Compounds targeting GAS41, GAS41 inhibitors and pharmaceutical compositions and uses thereof

Abstract

The present invention discloses a compound targeting GAS41, which is a compound represented by Formula (II) or Formula (VII), or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by Formula (II) or Formula (VII). The compound of the present invention can specifically bind to GAS41 and inhibit the expression or activity of GAS41, thereby inhibiting the activity of tumor cells in vivo and in vitro, and can effectively prevent and treat cancer or tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to a compound targeting GAS41, a GAS41 inhibitor, a pharmaceutical composition and uses thereof. Background Art

[0002] GAS41 (Glioma-amplified sequence 41) was first discovered in human glioma cells. Overexpression of this gene has been detected in 80% of grade I astrocytomas and early-stage cancer cells, making it a target gene for cancer development. The GAS41 gene is located on chromosome 12q13-15 and comprises seven exons. The protein encoded by GAS41 consists of 227 amino acids and includes an N-terminal YEATS domain and a C-terminal coiled-coil domain. As a subunit shared by the histone acetyltransferase Tip60 and the chromatin remodeling complex SRCAP in mammalian cells, GAS41 plays a critical role in cell survival and growth. Histone acetyltransferase Tip60, which has at least 16 subunits, plays important roles in DNA damage repair, transcriptional regulation, chromatin structure changes, cell migration, meiosis, and the interaction and expression regulation of tumor-related factors. GAS41, a key subunit of Tip60, has also been reported in recent years to be closely involved in the regulation of the p53 signaling pathway. During normal cell proliferation, GAS41 anchors to the promoter regions of the p14ARF and p21 genes, directly or indirectly recruiting transcription factors to inhibit the expression of these two genes, thereby blocking the p53 signaling pathway.

[0003] GAS41 is a key regulatory factor in tumorigenesis. Comparison of gene expression profiles in 15 drug-resistant and 11 sensitive ovarian cancer patients revealed that the transcription factors GAS41 and TFEB1 regulate the expression of downstream target genes, suggesting that these transcription factors influence the mechanism of drug resistance in ovarian cancer. Using siRNA to knock down GAS41 and TFEB1 by 70% individually restored the drug sensitivity of ovarian cancer cells by 20% to 30%. Simultaneous knockdown of both GAS41 and TFEB1 by siRNA reversed drug resistance in ovarian cancer cells by 35%. NuMA (nuclear mitotic apparatus protein) is a nuclear matrix protein that participates in spindle formation during mitosis and migrates from the interphase nuclear matrix to the spindle poles. Yeast two-hybrid analysis revealed that GAS41 binds to the C-terminal coiled-coil region of NuMA, suggesting an interaction between NuMA and GAS41. Overexpression of GAS41 leads to the formation of multiple spindles during mitosis in glioma cells, accompanied by irregular chromosome arrangement, suggesting that GAS41 overexpression contributes to karyotypic abnormalities in glioma cells. GAS41, a proto-oncogene essential for cancer cell proliferation and survival, is amplified in human non-small cell lung cancer (NSCLC) cells, with GAS41 transcript levels significantly elevated in all NSCLC subtypes. Soft agar colony formation assays have demonstrated that knockdown of GAS41 inhibits the proliferation of lung adenocarcinoma tumor cells. In mouse xenograft models, GAS41 deletion or mutation inhibits tumor growth, while ectopic expression of wild-type GAS41 restores proliferation and anchorage-independent growth in GAS41-deficient cells. GAS41 has been shown to be a histone acetylation reader that promotes histone H2A.Z deposition in NSCLC cells. Depletion of GAS41 or disruption of the interaction of the YEATS domain with acetylated histones affects the association of the histone variant H2A.Z with chromatin, thereby inhibiting cancer cell growth and survival in vitro and in vivo.

[0004] GAS41 is a histone acetylation reader. GAS41 binds to histone H3 acetylated at H3K27 and H3K14, acting as a histone acetylation "reader." While high-resolution cryo-EM structures of GAS41 and acetylated histone H3K27ac have been published, few inhibitors targeting GAS41 have been reported. Therefore, research is underway to develop a small molecule inhibitor targeting GAS41. Summary of the Invention

[0005] The present invention aims to at least partially address one of the technical problems existing in the prior art. To this end, the present invention provides a compound targeting GAS41, which can bind to GAS41 or further inhibit the expression or activity of GAS41, thereby inhibiting the activity of tumor cells in vivo and in vitro.

[0006] In one aspect of the present invention, the present invention provides a compound, which is a compound represented by formula (I) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (I):

[0007]

[0008] Among them, ring A is

[0009] Y1 is -C(O)-NR 1a R 1a 'or

[0010] R1 is H, -COOH, -C(O)-NR optionally substituted with one or more R6 1a R 1a '、 -C optionally substituted with one or more R6 1-10 alkyl, -C optionally substituted with one or more R6 1-10 Alkoxy, optionally substituted by one or more R6 -C 1-10 heteroalkyl, -C optionally substituted with one or more R6 2-10 Alkenyl, -C optionally substituted with one or more R6 2-10 Heteroalkenyl, -C optionally substituted with one or more R 2-10 Alkynyl or -C optionally substituted with one or more R6 2-10 heteroalkynyl;

[0011] R 1a and R 1a ' are each independently selected from H, -C(O)-OR optionally substituted with one or more R6 1a , -C optionally substituted with one or more R7 1-10 Alkyl, optionally substituted with one or more R7-C 1-10 Alkylene-R 1c , -C optionally substituted with one or more R7 1-10 Alkoxy, optionally substituted with one or more R7-C 1-10 Alkyleneoxy-R 1c , -C optionally substituted with one or more R7 1-10 heteroalkyl, -C optionally substituted with one or more R7 1-10Heteroalkylene-R 1c , -C optionally substituted with one or more R7 1-10 Alkenyl, optionally substituted with one or more R7-C 1-10 Alkenylene-R 1c , an aromatic ring group optionally substituted by one or more R7, a heteroaryl group optionally substituted by one or more R7, a saturated or unsaturated 4-10 membered cycloalkyl group optionally substituted by one or more R7, or a saturated or unsaturated 4-10 membered heterocycloalkyl group optionally substituted by one or more R7; or N and R 1a With R 1a ' are connected together to form a saturated or unsaturated 4-10 membered heterocyclic alkyl group containing R 1a and R 1a ' 4-10 membered heterocycloalkyl is optionally substituted by one or more R8;

[0012] R 1b -C(O)-4-10 membered heterocycloalkyl-C(O)-;

[0013] R 1c -OH, -N(R 1d )2、-C(O)-NR 1d R 1d '、-C(O)-OR 1d , a 5- to 10-membered aromatic ring group optionally substituted by one or more R9, a 5- to 10-membered heteroaryl group optionally substituted by one or more R9, a saturated or unsaturated 4- to 10-membered cycloalkyl group optionally substituted by one or more R9, or a saturated or unsaturated 4- to 10-membered heterocycloalkyl group optionally substituted by one or more R9;

[0014] R 1d and R 1d ' are each independently selected from H, -C 1-4 Alkyl, -C 1-4 Alkoxy or -C 1-4 Haloalkyl;

[0015] R2, R2', R3 and R3' are each independently halogen, H, -NO2, -CN, -C(O)-R 2a 、-C(O)-OR 2a 、-N(R 2b )-(CO) n -R 2a 、-N(R 2b )-(CO) n -OR 2a 、-N(R 2b )-(CO) n -NR 2a R 2a '、-S(O)mC0-10 Alkyl, -SO2NR 2a R 2a '、-NR 2a R 2a ', optionally one or more R 10 Substituted -C 1-10 Alkoxy-(CO) n -N(R 2b )-R 2a , optionally one or more R 10 Substituted-C(O)-C 1-10 Alkoxy, optionally substituted by one or more R 10 Substituted -C 1-10 Alkyl, optionally substituted by one or more R 10 Substituted -C 1-10 Alkoxy, optionally substituted by one or more R 10 Substituted -C 1-10 Heteroalkyl, optionally substituted by one or more R 10 Substituted -C 2-10 Alkenyl, optionally substituted by one or more R 10 Substituted -C 2-10 Alkynyl, optionally substituted with one or more R 10 Substituted 4-10 membered cycloalkyl, optionally substituted by one or more R 10 substituted 4-10 membered heterocycloalkyl, optionally substituted by one or more R 10 substituted 5-10 membered aromatic ring group or optionally substituted with one or more R 10 substituted 5- to 10-membered heteroaryl;

[0016] R4, R4', R5 and R5' are each independently halogen, H, -NO2, -CN, -C(O)-R 2a 、-C(O)-OR 2a 、-N(R 2b )-(CO) n -R 2a 、-N(R 2b )-(CO) n -OR 2a 、-N(R 2b )-(CO) n -NR 2a R 2a '、-S(O)mC 0-10 Alkyl, -SO2NR 2a R 2a '、-NR 2a R 2a ', optionally one or more R 12 Substituted -C 1-10 Alkoxy-N(R2b )-(CO) n -R 2a , optionally one or more R 12 Substituted-C(O)-C 1-10 Alkoxy, optionally substituted by one or more R 12 Substituted -C 1-10 Alkyl, optionally substituted by one or more R 12 Substituted -C 1-10 Alkoxy, optionally substituted by one or more R 12 Substituted -C 1-10 Heteroalkyl, optionally substituted by one or more R 12 Substituted -C 2-10 Alkenyl, optionally substituted by one or more R 12 Substituted -C 2-10 Alkynyl, optionally substituted with one or more R 12 substituted 4-10 membered cycloalkyl, optionally substituted by one or more R 12 substituted 4-10 membered heterocycloalkyl, optionally substituted by one or more R 12 substituted 5-10 membered aromatic ring group or optionally substituted with one or more R 12 substituted 5- to 10-membered heteroaryl;

[0017] R 2a and R 2a ' are each independently selected from H, -OH, -C optionally substituted with one or more R7 1-6 Alkyl, optionally substituted with one or more R7-C 1-6 Alkoxy, optionally substituted with one or more R7-C 1-6 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl optionally substituted by one or more R7, saturated or unsaturated 4-10 membered heterocycloalkyl optionally substituted by one or more R7, 5-10 membered aromatic ring group optionally substituted by one or more R7, or 5-10 membered heteroaryl optionally substituted by one or more R8;

[0018] n is 1, 2, or 3;

[0019] m is 0, 1, or 2;

[0020] Y2 and Y2' are each independently halogen, H, -NO2, optionally substituted by one or more R 13 Substituted -C 1-10 Alkoxy, optionally substituted by one or more R 13 Substituted -C 1-10 Alkyl, optionally substituted by one or more R 13 Substituted saturated or unsaturated 4-10 membered cycloalkyl, optionally substituted by one or more R 13Substituted saturated or unsaturated 4-7 membered heterocycloalkyl, optionally substituted by one or more R 13 substituted 5-10 membered aromatic ring group or optionally substituted with one or more R 13 a substituted 5- to 10-membered heteroaryl group; or, R4 or R5, together with Y2 and the atoms to which they are attached, form a saturated or unsaturated 4- to 7-membered heterocycloalkyl group and / or R4' or R5', together with Y2' and the atoms to which they are attached, form a saturated or unsaturated 4- to 7-membered heterocycloalkyl group;

[0021] R6 is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN or

[0022] R7 is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy, saturated or unsaturated 4-7 membered cycloalkyl or saturated or unsaturated 4-7 membered heterocycloalkyl;

[0023] R8 is halogen, -OH, -C(O)-R 11 , -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy, -C 1-4 Heteroalkyl, saturated or unsaturated 4-7 membered cycloalkyl or saturated or unsaturated 4-7 membered heterocycloalkyl;

[0024] R9 is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy or -C 1-4 Haloalkyl;

[0025] R 10 Halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy, -C 1-4 Haloalkyl, -C 1-4 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group;

[0026] R 11 is H, -OH, a saturated or unsaturated 4- to 7-membered cycloalkyl group, or a saturated or unsaturated 4- to 7-membered heterocycloalkyl group;

[0027] R 12 Halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C1-4 Alkyl, -C 1-4 Alkoxy, -C 1-4 Haloalkyl, -C 1-4 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group;

[0028] R 13 Halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -S(O)2-C 1-4 Alkyl, -C 1-6 Alkyl, -C 1-6 Alkoxy, -C 1-6 Haloalkyl, -C 1-6 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group;

[0029] X1 and X1' are each independently S or O;

[0030] X2 and X3 are each independently C or N.

[0031] The compounds of the embodiments of the present invention can bind to GAS41, or can further inhibit the expression or activity of GAS41, thereby inhibiting the activity of tumor cells in vivo and in vitro.

