each R ４ are each independently H, F, Cl, Br and CH ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

6. Each R ５ are each independently H, F, Cl, Br, I, or CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , OCH ３ , OCH ２ CH ３ , OCH ２ CH ２ CH ３ and OCH(CH ３ ) ２ wherein said CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ , CH(CH ３ ) ２ , OCH ３ , OCH ２ CH ３ , OCH ２ CH ２ CH ３ and OCH(CH ３ ) ２ each independently represents one, two or three R ｄ or each R ５ are each independently H, F, Cl, Br and CH ３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

7. R ６ is selected from ethynyl, propynyl, vinyl, and propenyl, wherein said ethynyl, propynyl, vinyl, and propenyl are each independently selected from one, two, or three R ｂ or optionally substituted by R ６ teeth, 【Chemistry 7】 4. The compound of claim 1 or 3, or a pharmaceutically acceptable salt thereof, selected from:

8. Structural Unit 【Chemistry 8】 Alternatively, the structural unit 【Chemistry 9】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

9. Ring A is selected from pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl, oxazolyl, triazolyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, oxacyclopentenyl, oxacyclohexenyl, azacyclopentenyl, and azacyclohexenyl, wherein said pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, furanyl, oxazolyl, triazolyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, oxacyclopentenyl, oxacyclohexenyl, azacyclopentenyl, and azacyclohexenyl each independently contain one, two, or three R ｃ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, optionally substituted by:

10. Structural Unit 【Chemistry 10】 teeth, 【Chemistry 11】 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

11. 11. The compound of any one of claims 1 to 10, selected from the following formulas: or a pharmaceutically acceptable salt thereof. 【Chemistry 12】 (however, Structural Unit 【Chemistry 13】 is selected from Ring A is selected from a 5- to 6-membered heteroaryl or a 5- to 6-membered heterocycloalkenyl, each of which independently has one, two, or three R ｃ is optionally replaced by T ３ , T ４ and T ５ are each independently CR ４ is selected from T ８ are each independently selected from CH and N; Each R １ are each independently H, halogen, or C １－３ alkyl, wherein C １－３ The alkyl may optionally have one, two or three R ａ is replaced by R ３ is H and C １－３ alkyl, wherein C １－３ The alkyl may optionally have one, two or three R ｅ is replaced by R ４ is H, halogen and C １－３ alkyl, wherein C １－３ The alkyl may optionally have one, two or three R ｄ is replaced by R ６ is C ２－４ Alkynyl and C ２－４ alkenyl, wherein C ２－４ Alkynyl and C ２－４ Alkenyl is each independently one, two or three R ｂ is optionally replaced by Each R ａ , each R ｃ , each R ｄ and each R ｅ are each independently H, D, halogen, OH, or NH ２ , C.H. ３ and CDs ３ is selected from Each R ｂ are each independently H, D, halogen, or C １－３ Alkyl, C １－３ alkylamino and 4- to 6-membered heterocycloalkyl; １－３ Alkyl, C １－３ Alkoxy and C １－３ alkylamino is optionally substituted by 1, 2 or 3 R, each independently; Each R is independently H, D, halogen, OH, or NH ２ , C.H. ３ , C.F. ３ and CDs ３ is selected from n is selected from 0 and 1, 2 and 3; m is selected from 0 and 1.

12. 11. The compound of any one of claims 1 to 10, selected from the following formulas: or a pharmaceutically acceptable salt thereof. 【Chemistry 14】 (however, Each R １ are each independently H, halogen, or C １－３ alkyl, wherein C １－３ The alkyl may optionally have one, two or three R ａ is replaced by R ２ is H, halogen and C １－３ alkyl, wherein C １－３ The alkyl may optionally have one, two or three R ｂ is replaced by E １ is selected from O, Structural Unit 【Chemistry 15】 is selected from Ring A is selected from 5- to 6-membered heteroaryl and 5- to 6-membered heterocycloalkenyl, each of which independently has one, two, or three R ｃ is optionally replaced by T ３ , T ４ and T ５ are each independently CR ４ is selected from T ６ and T ７ are each independently CH; T ８ is selected from CH and N; R ３ is H and C １－３ alkyl, wherein C １－３ The alkyl may optionally have one, two or three R ｅ is replaced by R ４ is H, halogen and C １－３ alkyl, wherein C １－３ The alkyl may optionally have one, two or three R ｄ is replaced by Each R ａ , each R ｂ , each R ｃ , each R ｄ and each R ｅ are each independently H, halogen, OH, or NH ２ , C.H. ３ and CDs ３ is selected from n is selected from 0, 1, 2 and 3; The "hetero" in the 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkenyl represents 1, 2, 3, or 4 heteroatoms or heteroatom groups independently selected from -O-, -NH-, -S-, and -N-.

13. A compound represented by the following formula or a pharmaceutically acceptable salt thereof: 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemical 21】 【Chemical 22】 【Chemical 23】 【Chemistry 24】 【Chemistry 25】 【Chemical 26】 【Chemical 27】 【Chemical Formula 28】 【Chemical 29】 【Chemistry 30】

14. 14. The compound of claim 13 selected from the following formulas: or a pharmaceutically acceptable salt thereof. 【Chemical Formula 31】 【Chemical 32】 【Chemical 33】 【Chemical 34】 【Chemistry 35】 【Chemical 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical Formula 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemical 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 【Chemistry 50】 【Chemistry 51】 【Chemistry 52】 【Chemistry 53】 【Chemical 54】 【Chemistry 55】 【Chemical 56】 【Chemical 57】 【Chemistry 58】 【Chemical Formula 59】 【Chemistry 60】 【Hua 61】 【Hua 62】 【Chemistry 63】 【Hua 64】 【Chemistry 65】

15. Use of the compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease associated with PRMT5.

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