[0032] In some optional embodiments of the present invention, the compound is a compound represented by formula (II) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (II):

[0033]

[0034] Wherein R1 is H, -COOH, -C(O)-OR optionally substituted by one or more R6 1a , -C(O)-NR optionally substituted with one or more R6 1a R 1a '、 -C optionally substituted with one or more R6 1-10 alkyl, -C optionally substituted with one or more R6 1-10 Alkoxy, optionally substituted by one or more R6 -C 1-10 heteroalkyl, -C optionally substituted with one or more R6 2-10 Alkenyl, -C optionally substituted with one or more R6 2-10 Heteroalkenyl, -C optionally substituted with one or more R 2-10 Alkynyl or -C optionally substituted with one or more R6 2-10 heteroalkynyl;

[0035] R 1a and R 1a ' are each independently selected from H, -C optionally substituted with one or more R7 1-10 Alkyl, optionally substituted with one or more R7-C 1-10 Alkylene-R 1c , -C optionally substituted with one or more R7 1-10 Alkoxy, optionally substituted with one or more R7-C 1-10 Alkyleneoxy-R 1c , -C optionally substituted with one or more R7 1-10 heteroalkyl, -C optionally substituted with one or more R7 1-10 Heteroalkylene-R 1c , -C optionally substituted with one or more R7 1-10 Alkenyl, optionally substituted with one or more R7-C 1-10 Alkenylene-R 1c , an aromatic ring group optionally substituted by one or more R7, a heteroaryl group optionally substituted by one or more R7, a saturated or unsaturated 4-10 membered cycloalkyl group optionally substituted by one or more R7, or a saturated or unsaturated 4-10 membered heterocycloalkyl group optionally substituted by one or more R7; or N and R 1a With R 1a ' are connected together to form a saturated or unsaturated 4-10 membered heterocyclic alkyl group containing R 1a and R 1a ' 4-10 membered heterocycloalkyl is optionally substituted by one or more R8;

[0036] R 1b -C(O)-4-10 membered heterocycloalkyl-C(O)-;

[0037] R 1c -OH, -N(R 1d )2、-C(O)-NR 1d R 1d '、-C(O)-OR 1d , a 5- to 10-membered aromatic ring group optionally substituted by one or more R9, a 5- to 10-membered heteroaryl group optionally substituted by one or more R9, a saturated or unsaturated 4- to 10-membered cycloalkyl group optionally substituted by one or more R9, or a saturated or unsaturated 4- to 10-membered heterocycloalkyl group optionally substituted by one or more R9;

[0038] Each R 1d and R 1d ' are each independently selected from H, -C 1-4 Alkyl, -C 1-4 Alkoxy or -C 1-4 Haloalkyl;

[0039] R2, R2', R3 and R3' are each independently halogen, H, -NO2, -CN, -C(O)-R 2a 、-C(O)-OR 2a 、-N(R 2b )-(CO) n -R 2a 、-N(R 2b )-(CO) n -OR 2a 、-N(R 2b )-(CO) n -NR 2a R 2a '、-S(O)mC 0-10 Alkyl, -SO2NR 2a R 2a '、-NR 2a R 2a ', optionally one or more R 10 Substituted -C 1-10 Alkoxy-(CO) n -N(R 2b )-R 2a , optionally one or more R 10 Substituted-C(O)-C 1-10 Alkoxy, optionally substituted by one or more R 10 Substituted -C 1-10 Alkyl, optionally substituted by one or more R 10 Substituted -C 1-10 Alkoxy, optionally substituted by one or more R 10 Substituted -C 1-10 Heteroalkyl, optionally substituted by one or more R 10 Substituted -C 2-10 Alkenyl, optionally substituted by one or more R 10 Substituted -C 2-10 Alkynyl, optionally substituted with one or more R 10 substituted 4-10 membered cycloalkyl, optionally substituted by one or more R 10 substituted 4-10 membered heterocycloalkyl, optionally substituted by one or more R 10 substituted 5-10 membered aromatic ring group or optionally substituted with one or more R 10 substituted 5- to 10-membered heteroaryl;

[0040] R4, R4', R5 and R5' are each independently halogen, H, -NO2, -CN, -C(O)-R 2a 、-C(O)-OR 2a 、-N(R 2b )-(CO) n-R 2a 、-N(R 2b )-(CO) n -OR 2a 、-N(R 2b )-(CO) n -NR 2a R 2a '、-S(O)mC 0-10 Alkyl, -SO2NR 2a R 2a '、-NR 2a R 2a ', optionally one or more R 12 Substituted -C 1-10 Alkoxy-N(R 2b )-(CO) n -R 2a , optionally one or more R 12 Substituted-C(O)-C 1-10 Alkoxy, optionally substituted by one or more R 12 Substituted -C 1-10 Alkyl, optionally substituted by one or more R 12 Substituted -C 1-10 Alkoxy, optionally substituted by one or more R 12 Substituted -C 1-10 Heteroalkyl, optionally substituted by one or more R 12 Substituted -C 2-10 Alkenyl, optionally substituted by one or more R 12 Substituted -C 2-10 Alkynyl, optionally substituted with one or more R 12 Substituted 4-10 membered cycloalkyl, optionally substituted by one or more R 12 substituted 4-10 membered heterocycloalkyl, optionally substituted by one or more R 12 substituted 5-10 membered aromatic ring group or optionally substituted with one or more R 12 substituted 5- to 10-membered heteroaryl;

[0041] R 2a and R 2a ' are each independently selected from H, -OH, -C optionally substituted with one or more R7 1-6 Alkyl, optionally substituted with one or more R7-C 1-6 Alkoxy, optionally substituted with one or more R7-C 1-6 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl optionally substituted by one or more R7, saturated or unsaturated 4-10 membered heterocycloalkyl optionally substituted by one or more R7, 5-10 membered aromatic ring group optionally substituted by one or more R7, or 5-10 membered heteroaryl optionally substituted by one or more R8;

[0042] n is 1, 2, or 3;

[0043] m is 0, 1, or 2;

[0044] Y2 and Y2' are each independently halogen, H, -NO2, optionally substituted by one or more R 13 Substituted -C 1-10 Alkoxy, optionally substituted by one or more R 13 Substituted -C 1-10 Alkyl, optionally substituted by one or more R 13 Substituted saturated or unsaturated 4-10 membered cycloalkyl, optionally substituted by one or more R 13 Substituted saturated or unsaturated 4-7 membered heterocycloalkyl, optionally substituted by one or more R 13 substituted 5-10 membered aromatic ring group or optionally substituted with one or more R 13 a substituted 5- to 10-membered heteroaryl group; or, R4 or R5, together with Y2 and the atoms to which they are attached, form a saturated or unsaturated 4- to 7-membered heterocycloalkyl group and / or R4' or R5', together with Y2' and the atoms to which they are attached, form a saturated or unsaturated 4- to 7-membered heterocycloalkyl group;

[0045] R6 is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN or

[0046] R7 is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy, saturated or unsaturated 4-7 membered cycloalkyl or saturated or unsaturated 4-7 membered heterocycloalkyl;

[0047] R8 is halogen, -OH, -C(O)-R 11 , -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy, -C 1-4 Heteroalkyl, saturated or unsaturated 4-7 membered cycloalkyl or saturated or unsaturated 4-7 membered heterocycloalkyl;

[0048] R9 is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy or -C 1-4 Haloalkyl;

[0049] R 10 Halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4Alkyl, -C 1-4 Alkoxy, -C 1-4 Haloalkyl, -C 1-4 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group;

[0050] R 11 is H, -OH, a saturated or unsaturated 4- to 7-membered cycloalkyl group, or a saturated or unsaturated 4- to 7-membered heterocycloalkyl group;

[0051] R 12 Halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy, -C 1-4 Haloalkyl, -C 1-4 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group;

[0052] R 13 Halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -S(O)2-C 1-4 Alkyl, -C 1-6 Alkyl, -C 1-6 Alkoxy, -C 1-6 Haloalkyl, -C 1-6 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group;

[0053] X1 and X1' are each independently S or O;

[0054] X2, X2', X3 and X3' are each independently C or N.

[0055] In some optional embodiments of the present invention, R1 is H, -COOH, -CH2OH, -CH(OH)CH3, -C(O)-OR 1a 、-C(O)-NR 1a R 1a '、-C 1-6 Alkyl, -C 1-6 Alkoxy, -C 1-6 Heteroalkyl, -C 2-6 Alkenyl, -C 2-6 Heteroalkenyl, -C 2-6 Alkynyl, -C 2-6 Heteroalkynyl,

[0056] In some optional embodiments of the present invention, R 1a H, -C 1-6 Alkyl, -C 1-6 Alkoxy, -C 1-3 Alkylene-saturated or unsaturated 4-7 membered cycloalkyl, -C 1-3 Alkylene-saturated or unsaturated 4-7 membered heterocycloalkyl, -C 1-3 Alkoxy-saturated or unsaturated 4-7 membered cycloalkyl, -C 1-3 Alkoxy-saturated or unsaturated 4-7 membered heterocyclic alkyl; or N and R 1a With R 1a 'Together connected to form a saturated or unsaturated 4 to 7 membered heterocyclic alkyl group; wherein the -C 1-6 Alkyl, -C 1-6 Alkoxy, saturated or unsaturated 4-7 membered cycloalkyl, saturated or unsaturated 4-7 membered heterocycloalkyl and R 1a With R 1a The saturated or unsaturated 4-7 membered heterocycloalkyl groups are each independently optionally substituted by one or more of the following groups: halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-2 Alkyl, -C 1-2 Halogenated alkyl, 4- to 7-membered cycloalkyl, saturated or unsaturated 4- to 7-membered heterocycloalkyl, -C(O)-saturated or unsaturated 4- to 7-membered cycloalkyl, -C(O)-saturated or unsaturated 4- to 7-membered heterocycloalkyl.

[0057] In some optional embodiments of the present invention, R1 is H, -COOH, -CH2OH, -CH(OH)CH3, -C(O)-OCH3, -C 1-6 Alkyl, -C 1-6 Alkoxy, -C 1-6 Heteroalkyl,

[0058] In some optional embodiments of the present invention, R 1b for

[0059] In some optional embodiments of the present invention, R2, R2', R3 and R3' are each independently halogen, H, -CH3, -NO2, -OCH3, -OCH2CH3, -C(O)-OR 2a 、-NH-(CO) n -R 2a 、-NH-(CO) n -OR 2a 、-NH-(CO)n -NR 2a R 2a ' or -C 1-3 Alkoxy-(CO) n -NH-R 2a .

[0060] In some optional embodiments of the present invention, R4, R4', R5 and R5' are each independently halogen, H, -CN, -NO2, -OCH3, -OCH2CH3, -C(O)-R 2a 、-C(O)-OR 2a 、-NH-(CO) n -R 2a 、-NH-(CO) n -OR 2a 、-NH-(CO) n -NR 2a R 2a ' or phenyl.

[0061] In some optional embodiments of the present invention, R 2a and R 2a ' are each independently selected from H, -OH, -C 1-6 Alkyl, saturated or unsaturated 4-7 membered cycloalkyl, saturated or unsaturated 4-7 membered heterocycloalkyl, 6-7 membered aromatic ring group or 6-7 membered heteroaryl group.

[0062] In some optional embodiments of the present invention, n is 1, 2 or 3, preferably 1 or 2.

[0063] In some optional embodiments of the present invention, Y2 and Y2' are each independently halogen, H, -NO2, -CH2OH, -C 1-3 Alkyl, phenyl, Alternatively, R4 or R5 is linked to Y2 and the atoms to which it is connected to form a phenyl group and / or R4' or R5' is linked to Y2' and the atoms to which it is connected to form a phenyl group; wherein, phenyl, Optionally substituted with one or more of the following groups: halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-6 Alkyl and -S(O)2-C 1-4 alkyl.

[0064] In some optional embodiments of the present invention, the compound is a compound represented by formula (III) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (III):

[0065]

[0066] X2 and X3 are each independently C or N, and X2 and X3 are not N at the same time;

[0067] X4, X5, X6 and X7 are each independently C or N;

[0068] R 14 、R 15 、R 16 、R 17 and R 18 Each is independently halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -S(O)2-C 1-4 Alkyl, -C 1-6 Alkyl, -C 1-6 Alkoxy, -C 1-6 Haloalkyl, -C 1-6 heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group.

[0069] In some optional embodiments of the present invention, X4 and X5 are C, X6 and X7 are each independently C or N, or X6 and X7 are C, X4 and X5 are each independently C or N.

[0070] In some optional embodiments of the present invention, R1 is -COOH, -CH2OH, -C(O)-OCH3,

[0071]

[0072] In some optional embodiments of the present invention, R 1b for

[0073] In some optional embodiments of the present invention, R2, R2', R3 and R3' are each independently H, -OCH3, -OCH2CH3, -C(O)-OCH3 or -OCH2-CO-NH-C 1-6 alkyl.

[0074] In some optional embodiments of the present invention, R4, R4', R5 and R5' are each independently halogen, H, -NO2, -OCH3, -OCH2CH3, -C(O)-OCH3 or phenyl.

[0075] In some optional embodiments of the present invention, R 14 '、R 15 '、R 16'、R 17 ' and R 18 'Each independently is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -S(O)2-C 1-4 Alkyl, -C 1-6 Alkyl, -C 1-6 Alkoxy, -C 1-6 Haloalkyl, -C 1-6 heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group.

[0076] In some optional embodiments of the present invention, the compound is a compound represented by formula (IV) or formula (V) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (IV) or formula (V):

[0077]

[0078] In some optional embodiments of the present invention, R1 is -C(O)-NR 1a R 1a ';R 1a and R 1a ' are each independently selected from H or -C 1-6 Alkyl; or N and R 1a With R 1a ' are connected together to form a saturated or unsaturated 4-7 membered heterocycloalkyl group, wherein each of the saturated or unsaturated 4-7 membered heterocycloalkyl groups is optionally substituted by one or more of the following groups: halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN or

[0079] In some optional embodiments of the present invention, R1 is

[0080] In some optional embodiments of the present invention, R2, R3, R4 and R5 are each independently selected from H, -C 1-6 Alkyl or -C 1-6 Alkoxy.

[0081] In some optional embodiments of the present invention, X1 is S.

[0082] In some optional embodiments of the present invention, X2 and X3 are C.

[0083] In some optional embodiments of the present invention, the compound is a compound represented by formula (VI) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (VI):

[0084]

[0085] In some optional embodiments of the present invention, R1 is -C(O)-NR 1a R 1a ';R 1a and R 1a ' are each independently selected from H or -C 1-6 Alkyl; or N and R 1a With R 1a ' are connected together to form a saturated or unsaturated 4-7 membered heterocycloalkyl group, wherein each of the saturated or unsaturated 4-7 membered heterocycloalkyl groups is optionally substituted by one or more of the following groups: halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN or

[0086] In some optional embodiments of the present invention, R1 is

[0087] In some optional embodiments of the present invention, R2, R3, R4 and R5 are each independently selected from H, -C 1-6 Alkyl or -C 1-6 Alkoxy.

[0088] In some optional embodiments of the present invention, X1 is S.

[0089] In some optional embodiments of the present invention, X2 is N, and X3 is C.

[0090] In some optional embodiments of the present invention, Y2 and Y2' are each independently halogen, H, -NO2, -CH2OH, optionally replaced by one or more R 13 Substituted -C 1-3 Alkoxy or optionally one or more R 13 Substituted -C 1-3 Alkyl; or, R4 or R5 and Y2 and the atoms to which they are connected are linked together to form a saturated or unsaturated 4-7 membered heterocycloalkyl and / or R4' or R5' and Y2' and the atoms to which they are connected are linked together to form a saturated or unsaturated 4-7 membered heterocycloalkyl; R 13 It is halogen, -OH, -COOH, -SH, -NH2, -NO2 or -CN.

[0091] In some optional embodiments of the present invention, R1 is H, -COOH, -CH2OH, -CH(OH)CH3, -C(O)-OCH3,

[0092]

[0093] In some optional embodiments of the present invention, R 1b for

[0094] In some optional embodiments of the present invention, R2, R2', R3 and R3' are each independently H, -CN, -CH3, -OCH3, -OCH2CH3, -NH-(CO) n -R 2a 、-C(O)-OCH3 or -OCH2-CO-NH-C 1-6 alkyl.

[0095] In some optional embodiments of the present invention, R4, R4', R5 and R5' are each independently halogen, H, -CN, -NO2, -CH3, -OCH3, -OCH2CH3, -C(O)-OCH3, -NH-(CO) n -NH-C 1-6 Alkyl, -NH-(CO) n -R 2a or phenyl; R 2a -OH, -C 1-6 Alkyl, -C 1-3 Alkoxy, -N(C 1-3 Alkyl)(C 1-3 alkyl) or

[0096] In some optional embodiments of the present invention, the compound is a compound represented by formula (VII) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (VII):

[0097]

[0098] Wherein, Y2 is halogen, H, -NO2, optionally replaced by one or more R 13 Substituted -C 1-10 Alkoxy, optionally substituted by one or more R 13 Substituted -C 1-10 Alkyl, optionally substituted by one or more R 13 Substituted saturated or unsaturated 4-10 membered cycloalkyl, optionally substituted by one or more R 13 Substituted saturated or unsaturated 4-7 membered heterocycloalkyl, optionally substituted by one or more R 13 substituted 5-10 membered aromatic ring group or optionally substituted with one or more R 13 substituted 5- to 10-membered heteroaryl;

[0099] Y3 is optionally replaced by one or more R 20 Substituted saturated or unsaturated 4- to 10-membered heterocycloalkyl;

[0100] R 13 Halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -S(O)2-C 1-4 Alkyl, -C 1-6 Alkyl, -C 1-6 Alkoxy, -C 1-6 Haloalkyl, -C 1-6 Heteroalkyl, saturated or unsaturated 4-10 membered cycloalkyl, saturated or unsaturated 4-10 membered heterocycloalkyl, 5-10 membered aromatic ring group or 5-10 membered heteroaryl group;

[0101] R 20 is halogen, -OH, -COOH, -SH, -NH2, -NO2 or -CN.

[0102] In some optional embodiments of the present invention, Y2 is halogen, H, -C 1-3 Alkyl, -C 1-3 Alkoxy or phenyl;

[0103] In some optional embodiments of the present invention, Y3 is

[0104] In some optional embodiments of the present invention, the compound is a compound represented by formula (VIII) or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound represented by formula (VIII):

[0105]

[0106] X2 is C or N; R3 is -C 1-3 Alkoxy; R 19 -C(O)-NR 1a R 1a ';R 1a and R 1a ' are each independently selected from H, -C optionally substituted with one or more R7 1-10 Alkyl, optionally substituted with one or more R7-C 1-10 Alkylene-R 1c , -C optionally substituted with one or more R7 1-10 Alkoxy, optionally substituted by one or more R7-C 1-10 Alkyleneoxy-R 1c , -C optionally substituted with one or more R7 1-10 heteroalkyl, -C optionally substituted with one or more R7 1-10 Heteroalkylene-R 1c , -C optionally substituted with one or more R7 1-10 Alkenyl, optionally substituted with one or more R7-C1-10 Alkenylene-R 1c , an aromatic ring group optionally substituted by one or more R7, a heteroaryl group optionally substituted by one or more R7, a saturated or unsaturated 4-10 membered cycloalkyl group optionally substituted by one or more R7, or a saturated or unsaturated 4-10 membered heterocycloalkyl group optionally substituted by one or more R7; or N and R 1a With R 1a ' are connected together to form a saturated or unsaturated 4-10 membered heterocyclic alkyl group containing R 1a and R 1a ' 4 to 10 membered heterocycloalkyl is optionally substituted by one or more R8; R 1c -OH, -N(R 1d )2、-C(O)-NR 1d R 1d '、-C(O)-OR 1d , a 5-10 membered aromatic ring group optionally substituted by one or more R9, a 5-10 membered heteroaryl group optionally substituted by one or more R9, a saturated or unsaturated 4-10 membered cycloalkyl group optionally substituted by one or more R9, or a saturated or unsaturated 4-10 membered heterocycloalkyl group optionally substituted by one or more R9; each R 1d and R 1d ' are each independently selected from H, -C 1-4 Alkyl, -C 1-4 Alkoxy or -C 1-4 Haloalkyl; R7 is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy, saturated or unsaturated 4-7 membered cycloalkyl or saturated or unsaturated 4-7 membered heterocycloalkyl; R9 is halogen, -OH, -COOH, -SH, -NH2, -NO2, -CN, -C 1-4 Alkyl, -C 1-4 Alkoxy or -C 1-4 Haloalkyl.

[0107] The inhibitors that can currently target GAS41 can specifically bind to GAS41 and inhibit the proliferation of tumor cells (such as non-small cell lung cancer cells (NSCLC)), showing anti-tumor activity against tumor cells, indicating that inhibiting the protein-protein interaction between GAS41 and acetylated histones may be an effective targeted therapy for lung cancer. However, such inhibitors did not show anti-tumor activity in animals, indicating that the inhibitors currently reported cannot effectively improve tumors caused by abnormal GAS41 expression in vivo and fail to show anti-cancer activity in vivo. The compounds of the embodiments of the present invention can not only bind to GAS41 (especially specifically bind), inhibit the activity of GAS41 and tumor cells, but also show good anti-tumor activity in vivo, thereby effectively preventing and / or treating tumors or cancer.

[0108] In some optional embodiments of the present invention, R1 is -C(O)-NH-C 1-6 Alkyl, -C(O)-NH-C 1-6 Alkyl-OH, -C(O)-NH-C 1-6 Alkyl-C(O)-NH2, -C(O)-NH-C 1-6 Alkyl-N(C 1-3 Alkyl)(C 1-3 alkyl), -C(O)-NH-C 1-6 Alkyl-NH(C 1-3 alkyl),

[0109]

[0110] In some optional embodiments of the present invention, R1 is -C(O)-NH-C 1-6 Alkyl, or

[0111] In another aspect of the present invention, the present invention provides a compound, which is a compound as shown below or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt of the compound as shown below:

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] The compounds of the present invention can bind to GAS41, or can further inhibit the expression or activity of GAS41, thereby inhibiting the activity of tumor cells in vivo and in vitro.

[0119] In another aspect of the present invention, a GAS41 inhibitor is provided. According to an embodiment of the present invention, the GAS41 inhibitor includes the aforementioned compound.

[0120] In another aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the aforementioned compound.

[0121] In some optional embodiments of the present invention, the pharmaceutical composition may further include pharmaceutically acceptable carriers, excipients, and vehicles.

[0122] In another aspect of the present invention, the present invention provides a use of the aforementioned compound in preparing a reagent for binding to GAS41.

[0123] In some optional embodiments of the present invention, the agent is used to bind to GAS41 and inhibit the activity or expression of GAS41.

[0124] In another aspect of the present invention, the present invention provides a use of the aforementioned compound, the aforementioned GAS41 inhibitor or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing or treating a disease associated with GAS41 overexpression.

[0125] In some optional embodiments of the present invention, the GAS41 overexpression-related disease includes tumors or cancer.

[0126] In another aspect of the present invention, the present invention provides a method for preventing or treating a disease related to GAS41 overexpression, comprising: administering a pharmaceutically acceptable dose of the aforementioned compound, the aforementioned GAS41 inhibitor or the aforementioned pharmaceutical composition to a subject.

[0127] The effective amount of the compound, GAS41 inhibitor or pharmaceutical composition of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to, the patient's age, weight, general health, sex, diet, time of administration, drug combination, severity of the disease to be treated, route of administration, etc. For example, depending on the needs of the treatment condition, several separate doses may be administered daily, for example, at a dose of four times a day, three times a day, twice a day, once a day, or every other day, or several doses administered daily may be proportionally reduced; alternatively, the pharmaceutical composition or gel of the present invention is administered for at least one year or longer, preferably for at least one month, more preferably for at least one week, and most preferably for at least one day, to achieve continuous relief of tumors or cancers.

[0128] The compounds, GAS41 inhibitors, or pharmaceutical compositions of the present invention can be incorporated into suitable pharmaceuticals, which can be prepared in various forms, such as liquid, semisolid, and solid dosage forms, including but not limited to solid dosage forms, semisolid dosage forms, liquid dosage forms, and gaseous dosage forms. Various routes of administration of the compounds, GAS41 inhibitors, pharmaceutical compositions, or pharmaceuticals of the present invention are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, nasal, pulmonary, rectal, and topical administration, but the present invention is not limited to these exemplified routes of administration.

[0129] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0131] Figure 1 This is the SPR test result diagram of compound LH-145B in the test example of the present invention;

[0132] Figure 2 This is the SPR test result diagram of compound LH-143 in the test example of the present invention;

[0133] Figure 3 The CCK8 cell toxicity test results of compounds LH-145B (top) and LH-2-169 (bottom) in the test examples of the present invention are shown;

[0134] Figure 4 is the cell inhibition rate of the compound LH-145B in the test example of the present invention in the DIPG17 tumor cell line;

[0135] Figure 5 is the cell inhibition rate of the compound LH-145B in the test example of the present invention in the U-118MG tumor cell line;

[0136] Figure 6 is the cell inhibition rate of the compound LH-145B in the test example of the present invention in the PPC embryonic pons progenitor cell line;

[0137] Figure 7 The results of the cell proliferation inhibition experiment of the compound LH-145B (5.0 μM) in the test example of the present invention in the DIPG17 tumor cell line;

[0138] Figure 8 The results of the effect of compound LH-145B on MEF cell viability in the test example of the present invention;

[0139] Figure 9 Results of the CCK8 cytotoxicity test of compounds LH-145B (left) and LH-2-169 (right) on the viability of U87-LUC cells in the test examples of the present invention;

[0140] Figure 10 Results of the CCK8 cytotoxicity test of compounds in the test examples of the present invention on the effects of compound LH-145B (left) and compound LH-2-169 (right) on HCMEC / D3 cell viability;

[0141] Figure 11 Results of the CCK8 cytotoxicity test of compounds in the test examples of the present invention on the effects of compound LH-145B (left) and compound LH-2-169 (right) on the viability of M231 cells;

[0142] Figure 12 The inhibitory effects of compounds LH-143 and LH-145B on spheroidization of DIPG tumor cells in the test examples of the present invention are shown;

[0143] Figure 13 The figures show the concentration changes of compound LH-145B in plasma, brain, and cerebrospinal fluid over 24 hours after intraperitoneal injection of compound LH-145B in Balb / c mice in the test examples of the present invention. Specifically, (A) the concentration change curve of LH-145B in plasma within 24 hours after intraperitoneal injection of Balb / c mice, (B) the concentration change curve of LH-145B in the brain within 24 hours, and (C) the concentration curve of compound LH-145B in cerebrospinal fluid (CSF). DETAILED DESCRIPTION

[0144] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0145] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0146] It should be noted that, for the structural formula and chemical formula descriptions in the embodiments or embodiments of the present invention, the present invention is intended to cover all replacements, modifications and equivalent technical solutions, which are all within the scope of the present invention as defined in the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described in the present invention can be used to practice the present invention. The present invention is in no way limited to the methods and materials described in the present invention. In the event that one or more of the combined documents, patents and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present invention shall prevail.

[0147] It will be further appreciated that certain features of the invention, which for clarity are described in the context of separate embodiments or implementations, may also be provided in combination in a single embodiment or implementation. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment or implementation, may also be provided separately or in any suitable subcombination.

[0148] Unless otherwise specified, technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention belongs, and unless otherwise specified, all patent publications cited in the entire disclosure of the present invention are incorporated herein by reference in their entirety.

[0149] The following definitions apply to the present invention unless otherwise indicated. For purposes of the present invention, the chemical elements are defined according to the Periodic Table of the Elements, CAS version, and the Chemical Handbook, 75th Ed, 1994. Additionally, general principles of organic chemistry are found in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, all of which are hereby incorporated by reference herein.

[0150] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0151] As used herein, the compounds of the present invention also include isotopically labeled compounds of the present invention, which are identical to those described herein except for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Exemplary isotopes that may also be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as 2 H. 3 H. 13 C. 14 C. 15 N. 16 O. 17 O. 31 P. 32 P. 36 S. 18 F and 37 Cl.

[0152] Compounds of the invention containing the aforementioned isotopes and / or other isotopes of other atoms, as well as pharmaceutically acceptable salts of the compounds, are encompassed within the scope of the invention. Isotopically labeled compounds of the invention, for example radioactive isotopes, such as 3 H and 14 C is incorporated into the compounds of the present invention for drug and / or substrate tissue distribution analysis. 3 H, and carbon-14, i.e. 14 C, isotopes are particularly preferred. In addition, heavy isotopes, such as deuterium, 2 H substitutions may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0153] The stereochemical definitions and conventions used herein are generally in accordance with SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers, and mixtures thereof, such as racemic mixtures, are encompassed by the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule with respect to the chiral center(s) in the molecule. The prefixes d and l, or (+) and (-), are symbols used to designate the rotation of plane-polarized light caused by a compound, where (-) or l indicates that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. With respect to a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. Specific stereoisomers may also be referred to as enantiomers, and a mixture of such isomers is often referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.

[0154] Depending on the choice of starting materials and process, the compounds of the present invention may exist as one of the possible isomers or as a mixture thereof, for example as pure optical isomers, or as a mixture of isomers, such as a racemic and diastereomeric mixture, depending on the number of asymmetric carbon atoms. Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituents may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be in the cis or trans configuration.

[0155] The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers and geometric (or conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention.

[0156] Unless otherwise indicated, structures depicted herein are also intended to include all isomeric (e.g., enantiomers, diastereomeric atropisomers, and geometric (or conformational)) forms of such structures; for example, R and S configurations at various asymmetric centers, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, individual stereochemical isomers as well as enantiomeric mixtures, diastereomeric mixtures, and geometric (or conformational) mixtures of the present compounds are within the scope of the present invention.

[0157] Any asymmetric atom (e.g., carbon, etc.) of the compounds of the present invention may exist in a racemic or enantiomerically enriched form, such as in the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in terms of (R)- or (S)-configuration. Substituents on atoms with unsaturated double bonds may exist in cis-(Z)- or trans-(E)-form, if possible.

[0158] Thus, as described herein, the compounds of the invention may exist in the form of one of the possible isomers, rotamers, atropisomers, tautomers or mixtures thereof, for example, as substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0159] Any resulting mixtures of isomers can be separated on the basis of the physicochemical differences of the constituents into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and / or fractional crystallization.

[0160] Any racemate of the resulting final product or intermediate can be resolved into its optical antipodes by methods familiar to those skilled in the art using known methods, such as by separation of the resulting diastereoisomer salts thereof. Racemic products can also be separated by chiral chromatography, such as high pressure liquid chromatography (HPLC) using a chiral adsorbent. In particular, enantiomers can be prepared by asymmetric synthesis (e.g., Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0161] As used herein, the term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be converted into each other through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be reached. For example, proton tautomers (also known as prototropic tautomers) include interconversions that occur through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions that occur through the reorganization of some bonding electrons. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0162] As used herein, the term "solvate" refers to an association formed between one or more solvent molecules and a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed when the solvent molecule is water.

[0163] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0164] As used herein, the term "pharmaceutically acceptable salt" refers to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Pharmaceutically acceptable salts formed by non-toxic acids include, but are not limited to, inorganic acid salts (such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates) formed by reaction with amino groups, and organic acid salts (such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates), or other methods described in the literature, such as ion exchange methods, to obtain these salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1～4 The present invention also contemplates quaternary ammonium salts formed by any compound containing a N group. Water-soluble or oil-soluble or dispersible products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include appropriate, non-toxic ammonium, quaternary ammonium salts and amine cations formed by counter ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1～8 Sulfonates and aromatic sulfonates.

[0165] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0166] As used herein, the terms "optionally substituted," "optionally substituted," and "substituted or unsubstituted" are used interchangeably. Generally, the term "optionally," whether or not preceded by the term "substituted," indicates that one or more hydrogen atoms in a given structure are replaced by a specified substituent. Unless otherwise indicated, an optional substituent group may be substituted at each substitutable position of the group. When more than one position in a given structural formula can be substituted by one or more substituents selected from a specified group, the substituents may be the same or different at each position. Such substituents may include, but are not limited to, F, Cl, Br, CN, OH, NH2, NO2, and the like.

[0167] As used herein, the term "one or more" (e.g. in the definition of substituents of compounds of the general formula of the invention) means "one, two, three, four or five, in particular one, two, three or four, more in particular one, two or three, even more in particular one or two".

[0168] In addition, it should be noted that, unless otherwise explicitly stated, the description methods used in the present invention such as "each...independently is" and "...each independently is" and "...independently is" can be interchanged and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0169] As used herein, the term "halogen" refers to a fluorine, chlorine, bromine or iodine atom.

[0170] In this document, the minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～b Refers to a carbon atom containing "a" to "b". For example, "C 1～n ” refers to a linear or branched saturated / unsaturated carbon chain containing 1, 2, 3, 4, 5, ... or n carbon atoms; it is further understood that “C 1～n " shall be interpreted as including any sub-ranges therein, such as C 1～10 Including C 1～10 、C 1～8 、C 1～6 、C 1～5 、C 2～10 、C 2～8 、C 2～6 、C3～10 、C 3～8 、C 4～10 .

[0171] In this article, the term “C 1～10 "Alkyl" refers to a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, ... or 10 carbon atoms, such as C 1～10 Alkyl, C 1～8 Alkyl, C 1～6 Alkyl, C 1～5 Alkyl, C 1～4 Alkyl, C 1～3 Alkyl, C 2～10 Alkyl, C 2～8 Alkyl, C 2～6 Alkyl. These include, but are not limited to, methyl, ethyl, n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -C(CH3)3), n-pentyl (-CH2CH2CH2C H2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl ( -CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), wherein the alkyl groups may independently be unsubstituted or substituted with one or more substituents described herein.

[0172] In this article, the term “C 2～10"Alkenyl" refers to a linear or branched monovalent hydrocarbon group having 2, 3, 4, 5, ... or 10 carbon atoms, wherein at least one position CC is an sp2 double bond unsaturated state, wherein the alkenyl group can be independently unsubstituted or substituted with one or more substituents described in the present invention, including groups with "cis", "trans" or "Z" or "E" positioning, wherein specific examples include but are not limited to alkenylethyl (-CH=CH2), allyl (-CH2CH=CH2), etc. For example, C 2～10 Alkenyl, C 2～8 Alkenyl, C 2～7 Alkenyl, C 3～10 Alkenyl, C 3～8 Alkenyl, C 3～7 Alkenyl.

[0173] In this article, the term “C 2～10 "Alkynyl" refers to a linear or branched monovalent hydrocarbon group having 2, 3, 4, 5, ... or 10 carbon atoms, wherein at least one position CC is in an unsaturated sp triple bond state, wherein the alkynyl group may be independently unsubstituted or substituted with one or more substituents described in the present invention, specific examples include but are not limited to alkynylethyl (-C≡CH2), propargyl (-CH2C≡CH), 1-propynyl (-C≡C-CH3), etc. For example, C 2～10 Alkynyl, C 2～8 Alkynyl, C 2～6 Alkynyl, C 3～10 Alkynyl, C 3～8 Alkynyl, C 3～6 Alkynyl.

[0174] In this article, the term “C 1～10 "Alkoxy" refers to a C 1～10 Alkyl, wherein the term "alkyl" is as defined above. For example: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, pentyloxy, isopentyloxy and n-hexyloxy, or isomers of the above groups. In particular, the "C 1～10 The "alkoxy" group may contain 1, 2, 3, 4 or 5 carbon atoms ("C 1～5 Alkoxy”), preferably, may contain 1, 2, 3 or 4 carbon atoms (“C 1～4 alkoxy").

[0175] "Carbocyclyl", "cycloalkyl" and "cycloalkane" described in the present invention all refer to saturated or partially saturated cyclic groups having multiple carbon atoms and no ring heteroatoms and having a single ring or multiple rings (fused, bridged). For polycyclic systems with aromatic and non-aromatic rings without ring heteroatoms. The term "carbocyclyl" includes cycloalkenyl groups, such as cyclohexenyl. Examples of carbocyclyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl and cyclohexenyl. Examples of carbocyclyl groups including polybicycloalkyl ring systems are bicyclohexyl, bicyclopentyl, bicyclooctyl and the like. Two such bicycloalkyl polycyclic structures are exemplified and named below: Biscyclohexyl and The saturated or unsaturated 3-10 membered carbocyclic group of the present invention refers to a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered saturated or unsaturated carbocyclic group, and the unsaturated 3-10 membered carbocyclic group is preferably a 5-10 membered unsaturated carbocyclic group, a 6-10 membered unsaturated carbocyclic group, a 7-10 membered unsaturated carbocyclic group, an 8-10 membered unsaturated carbocyclic group or a 9-10 membered unsaturated carbocyclic group.

[0176] Further, the "heterocycloalkyl", "heterocyclyl", "heterocycle" and "heterocycloalkane" described in the present invention all refer to a saturated or unsaturated ring containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom, etc. It usually refers to a monovalent saturated or partially unsaturated monocyclic or bicyclic ring system with multiple ring atoms, which contains 1, 2 or 3 ring heteroatoms selected from N, O and S, and the remaining ring atoms are carbon. Bicyclic means composed of two rings with two ring atoms in common, that is, the bridge separating the two rings is a single bond or a chain of one or two ring atoms. Examples of monocyclic saturated heterocycloalkyl are oxetanyl, azetidinyl, pyrrolidinyl, 2-oxo-pyrrolidin-3-yl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, Examples of bicyclic saturated heterocycloalkyl groups are 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl. Examples of partially unsaturated heterocycloalkyl groups are dihydrofuranyl, imidazolinyl, tetrahydro-pyridyl or dihydropyranyl. The unsaturated heterocyclic group may or may not include heteroaryl groups, which can be freely selected by those skilled in the art.

[0177] As used herein, "aromatic ring group" and "aromatic ring" refer to aromatic hydrocarbon groups having multiple carbon atoms. An aryl group is typically a monocyclic, bicyclic, or tricyclic aromatic group having multiple carbon atoms. Furthermore, the term "aryl" as used herein refers to an aromatic substituent that can be a single aromatic ring or multiple aromatic rings fused together. Non-limiting examples include phenyl, naphthyl, or tetrahydronaphthyl.

[0178] As used herein, "heteroaryl" and "aromatic heterocycle" refer to an aromatic unsaturated ring containing at least one heteroatom; a heteroatom includes nitrogen, oxygen, sulfur, and the like. Typically, the rings contain multiple heteroatoms, one or more of which are selected from O, N, and S. Preferably, there are one to three heteroatoms. Examples of heteroaryl include pyridyl, indolyl, quinoxalinyl, quinolyl, isoquinolyl, benzothiophenyl, benzofuranyl, benzothiophenyl, benzopyranyl, benzothiapyranyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, and benzoxazolyl.

[0179] In this article, the term “C 4～10 "Cycloalkyl", "4-10 membered cycloalkyl" or "4 to 10 membered cycloalkyl" refers to a saturated monovalent mono- or bicyclic hydrocarbon ring containing 4, 5, 6, ... or 10 carbon atoms. 4～10 Cycloalkyl is a monocyclic hydrocarbon ring, for example: cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0180] As used herein, the term "4-10 membered heterocycloalkyl" or "4 to 10 membered heterocycloalkyl" refers to a 4-, 5-, 6-, ... or 10-membered saturated or unsaturated heterocyclic ring, wherein the unsaturated refers to a group or molecule containing a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-oxygen double bond, a carbon-sulfur double bond, a carbon-nitrogen triple bond, etc.

[0181] In this context, the term "alkylene" refers to a group formed by removing a hydrogen atom from an "alkyl" group, wherein "C 1～10 "Alkylene" includes methylene, ethylene, propylene, isopropylene (such as ), butylene (such as ), pentylene (such as ), hexamethylene (such as )wait.

[0182] As used herein, the term "cycloalkylene" refers to a group formed by removing a hydrogen atom from a "cycloalkyl" group.

[0183] As used herein, the terms "-OR", "-NRR" and the like refer to an R group connected to an oxygen atom or a nitrogen atom via a single bond.

[0184] The group description of the present invention It is used to describe the position of group substitution.

[0185] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent, dispersion medium, coating material, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonic agent, salt, pharmaceutical stabilizer, binder, excipient, dispersant, lubricant, sweetener, flavoring agent, colorant, or combination thereof, which are known to those skilled in the art (e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except in the case where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is encompassed.

[0186] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The compounds or pharmaceutical compositions of the present invention may be administered by any common route, as long as they reach the desired tissue. Various modes of administration are contemplated, including oral administration, peritoneal administration, intravenous injection, intramuscular injection, subcutaneous injection, and the like, but the present invention is not limited to these exemplified modes of administration.

[0187] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0188] As used herein, the term "cancer" or "tumor" may be any unregulated cell growth. Exemplary cancers include non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer, among others.

[0189] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0190] Example 1: Synthesis of Compound LH-3

[0191]

[0192] Synthesis of intermediate compound LH-2 (5-(pyridine-2-)furan-2-carbaldehyde): To a mixture containing 2-bromopyridine (316 mg, 2.0 mmol), (5-formylfuran-2-)boronic acid (280 mg, 2.0 mmol), Pd(PPh3)2Cl2 (70 mg, 0.1 mmol), and 2M aqueous sodium carbonate solution (6 mL) were added ethanol (4 mL) and ethylene glycol dimethyl ether (6 mL) to obtain a mixed solution. The resulting mixed solution was stirred at 50°C under argon protection for 2 h, then concentrated under reduced pressure, diluted with water, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and then concentrated. The crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 6 / 1) to obtain compound LH-2 (182 mg, 52%) as a yellow oil. Compound LH-2. 1 H NMR (400MHz, CDCl3) δ9.74 (s, 1H), 8.68 (d, J = 4.5Hz, 1H), 7.96 (d, J = 7.9Hz, 1H), 7.87–7.77 (m, 1H), 7.38 (d, J = 3.7Hz, 1H), 7.35–7.24 (m, 2H).

[0193] Synthesis of Compound LH-3 ((5-(pyridine-2-)furan-2-)methanol): Compound LH-2 (54 mg, 0.3 mmol) obtained above was dissolved in methanol (5 mL) under an ice-water bath. NaBH4 (12 mg, 0.3 mmol) was added portionwise to the solution, and the resulting mixture was stirred at room temperature for 10 min. Water was added to the reaction mixture portionwise and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (hexane / ethyl acetate = 3 / 1) to obtain Compound LH-3 (32 mg, 67%) as a yellow solid.

[0194] Compound LH-3. 1H NMR (400MHz, CDCl3) δ8.55 (d, J = 4.3 Hz, 1H), 7.73–7.61 (m, 2H), 7.19–7.07 (m, 1H), 6.98 (d, J = 3.1 Hz, 1H), 6.39 (d, J = 3.1 Hz, 1H), 4.68 (s, 2H).

[0195] Example 2: Synthesis of Compound LH-7

[0196]

[0197] Synthesis of intermediate compound LH-4 (1-(5-(6-methylpyridine-2-)thiophene-2-)ethane-1-one): 2-bromo-6-methylpyridine (344 mg, 2.0 mmol), (5-acetylthiophene-2-)boric acid (340 mg, 2.0 mmol), ethanol (4 mL), ethylene glycol dimethyl ether (6 mL), 2M aqueous sodium carbonate solution (6 mL) and Pd(PPh3)2Cl2 (70 mg, 0.1 mmol) were added to a flask at room temperature, and the resulting mixture was stirred at 50°C under argon protection for 2 hours. The mixture was concentrated under reduced pressure and extracted with water and ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a yellow oil (260 mg, 62%). Intermediate compound LH-4 was used directly in the next reaction without column chromatography purification.

[0198] Synthesis of compound LH-7 (1-(5-(6-methylpyridine-2-)thiophene-2-)ethane-1-ol): Compound LH-4 (63 mg, 0.3 mmol) obtained above was dissolved in methanol (5 ml) under an ice-water bath. NaBH4 (12 mg, 0.3 mmol) was added portionwise to the solution, and the resulting mixture was stirred at room temperature for 10 min. Water was added portionwise to the reaction mixture and extracted with EtOAc. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (PE / EtOAc = 3 / 1) to obtain compound LH-7 (40 mg, 63%) as a yellow solid.

[0199] Compound LH-7. 1 H NMR (400MHz, CDCl3) δ7.60–7.50(m,1H),7.42(d,J=8.0Hz,2H),7.07–6.89(m,2H),5.12(d,J=6.1Hz,1H),2.57(s,3H),1.62(d,J=6.3Hz,3H).

[0200] Example 3: Synthesis of compounds LH-17, LH-29 to LH-36, LH-38 and LH-39

[0201] 1. Synthesis of compound LH-29

[0202]

[0203] Synthesis of intermediate compound LH-6 (5-(6-methylpyridine-2-)thiophene-2-aminocarbaldehyde): To a mixture containing 2-bromo-6-methylpyridine (344 mg, 2.0 mmol) and (5-formylthiophene-2-)boronic acid (312 mg, 2.0 mmol) were added ethanol (4 mL), ethylene glycol dimethyl ether (6 mL), Pd(PPh3)2Cl2 (70 mg, 0.1 mmol) and 2M aqueous sodium carbonate solution (6 mL) to obtain a mixed solution. The resulting mixed solution was stirred at 50°C under argon for 2 h, concentrated under reduced pressure, then diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain intermediate compound LH-6 (277 mg, 68%) as a yellow oil. Intermediate compound LH-6 was used directly in the next reaction without purification.

[0204] Synthesis of compound LH-29 ((5-(6-methylpyridine-2-)thiophene-2-)methanol): Compound LH-6 (50 mg, 0.3 mmol) was dissolved in methanol (5 mL) under an ice-water bath. NaBH4 (12 mg, 0.3 mmol) was added portionwise to the solution and the resulting mixture was stirred at room temperature for 10 min. After the reaction was completed, water was added portionwise to the reaction mixture and extracted with EtOAc. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE / EtOAc = 3 / 1) to obtain compound LH-29 (45 mg, 68%) as a yellow solid.

[0205] Compound LH-29. 1 H NMR (400MHz, CDCl3) δ7.62–7.50(m,1H),7.50–7.36(m,2H),7.06–6.91(m,2H),4.82(s,2H),2.57(s,3H).

[0206] 2. The preparation of compounds LH-17, LH-30, LH-31, LH-32, LH-33, LH-34, LH-35, LH-36, LH-38, and LH-39 followed the synthesis of compound LH-29 in step 1 of this example. The structures and NMR characterization results of compounds LH-17, LH-30, LH-31, LH-32, LH-33, LH-34, LH-35, LH-36, LH-38, and LH-39 are shown in Table 1.

[0207] Table 1: Structural formulas and NMR characterization results of compounds LH-17, LH-30~LH-36, LH-38 and LH-39

[0208]

[0209] Example 4: Synthesis of Compound LH-37

[0210]

[0211] Synthesis of intermediate compound LH-19 (5-(quinolin-2-yl)thiophene-2-aminocarbaldehyde): To a mixture containing 2-bromoquinoline (312 mg, 1.5 mmol) and (5-formylthiophene-2-yl)boronic acid (234 mg, 1.5 mmol) was added 1.5 M aqueous sodium carbonate solution (6.0 mL), Pd(PPh3)2Cl2 (70 mg, 0.1 mmol), ethanol (4 mL) and DME (6 mL) at room temperature. The mixture was protected by argon and stirred at 110°C for 5 hours. After completion of the reaction, the mixture was extracted with water and ethyl acetate, dried and concentrated to obtain a yellow solid (184 mg, 51%). Intermediate compound LH-19 was used directly in the next reaction without purification.

[0212] Synthesis of Compound LH-37 ((5-(quinolin-2-yl)thiophene-2-)methanol): Compound LH-19 (70 mg, 0.29 mmol) was dissolved in methanol (5 ml) under an ice-water bath. NaBH4 (15 mg, 0.4 mmol) was added and stirred for 10 min. The mixture was extracted with water and EtOAc. The organic layer was dried and concentrated, and purified by silica gel column chromatography (PE / EtOAc = 10 / 1) to afford Compound LH-37 (52 mg, 74%) as a white solid.

[0213] Compound LH-37. 1 H NMR (400MHz, CDCl3) δ8.23–7.99(m,2H),7.86–7.58(m,4H),7.50(d,J=6.8Hz,1H),7.06(s,1H),4.88(s,2H).

[0214] Example 5: Synthesis of Compounds LH-40 and LH-41

[0215] 1. Synthesis of compound LH-40

[0216]

[0217] Synthesis of compound LH-40 ((5-(thiophen-2-yl)pyridin-3-yl)methanol): Pd(PPh3)4 (22 mg, 0.02 mmol) was added to a mixture containing (5-bromopyridine-3-)methanol (360 mg, 1.9 mmol), thiophene-2-boronic acid (250 mg, 1.9 mmol), 10 mL of toluene, 2.5 mL of methanol, and 4 mL of 2M aqueous sodium carbonate solution. The resulting mixture was stirred at 70°C for 20 hours under argon protection. The mixture was then concentrated under reduced pressure, diluted with water, and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated. The residue was purified by silica gel column chromatography (PE / EA = 1 / 3) to give compound LH-40 (236 mg, 65%) as a yellow solid.

[0218] Compound LH-40. 1 H NMR (400MHz, CDCl3) δ8.72(s,1H),8.42(s,1H),7.92(s,1H),7.62(d,J=4.0Hz,2H),7.44(d,J=2.0Hz,1H),7.14–7.05(m,1H),4.75(s,2H).

[0219] 2. The preparation of compound LH-41 refers to the synthesis of compound LH-40 in step 1 of this example. The structural formula of compound LH-41 is The NMR characterization results are: 1 H NMR (400MHz, DMSO) δ8.85 (s, 1H), 8.45 (s, 1H), 8.03 (s, 2H), 7.79–7.57 (m, 2H), 5.37 (t, J = 5.6Hz, 1H), 4.59 (d, J = 5.6Hz, 2H).

[0220] Example 6: Synthesis of Compound LH-54

[0221]

[0222] Synthesis of intermediate compound LH-50 (5-(pyrimidin-2-yl)furan-2-aminocarbaldehyde): Pd(PPh3)2Cl2 (70 mg 0.1 mmol) was added to a mixture of 2-bromopyrimidine (318 mg, 2.0 mmol), (5-formylfuran-2-yl)boronic acid (280 mg, 2.0 mmol), 2M aqueous sodium carbonate solution (6 mL), ethanol (4 mL), and DME (6 mL). The mixture was stirred at 50°C for 3 hours under argon protection. The mixture was extracted with water and ethyl acetate and concentrated. The residue was purified by column chromatography (PE / EA = 5 / 1) to obtain compound LH-50 (109 mg, 31%) as a yellow solid. Compound LH-50.1 H NMR (400MHz, DMSO) δ10.56(s,1H),8.56–8.43(m,1H),8.00(d,J=8.9Hz,1H),7.88(d,J=4.0Hz,1H),7.73(d,J=4.0Hz,1H),7.67(d,J=9.1Hz,1H).

[0223] Synthesis of compound LH-54 ((5-(pyrimidin-2-yl)furan-2-yl)methanol): Compound LH-50 (30 mg, 0.17 mmol) was dissolved in methanol (5 ml) under an ice-water bath. NaBH4 (15 mg, 0.4 mmol) was added portionwise to the solution and the resulting mixture was stirred at room temperature for 10 min. Water was added portionwise to the reaction mixture and extracted with EtOAc. The organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (PE / EtOAc = 1 / 2) to obtain compound LH-54 (20 mg, 67%) as a white solid.

[0224] Compound LH-54. 1 H NMR (400MHz, DMSO) δ 8.80 (d, J = 4.8 Hz, 2H), 7.43–7.31 (m, 1H), 7.25 (d, J = 3.2 Hz, 1H), 6.51 (d, J = 3.1 Hz, 1H), 5.48–5.36 (m, 1H), 4.50 (d, J = 5.7 Hz, 2H).

[0225] Example 7: Synthesis of compounds LH-1, LH-25, LH-26, LH-27, LH-45 and LH-46

[0226] 1. Synthesis of compound LH-1

[0227]

[0228] Synthesis of compound LH-1 (5-(pyridin-2-yl)thiophene-2-carboxylic acid): Pd(PPh3)4 (87 mg, 0.075 mmol) was added to a mixture containing 2-bromopyridine (285 mg, 1.8 mmol), 5-carboxythiophene-2-boronic acid (258 mg, 1.5 mmol), sodium carbonate (318 mg, 3.0 mmol), MeCN (8 mL) and H2O (8 mL) at room temperature. Under argon protection, the mixture was stirred at 80°C for 2 h. The product was extracted with water and EtOAc, and the product dissolved in the aqueous phase. The aqueous phase was adjusted to pH = 2-3 with 2M HCl aqueous solution and extracted with EtOAc. The product was in the organic layer. The product was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. Purification by silica gel column chromatography (DCM / MeOH = 10 / 1) gave compound LH-1 (191 mg, 62%) as a white solid.

[0229] Compound LH-1. 1 H NMR (400MHz, DMSO) δ8.60(d,J=4.7Hz,1H),8.05(d,J=8.0Hz,1H),8.00–7.93(m,1H),7.90(d,J=4.0Hz,1H),7.74(d,J=3.9Hz,1H),7.47–7.39(m,1H). 13 C NMR (101MHz, DMSO) δ163.28,150.47,149.43,149.21,139.03,136.41,134.57,126.89,124.30,120.54.

[0230] 2. The preparation of compounds LH-25, LH-26, LH-27, LH-45, and LH-46 followed the synthesis of compound LH-1 in step 1 of this example. The structures and NMR characterization results of compounds LH-25, LH-26, LH-27, LH-45, and LH-46 are shown in Table 2.

[0231] Table 2: Structural formulas and NMR characterization results of compounds LH-25, LH-26, LH-27, LH-45, and LH-46

[0232]

[0233]

[0234] Example 8: Synthesis of Compounds LH-47 and LH-48

[0235] 1. Synthesis of compound LH-48

[0236]

[0237] Synthesis of compound LH-47 (5-(quinoline-2-)thiophene-2-carboxylic acid): Pd(PPh3)4 (35 mg, 0.03 mmol) was added to a mixture of 2-bromoquinoline (150 mg, 0.72 mmol), 5-carboxythiophene-2-boronic acid (103 mg, 0.6 mmol), sodium carbonate (127 mg, 1.2 mmol), MeCN (3 mL) and H2O (3.0 mL) at room temperature. Under argon protection, the resulting mixture was stirred at 90°C for 5 hours. The mixture was extracted with water and EtOAc, and the product was dissolved in the aqueous phase. The aqueous phase was adjusted to pH = 2-3 with 2M HCl aqueous solution and extracted with EtOAc. The product was dissolved in the organic layer. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (DCM / MeOH = 10 / 1) to give compound LH-47 (62 mg, 47%) as a white solid.

[0238] Compound LH-47. 1 H NMR (400MHz, DMSO) δ8.46(d,J=8.6Hz,1H),8.17(d,J=8.7Hz,1H),8.05–7.97(m,3H),7.83–7.75(m,1H),7.69(d,J=3.7Hz,1H),7.66–7.58(m,1H).

[0239] 2. The preparation of compound LH-48 refers to the synthesis of compound LH-47 in step 1 of this example. The structural formula of compound LH-48 is The NMR characterization results are: 1 H NMR (400MHz, DMSO) δ8.87(d,J=4.9Hz,2H),7.95(d,J=3.8Hz,1H),7.76(d,J=3.9Hz,1H),7.53–7.38(m,1H).

[0240] Example 9: Synthesis of Compound LH-51

[0241]

[0242] Synthesis of Compound LH-51 ((((5-(pyridin-2-yl)furan-2-yl)methylene)amino)imidazoline-2,4-dione): 1-Aminohydantoin (33 mg, 0.22 mmol), 0.67 M HCl (0.3 mL), and Compound LH-2 (35 mg, 0.2 mmol) were dissolved in DMF (1 mL). The mixture was stirred at room temperature for 1 hour. Water (5 mL) was added, and the precipitate was filtered. The collected solid was dried to obtain Compound LH-51 (50 mg, 92%) as a yellow solid.

[0243] Compound LH-51. 1 H NMR (400MHz, DMSO) δ11.31(s,1H),8.63(d,J=4.4Hz,1H),7.95(s,1H),7.85(d,J=7.9Hz,1H) ,7.77(s,1H),7.43–7.35(m,1H),7.31(d,J=3.4Hz,1H),7.02(d,J=3.5Hz,1H),4.37(s,2H).

[0244] Example 10: Synthesis of Compound LH-55

[0245]

[0246] Synthesis of intermediate compound LH-50 (5-(pyrimidin-2-yl)furan-2-carbaldehyde): Pd(PPh3)2Cl2 (70 mg 0.1 mmol) was added to a mixture of 2-bromopyrimidine (318 mg, 2.0 mmol), (5-formylfuran-2-yl)boronic acid (280 mg, 2.0 mmol), 2M aqueous sodium carbonate solution (6 mL), ethanol (4 mL), and DME (6 mL). Under argon, the mixture was stirred at 50°C for 3 hours. The mixture was concentrated under reduced pressure, extracted with water and ethyl acetate, and concentrated. Purification by silica gel column chromatography (PE / EA = 5 / 1) afforded intermediate compound LH-50 (109 mg, 31%) as a yellow solid. Compound LH-50. 1 H NMR (400MHz, DMSO) δ10.56(s,1H),8.56–8.43(m,1H),8.00(d,J=8.9Hz,1H),7.88(d,J=4.0Hz,1H),7.73(d,J=4.0Hz,1H),7.67(d,J=9.1Hz,1H).

[0247] Synthesis of compound LH-55 ((1-((5-(pyrimidin-2-yl)furan-2-yl)methylene)amino)imidazoline-2,4-dione): 1-aminohydantoin (60 mg, 0.4 mmol), 0.67 M HCl (0.6 mL), and compound LH-50 (50 mg, 0.3 mmol) were dissolved in DMF (2 mL) under an ice-water bath, and the mixture was stirred at room temperature for 1 h. Water (5 mL) was added, and the precipitate was filtered and rinsed with water. The collected solid was dried to give compound LH-55 (63 mg, 80%) as a yellow solid.

[0248] Compound LH-55. 1 H NMR (400MHz, DMSO) δ11.33(s,1H),8.82(d,J=14.6Hz,2H),7.89–7.66(m,1H),7.37(d,J=27.9Hz,2H),7.01(s,1H),4.35(s,2H).

[0249] Example 11: Synthesis of compounds LH-49, LH-56, LH-57 and LH-60

[0250] 1. Synthesis of compound LH-49

[0251]

[0252] Synthesis of compound LH-49 ((1-((5-(5-fluoropyridin-2-)thiophene-2-)methylene)amino)imidazoline-2,4-dione): 1-Aminohydantoin (33 mg, 0.22 mmol), 0.67 M HCl (0.3 mL), and 5-(5-fluoropyridin-2-yl)thiophene-2-aminocarbaldehyde (compound LH-22) (38 mg, 0.18 mmol) were dissolved in DMF (1 mL), and the mixture was stirred at room temperature for 1 h. Water (5 mL) was added to the mixture, and the precipitate was filtered to obtain the product. The solid was dried and collected to obtain compound LH-49 as a yellow solid (51 mg, 93%).

[0253] Compound LH-49. 1 H NMR (400MHz, DMSO) δ11.29(s,1H),8.57(s,1H),8.08–8.02(m,1H),8.00(s,1H),7.87–7.73(m,2H),7.42(d,J=3.7Hz,1H),4.35(s,2H).

[0254] 2. The preparation of compounds LH-56, LH-57, and LH-60 was carried out by referring to the synthesis of compound LH-49 in step 1 of this example. The structural formulas and NMR characterization results of compounds LH-56, LH-57, and LH-60 are shown in Table 3.

[0255] Table 3: Structural formula and NMR characterization results of compounds LH-56, LH-57 and LH-60

[0256]

[0257] Example 12: Synthesis of Compound LH-58

[0258]

[0259] Synthesis of compound LH-58 (1-(((5-(quinolin-2-yl)thiophene-2-)methylene)amino)imidazoline-2,4-dione): 1-Aminohydantoin (33 mg, 0.22 mmol), 0.67 M HCl (0.3 mL), and compound LH-19 (45 mg, 0.19 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 1 h. 10 mL of water was added to the mixture, and the precipitate was filtered to obtain a precipitate. The precipitate was collected, washed with water, and dried to obtain compound LH-58 (40 mg, 85%) as a yellow solid.

[0260] Compound LH-58. 1 H NMR (400MHz, DMSO) δ11.31(s,1H),8.44(d,J=8.7Hz,1H),8.15(d,J=8.5Hz,1H),8.04(s,1H),7. 99(d,J=9.0Hz,3H),7.84–7.74(m,1H),7.64–7.54(m,1H),7.48(d,J=3.7Hz,1H),4.37(s,2H).

[0261] Example 13: Synthesis of compounds LH-61 to LH-64, LH-66, LH-67, LH-87 to LH-95, LH-98, LH-99 and LH-102

[0262] 1. Synthesis of compound LH-61

[0263]

[0264] Synthesis of compound LH-61 (N-cyclohexyl-5-(pyridine-2-)thiophene-2-carboxamide): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 171 mg, 0.45 mmol), compound LH-1 (5-(pyridine-2-)thiophene-2-carboxylic acid) (62 mg, 0.3 mmol), cyclohexylamine (30 mg, 0.3 mmol), and N,N-diisopropylethylamine (DIPEA; 58 mg, 0.45 mol) were added to DMF (1.0 mL), and the mixture was stirred at room temperature for 12 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate, and the product dissolved in the organic layer. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA=3 / 1) to obtain white solid compound LH-61 (73 mg, 85%).

[0265] Compound LH-61. 1 H NMR (400MHz, CDCl3) δ8.68–8.47(m,1H),7.80–7.63(m,2H),7.60–7.45(m,2H),7.25–7.14(m,1H),5.87 (s,1H),4.12–3.80(m,1H),2.13–1.93(m,2H),1.81–1.58(m,3H),1.50–1.34(m,2H),1.34–1.08(m,4H). 13 C NMR (101MHz, DMSO) δ160.54,151.72,150.02,148.35,141.94,137.74,129.18,125.80,123.51,119.45,48.87,32.95,25.70,25.41.

[0266] 2. The preparation of compounds LH-62, LH-63, LH-64, LH-66, LH-67, LH-87, LH-88, LH-89, LH-90, LH-91, LH-92, LH-93, LH-94, LH-95, LH-98, LH-99, and LH-102 was carried out by referring to the synthesis of compound LH-41 in step 1 of this example. The structures and NMR characterization results of compounds LH-62, LH-63, LH-64, LH-66, LH-67, LH-87, LH-88, LH-89, LH-90, LH-91, LH-92, LH-93, LH-94, LH-95, LH-98, LH-99, and LH-102 are shown in Table 4.

[0267] Table 4: Structural formulas and NMR characterization results of compounds LH-62 to LH-64, LH-66, LH-67, LH-87 to LH-95, LH-98, LH-99, and LH-102

[0268]

[0269]

[0270]

[0271] Example 14: Synthesis of compounds LH-2-5, LH-2-6, LH-2-7, LH-2-10, LH-2-12, LH-2-15, LH-2-16, LH-2-17, LH-159, LH-165, LH-168, LH-169, LH-2-169, LH-2-170, LH-2-174, and LH-2-187

[0272] 1. Synthesis of compound LH-159

[0273]

[0274] Synthesis of intermediate compound LH-2-139 (5-bromo-2-iodo-3-methoxypyridine): 5-Bromo-2-iodopyridin-3-ol (650 mg, 2.2 mmol), t-BuONa (250 mg, 2.6 mmol), and MeI (0.47 mL, 7.6 mmol) were dissolved in DMF (10 mL) and stirred at room temperature for 12 h. After completion of the reaction, water was added portionwise to the mixture and extracted with EtOAc. The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE / EtOAc = 30 / 1) to obtain compound LH-2-139 (457 mg, 67%) as a white solid. Compound LH-2-139. 1 H NMR (400MHz, CDCl3) δ8.10(s,1H),7.12(s,1H),3.92(s,3H).

[0275] Synthesis of intermediate compound LH-2-157 (5-(5-bromo-3-methoxypyridine-2-)thiophene-2-carboxylic acid): Pd(PPh3)4 (70 mg, 0.06 mmol) was added to a mixture of 5-bromo-2-iodo-3-methoxypyridine (compound LH-2-139) (314 mg, 1.0 mmol), 5-boronic acid-thiophene-2-carboxylic acid (189 mg, 1.1 mmol), sodium carbonate (424 mg, 4.0 mmol), MeCN (20 mL) and H2O (10 mL). Under argon protection, the resulting mixture was stirred at 80°C for 12 h. The mixture was then extracted with water and EtOAc, and the product was dissolved in the aqueous phase. The aqueous phase was adjusted to pH = 2-3 with 2M HCl aqueous solution and extracted with EtOAc. The product was dissolved in the organic layer. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (PE / EA=1 / 2) to obtain yellow solid compound LH-2-157 (188 mg, 68%). 1 H NMR (400MHz, DMSO) δ13.18(s,1H),8.35(d,J=1.8Hz,1H),7.98–7.88(m,2H),7.72(d,J=4.0Hz,1H),4.04(s,3H).

[0276] Intermediate compound LH-2-162 (methyl 5-(5-bromo-3-methoxypyridine-2-)thiophene-2-carboxylate): Compound LH-2-157 (100 mg, 0.3 mmol) was dissolved in MeOH (10 mL), concentrated sulfuric acid (2.0 mL) was slowly added, and the mixture was stirred at 60°C for 3 h. After completion of the reaction, the mixture was adjusted to neutrality with solid NaOH. Water was added portionwise to the reaction mixture, diluted, and then extracted with EtOAc. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE / EtOAc = 1 / 1) to obtain compound LH-2-162 (86 mg, 83%) as a yellow solid. Compound LH-2-162. 1 H NMR (400MHz, CDCl3) δ8.28(d,J=1.6Hz,1H),7.92(d,J=4.0Hz,1H),7.77(d,J=4.1Hz,1H),7.43(d,J=1.5Hz,1H),4.01(s,3H),3.90(s,3H).

[0277] Intermediate compound LH-2-163 (methyl 5-(3-methoxy-5-phenylpyridine-2-)thiophene-2-carboxylate): To a mixture of compound LH-2-162 (164 mg, 0.5 mmol), phenylboronic acid (122 mg, 1.0 mmol), potassium carbonate (553 mg, 4.0 mmol), PhMe (5 mL), 1,4-dioxane (5 mL) and H2O (2 mL) was added Pd(PPh3)4 (70 mg, 0.06 mmol). Under argon protection, the resulting mixture was stirred at 100°C for 12 h. After completion of the reaction, the mixture was extracted with water and EtOAc, and the product was dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA=5 / 1) to obtain compound LH-2-163 (117 mg, 72%) as a yellow solid. Compound LH-2-163. 1 H NMR(400MHz,DMSO)δ8.59(d,J=1.7Hz,1H),7.99(d,J=4.0Hz,1H),7.93–7.8 0(m,4H),7.59–7.52(m,2H),7.51–7.42(m,1H),4.14(s,3H),3.86(s,3H).

[0278] Synthesis of Compound LH-159 (5-(3-methoxy-5-phenylpyridine-2-)thiophene-2-carboxylic acid): Compound LH-2-163 (60 mg, 0.18 mmol) and NaOH (80 mg, 2 mmol) were dissolved in a mixture of MeOH (10 mL), THF (10 mL), and H₂O (3 mL) and stirred at room temperature for 3 h. After completion of the reaction, the pH of the mixture was adjusted to 2-3 with 2M HCl solution. Water was added portionwise to the reaction mixture, and the mixture was extracted with EtOAc. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE / EtOAc = 1 / 2) to yield Compound LH-159 (33 mg, 53%) as a yellow solid.

[0279] Compound LH-159. 1 H NMR (400MHz, DMSO) δ13.12(s,1H),8.58(d,J=1.8Hz,1H),7.96(d,J=4.0Hz,1H),7.89– 7.81(m,3H),7.74(d,J=4.0Hz,1H),7.58–7.50(m,2H),7.49–7.43(m,1H),4.13(s,3H). 13C NMR (101MHz, DMSO) δ163.65,153.03,147.75,139.58,139.06,136.89,136.80,134.11,129.57,128.98,128.33,127.55,118.22,56.55.

[0280] 2. Synthesis of compound LH-165

[0281]

[0282] Synthesis of compound LH-165 (5-(3-methoxy-5-phenylpyridine-2-)-N-pentylthiophene-2-carboxamide): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 171 mg, 0.45 mmol), compound LH-159 (5-(pyridine-2-)thiophene-2-carboxylic acid) (62 mg, 0.3 mmol), n-pentylamine (30 mg, 0.3 mmol), and N,N-diisopropylethylamine (DIPEA; 58 mg, 0.45 mol) were added to DMF (1.0 mL), and the mixture was stirred at room temperature for 12 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate, and the product dissolved in the organic layer. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA=3 / 1) to obtain compound LH-165 (32 mg, 62%) as a white solid.

[0283] Compound LH-165. 1 H NMR(400MHz,MeOD)δ8.37(s,1H),7.89(s,1H),7.79–7.55(m,4H),7.55–7.29(m,3H),4. 06(s,3H),3.38–3.30(m,2H),1.79–1.50(m,2H),1.40–1.24(m,4H),0.99–0.84(m,3H). 13 C NMR(101MHz,MeOD)δ163.22(s),152.73(s),145.22(s),139.53(s),139.32(s),138.69(s),136.95(s),136.85(s),128.78(s) ,128.26(s),128.06(s),127.62(s),126.66(s),116.95(s),54.85(s),39.56(s),28.93(s),28.92(s),22.09(s),12.99(s)..

[0284] 3. The preparation of compounds LH-2-5, LH-2-6, LH-2-7, LH-2-10, LH-2-12, LH-2-15, LH-2-16, LH-2-17, LH-168, LH-169, LH-2-169, LH-2-170, LH-2-174 and LH-2-187 refers to the synthesis of compound LH-165 in step 2 of this example. The structures and NMR characterization results of compounds LH-2-5, LH-2-6, LH-2-7, LH-2-10, LH-2-12, LH-2-15, LH-2-16, LH-2-17, LH-168, LH-169, LH-2-169, LH-2-170, LH-2-174 and LH-2-187 are shown in Table 5.

[0285] Table 5: Structural formula and NMR characterization results of each compound

[0286]

[0287]

[0288] Example 15: Synthesis of Compound LH-2-180

[0289]

[0290] Synthesis of intermediate compound LH-68 (tert-butyl 4-(5-methoxypyridine-3-)piperazine-1-carboxylate): Pd(PPh3)4 (22 mg, 0.02 mmol) was added to a mixture of 3-bromo-5-methoxypyridine (1.06 g, 5.6 mmol), tert-butyl piperazine-1-carboxylate (1.04 g, 5.6 mmol), potassium tert-butoxide (1.24 g, 11.2 mmol) and toluene (8 mL) at room temperature. Under argon protection, the resulting mixture was stirred at 80°C for 3 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, diluted with water, and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated. The residue was purified by silica gel column chromatography (PE / EA=2 / 1) to obtain white solid compound LH-68 (520 mg, 32%). Compound LH-68. 1 H NMR (400MHz, CDCl3) δ7.94(s,1H),7.83(s,1H),6.69(s,1H),3.83(s,3H),3.65–3.49(m,4H),3.25–3.05(m,4H),1.47(s,10H).

[0291] Synthesis of intermediate compound LH-79 (tert-butyl 4-(6-bromo-5-methoxypyridine-3-)piperazine-1-carboxylate): Compound LH-68 (432 mg, 1.4 mmol) was dissolved in MeCN (10 ml) at room temperature. NBS (249 mg, 1.4 mmol) was added portionwise to the solution and the resulting mixture was stirred at room temperature for 1 hour. After the reaction was complete, water was added portionwise and the reaction mixture was extracted with EtOAc. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE / EtOAc = 3 / 1) to obtain compound LH-79 (349 mg, 67%) as a white solid. Compound LH-79. 1 H NMR (400MHz, DMSO) δ7.65(d,J=2.2Hz,1H),7.03(d,J=2.1Hz,1H),3.86(s,3H),3.52–3.42(m,5H),3.27–3.18(m,4H),1.42(s,9H).

[0292] Synthesis of intermediate compound LH-83 (5-(5-(4-(tert-butoxycarbonyl)piperazine-1-)-3-methoxypyridin-2-yl)thiophene-2-carboxylic acid): To a mixture of compound LH-79 (300 mg, 0.80 mmol), 5-borothiophene-2-carboxylic acid (115 mg, 0.67 mmol), sodium carbonate (142 mg, 1.3 mmol), MeCN (4 mL) and H2O (4 mL) was added Pd(PPh3)4 (35 mg, 0.03 mmol) to room temperature. Under argon protection, the resulting mixture was stirred for 12 h to 90°C. After the reaction was completed, the mixture was extracted with water and EtOAc, and the product was dissolved in the aqueous phase. The aqueous phase was adjusted to pH = 2-3 with 2M HCl aqueous solution and extracted with EtOAc. The product was dissolved in the organic layer. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (PE / EA=1 / 1) to obtain yellow solid compound LH-83 (188 mg, 68%). 1 H NMR (400MHz, DMSO) δ7.97(d,J=2.0Hz,1H),7.69(d,J=4.0Hz,1H),7.65(d,J=3.9Hz ,1H),7.04(s,1H),3.98(s,3H),3.50–3.46(m,4H),3.34–3.34(m,5H),1.43(s,9H). 13CNMR (101MHz, DMSO) δ163.34,153.86,153.19,148.76,147.53,133.74,132.40,130.43,129.06,125.08,105.41,79.15,55.81,47.15,28.10.

[0293] Synthesis of intermediate compound LH-86 (tert-butyl 4-(6-(5-(butylcarbamoyl)thiophene-2-)-5-methoxypyridine-3-)piperazine-1-carboxylate): HATU (171 mg, 0.45 mmol), LH-83 (50 mg, 0.12 mmol), n-butylamine (30 mg, 0.41 mmol) and DIPEA (58 mg, 0.45 mol) were added to DMF (1.0 mL), and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, concentrated, and the residue was washed with petroleum ether to obtain white solid compound LH-86 (31 mg, 55%). Compound LH-86 was used directly in the next reaction without purification.

[0294] Synthesis of compound LH-2-180 (n-butyl-5-(3-methoxy-5-(piperazine-1-)pyridin-2-yl)thiophene-2-carboxamide): Compound LH-86 (31 mg, 0.07 mmol) was dissolved in 1,4-dioxane 4M HCl solution (10 mL), and the mixture was stirred at room temperature for 12 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 1 / 1) to obtain compound LH-2-180 (18 mg, 69%) as a white solid.

[0295] Compound LH-2-180. 1 H NMR (400MHz, MeOD) δ7.99(d,J=2.3Hz,1H),7.88(d,J=4.2Hz,1H),7.76(d,J=4.2Hz,1H),7.61(d,J=2.1Hz,1H),4.17(s,3H) ,3.87–3.72(m,4H),3.49–3.40(m,4H),3.38(t,J=7.2Hz,2H),1.67–1.54(m,2H),1.48–1.35(m,2H),0.98(t,J=7.4Hz,3H). 13C NMR(101MHz,MeOD)δ162.29,154.60,147.52,142.64,135.07,128.26,127.79 ,127.60,121.57,110.94,56.44,44.33,42.75,39.35,31.26,19.80,12.76..

[0296] Example 16: Synthesis of Compounds LH-3-8 and LH-3-45

[0297] 1. Synthesis of compound LH-3-8

[0298]

[0299] Synthesis of intermediate compound LH-2-141 (5-bromo-2-iodo-3-ethoxypyridine): 5-Bromo-2-iodopyridin-3-ol (650 mg, 2.2 mmol), t-BuONa (250 mg, 2.6 mmol), and iodoethane (620 mg, 4.0 mmol) were dissolved in DMF (10 mL) and stirred at room temperature for 12 h. After completion of the reaction, water was added portionwise to the reaction mixture, and the mixture was extracted with EtOAc. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE / EtOAc = 20 / 1) to yield compound LH-2-141 as a white solid (439 mg, 61%). Compound LH-2-141. 1 H NMR (400MHz, MeOD) δ8.05 (d, J = 19.0Hz, 1H), 7.43 (s, 1H), 4.15 (q, J = 6.9Hz, 2H), 1.46 (t, J = 6.9Hz, 3H).

[0300] Synthesis of intermediate compound LH-2-143 (5-(5-bromo-3-ethoxypyridine-2-)thiophene-2-carboxylic acid): The synthesis procedure was the same as that for compound LH-2-157 in Step 1 of Example 14, yielding intermediate compound LH-2-143 as a yellow solid (31 mg, 59%). Intermediate compound LH-2-143 was used directly in the next reaction without column chromatography.

[0301] Synthesis of intermediate compound LH-2-145 (methyl 5-(5-bromo-3-ethoxypyridine-2-)thiophene-2-carboxylate): The synthesis procedure was the same as that for compound LH-2-162 in Step 1 of Example 14. Purification by silica gel column chromatography (PE / EtOAc = 3 / 1) afforded compound LH-2-145 as a yellow solid (152 mg, 83%). 1H NMR (400MHz, CDCl3) δ8.26(d,J=1.7Hz,1H),7.95(d,J=4.0Hz,1H),7.78(d,J=4.1Hz,1 H),7.41(d,J=1.6Hz,1H),4.22(q,J=7.0Hz,2H),3.90(s,3H),1.60(t,J=7.0Hz,3H).

[0302] Synthesis of intermediate compound LH-3-3 (methyl 5-(3-ethoxy-5-phenylpyridine-2-)thiophene-2-carboxylate): The synthesis procedure was the same as that for compound LH-2-163 in Step 1 of Example 14. Purification by silica gel column chromatography (PE / EtOAc = 2 / 1) afforded compound LH-3-3 (170 mg, 65%) as a white solid. 1 H NMR (400MHz, CDCl3) δ8.46(d,J=1.4Hz,1H),8.01(d,J=4.0Hz,1H),7.81(d,J=4.0Hz,1H),7.61(d,J=7.3 Hz,2H),7.54–7.46(m,2H),7.46–7.38(m,2H),4.30(q,J=6.9Hz,2H),3.91(s,3H),1.63(t,J=6.9Hz,3H).

[0303] Synthesis of intermediate compound LH-3-6 (5-(3-ethoxy-5-phenylpyridine-2-)thiophene-2-carboxylic acid): The synthesis procedure was the same as that for compound LH-159 in Step 1 of Example 14, yielding LH-3-6 as a white solid (86 mg, 57%). Compound LH-3-6 was used directly in the next reaction without column chromatography.

[0304] Synthesis of compound LH-3-8 (tert-butyl 4-(5-(3-ethoxy-5-phenylpyridin-2-yl)thiophene-2-carbonyl)piperazine-1-carboxylate): The synthesis steps were the same as the synthesis steps of compound LH-65 in step 2 of Example 14 to obtain white solid compound LH-3-8 (57 mg, 76%).

[0305] Compound LH-3-8. 1H NMR(400MHz, CDCl3)δ8.45(s,1H),7.97(d,J=3.6Hz,1H),7.60(d,J=7.4Hz,2H),7.53–7.39(m,4H),7.3 6(d,J=3.6Hz,1H),4.29(q,J=6.8Hz,2H),3.76(s,4H),3.51(s,4H),1.61(t,J=6.8Hz,3H),1.48(s,9H). 13 C NMR (101MHz, CDCl3) δ164.26,154.60,151.77,144.81,139.98,139.53,137.43,136.88,136 .56,130.22,129.15,128.33,127.11,127.06,117.56,80.35,64.56,29.71,28.40,14.81.

[0306] 2. The preparation of compound LH-3-45 refers to the synthesis of compound LH-3-8 in step 1 of this example. The structural formula of compound LH-3-45 is The NMR characterization results are: 1 H NMR (400MHz, DMSO) δ8.36(d,J=1.6Hz,1H),7.76(d,J=3.9Hz,1H),7.68–7.62(m,3H),7.39–7.32(m,2H),7.32– 7.23(m,2H),4.22(q,J=6.9Hz,2H),3.57–3.51(m,4H),2.79–2.61(m,4H),1.74(s,1H),1.34(t,J=6.9Hz,3H). 13 C NMR(101MHz,DMSO)δ163.03,151.94,144.62,139.40,139.26,137.90,136.92,1 36.34,130.36,129.57,128.89,127.48,127.35,118.58,64.98,44.97,15.00.

[0307] Example 17: Synthesis of compounds LH-2-54, LH-2-55, LH-2-56, LH-2-64 and LH-2-83

[0308] 1. Synthesis of compound LH-2-54

[0309]

[0310] Synthesis of the intermediate compound LH-2-22 (5-(3-methoxy-6-nitropyridin-2-yl)thiophene-2-carboxylic acid): Refer specifically to the synthesis steps of compound LH-2-157 in Step 1 of Example 14 to obtain a yellow solid compound LH-2-22 (1072 mg, 88%).

[0311] Compound LH-2-22. 1 H NMR (400MHz, DMSO) δ8.36(d,J=8.9Hz,1H),8.05(d,J=4.0Hz,1H),7.95(d,J=9.0Hz,1H),7.89( d,J=4.9Hz,1H),7.78(d,J=3.7Hz,1H),7.73(d,J=3.6Hz,1H),7.23–7.16(m,1H),4.17(s,3H).

[0312] Synthesis of intermediate compound LH-2-33 (5-(3-methoxy-6-nitropyridin-2-yl)-N-pentylthiophene-2-carboxamide): Refer to the synthesis procedure for compound LH-61 in Step 1 of Example 13. Compound LH-2-22 and n-pentylamine were used as starting materials. Purification by silica gel column chromatography (PE / EtOAc = 2 / 1) afforded compound LH-2-33 as a yellow oil (460 mg, 58%). Compound LH-2-33. 1 H NMR (400MHz, DMSO) δ8.54–8.40(m,1H),7.80–.67(m,2H),7.20–7.08(m,1H),3.35(s, 3H),3.27–3.16(m,2H),1.58–1.43(m,2H),1.37–1.20(m,4H),0.88(t,J=6.9Hz,3H).

[0313] Synthesis of intermediate compound LH-2-41 (5-(6-amino-3-methoxypyridin-2-yl)-N-pentylthiophene-2-carboxamide): Compound LH-2-23 (200 mg, 0.57 mmol), iron powder (560 mg, 10 mmol) and H2O (1.0 mL) were added to AcOH (15 mL) and EtOH (20 mL) and stirred at 80°C for 2 h. The mixture was then extracted with water and EtOAc, and the product was dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to obtain a yellow solid and a yellow oil (203 mg, 67%). Intermediate compound LH-2-41 was used directly in the next reaction without purification.

[0314] Synthesis of intermediate compound LH-2-50 (ethyl 3-((5-methoxy-6-(5-(pentylcarbamoyl)thiophen-2-yl)pyridin-2-yl)amino)-3-oxopropanoate): Compound LH-2-41 (190 mg, 0.6 mmol), TEA (0.5 mL), and ethyl chloroformyl acetate (150 mg, 1.0 mmol) were added to anhydrous DCM (10 mL) at 0°C, maintained in an ice-water bath, and stirred for 2 h. The mixture was extracted with water and EtOAc, and the product dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 1 / 1) to obtain compound LH-2-50 (137 mg, 53%) as a yellow solid. Compound LH-2-50. 1 H NMR (400MHz, DMSO) δ10.56(s,1H),8.57–8.44(m,1H),8.00(d,J=8.9Hz,1H),7.88(d,J=4.0Hz,1H),7.73(d,J=4.0Hz,1H),7.67(d,J=9.1Hz,1H),4 .17–4.09(m,2H),3.98(s,3H),3.57(s,2H),3.28–3.19(m,2H),1.61–1.4 5(m,2H),1.36–1.28(m,4H),1.22(t,J=7.1Hz,3H),0.89(t,J=6.8Hz,3H).

[0315] Synthesis of compound LH-2-54 (3-((5-methoxy-6-(5-(pentylcarbamoyl)thiophen-2-yl)pyridin-2-yl)amino)-3-oxopropanoic acid): For the specific synthesis method, refer to the synthesis step of compound LH-159 in Step 1 of Example 14 to obtain yellow oily compound LH-2-54 (41 mg, 76%).

[0316] Compound LH-2-54. 1 H NMR (400MHz, Acetone) δ8.13(d,J=8.7Hz,1H),7.93–7.81(m,2H),7.69–7.55(m,2H),4.04(d,J=6.4Hz,3H ),3.61(s,1H),3.41–3.34(m,3H),2.20(s,1H),1.68–1.53(m,2H),1.41–1.30(m,4H),0.94–0.85(m,3H).

[0317] 2. The preparation of compound LH-2-83 refers to the synthesis of compound LH-2-54 in step 1 of this example. The structural formula of compound LH-2-83 is The NMR characterization results are: 1 H NMR (400MHz, DMSO) δ10.63(s,1H),8.05(d,J=8.6Hz,1H),7.96–7.83(m,1H),7.70(d,J=9.0Hz,2H),3.98(s,3H),3.45(s,3H),3.39–3.31(m,8H).

[0318] 3. Synthesis of compound LH-2-55

[0319]

[0320] Compound LH-2-55(N 1 -(tert-Butyl)-N 3 -(5-methoxy-6-(5-(pentylcarbamoyl)thiophene-2-)pyridine-2-)malonamide): Using compound LH-2-54 as raw material, refer to the synthesis steps of compound LH-61 in step 1 of Example 13 for details, and obtain yellow solid compound LH-2-55 (45 mg, 52%).

[0321] Compound LH-2-55. 1 H NMR (400MHz, DMSO) δ10.44(s,1H),8.53(s,1H),8.11–7.96(m,1H),7.95–7.56(m,4H),3. 97(s,3H),3.35–3.12(m,4H),1.63–1.42(m,2H),1.41–1.14(m,13H),0.96–0.75(m,3H). 13 C NMR (101MHz, DMSO) δ166.75,161.58,149.24,144.80,144.44,141.21,137.82,128.5 7,128.37,122.97,114.24,56.67,50.82,45.07,29.35,29.18,28.90,22.37,14.42.

[0322] 4. The preparation of compounds LH-2-56 and LH-2-64 was carried out by referring to the synthesis of compound LH-2-55 in step 3 of this example. The structural formulas and NMR characterization results of compounds LH-2-56 and LH-2-64 are shown in Table 6.

[0323] Table 6: Structural formula and NMR characterization results of compounds LH-2-56 and LH-2-64

[0324]

[0325] Example 18: Synthesis of Compound LH-2-34

[0326]

[0327] Synthesis of compound LH-2-34 (N-(5-methoxy-6-(5-(morpholine-4-carbonyl)thiophene-2-)pyridine-2-)butanamide): Refer to the synthesis steps of compound LH-2-50 in Example 10 for details. Use n-butyryl chloride to react with compound LH-2-27 to obtain compound LH-2-34 (46 mg, 57%) as a light yellow oil.

[0328] Compound LH-2-34. 1 H NMR (400MHz, DMSO) δ10.24(s,1H),8.04(d,J=8.7Hz,1H),7.85(d,J=3.9Hz,1H),7.65(d,J=9.1Hz,1H),7.43(d, J=3.9Hz,1H),3.96(s,3H),3.72–3.56(m,8H),2.39(t,J=7.3Hz,2H),1.68–1.55(m,2H),0.92(t,J=7.4Hz,3H). 13 C NMR(101MHz,DMSO)δ172.19,162.97,149.01,144.96,144.03,138.02,137 .59,130.24,127.55,123.01,114.58,66.62,56.72,38.32,18.92,14.12..

[0329] Example 19: Synthesis of compounds LH-2-106, LH-2-109, LH-2-110, LH-2-112, LH-2-113, LH-2-114, LH-2-130 and LH-2-133

[0330] 1. Synthesis of compound LH-2-110

[0331]

[0332] Synthesis of intermediate compound LH-2-90 (2,6-dibromo-3-methoxypyridine): K2CO3 (980 mg, 7.1 mmol), 2,6-dibromo-3-hydroxypyridine (2000 mg, 10.0 mmol) and iodomethane MeI (1.68 mL, 27 mmol) were added to DMSO (5.0 mL), and the mixture was stirred at 70 ° C for 12 h. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA=3 / 1) to obtain yellow solid compound LH-2-90 (1090 mg, 52%). Compound LH-2-90. 1 H NMR (400MHz, CDCl3) δ7.38 (d, J = 8.4Hz, 1H), 7.04 (d, J = 8.4Hz, 1H), 3.91 (s, 3H).

[0333] Synthesis of intermediate compound LH-2-91 (2,6-dibromo-3-methoxy-5-nitropyridine): Compound LH-2-90 (490 mg, 1.87 mmol) was added portionwise to fuming nitric acid (2.3 mL) and concentrated sulfuric acid (2.3 mL) in an ice-water bath, heated to 65°C, and stirred for 2 h. The mixture was diluted with ice water and extracted with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate and concentrated. Purification by silica gel column chromatography (PE / EA = 20 / 1) gave compound LH-2-91 (280 mg, 47%) as a yellow solid. Compound LH-2-91. 1 H NMR (400MHz, CDCl3) δ7.66 (s, 1H), 4.04 (s, 3H).

[0334] Synthesis of intermediate compound LH-2-92 (2-bromo-5,6-dimethoxy-3-nitropyridine): NaOMe (60 mg, 1.1 mmol) and LH-2-91 (325 mg, 1.0 mmol) were added to MeOH (4.0 mL), and the mixture was stirred at room temperature for 12 h. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-2-92 (262 mg, 85%) as a yellow solid. Compound LH-2-92. 1 H NMR (400MHz, DMSO) δ7.99(s,1H),3.99(s,3H),3.90(s,3H).

[0335] Synthesis of intermediate compound LH-2-100 (5-(5,6-dimethoxy-3-nitropyridine-2-)thiophene-2-carboxylic acid): Refer to the synthesis method for compound LH-2-157 in Step 1 of Example 14. Using compound LH-2-92 as the starting material, a yellow solid compound LH-2-100 (105 mg, 39%) was obtained. Compound LH-2-100. 1 H NMR (400MHz, DMSO) δ13.31(s,1H),7.89(s,1H),7.68(d,J=3.9Hz,1H),7.30(d,J=3.9Hz,1H),4.02(s,3H),3.92(s,3H). 13 C NMR (101MHz, DMSO) δ163.14,154.55,144.73,144.08,139.18,136.98,134.11,132.09,127.93,114.99,57.22,54.95.

[0336] Synthesis of intermediate compound LH-2-107 (5-(5,6-dimethoxy-3-nitropyridine-2-)-N-pentylthiophene-2-carboxamide): 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 380 mg, 1.0 mmol), compound LH-2-100 (5-(5,6-dimethoxy-3-nitropyridine-2-)thiophene-2-carboxylic acid) (150 mg, 0.5 mmol), n-pentylamine (87 mg, 1.0 mmol), and N,N-diisopropylethylamine (DIPEA; 130 mg, 1.0 mol) were added to DMF (1.0 mL), and the mixture was stirred at room temperature for 12 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate, and the product dissolved in the organic layer. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. Purification by column chromatography (PE / EA=2 / 1) gave compound LH-2-107 (152 mg, 80%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ7.48 (s, 1H), 7.43–7.35 (m, 2H), 4.12 (d, J = 11.5Hz, 3H), 3.97 ( s,3H),3.54–3.37(m,2H),1.39–1.33(m,4H),1.31–1.22(m,2H),0.95–0.89(m,3H).

[0337] Synthesis of intermediate compound LH-2-108 (5-(3-amino-5,6-dimethoxypyridine-2-)-N-pentylthiophene-2-carboxamide): Compound LH-2-107 (5-(5,6-dimethoxy-3-nitropyridine-2-)-N-pentylthiophene-2-carboxamide) (133 mg, 0.35 mmol), iron powder (560 mg, 10 mmol), and H2O (1.0 mL) were added to AcOH (15 mL) and EtOH (20 mL) and stirred at 80 ° C for 2 h. The mixture was then extracted with water and EtOAc, and the product was dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give a yellow oily compound LH-2-108 (80 mg, 65%). The intermediate compound LH-2-108 was used directly in the next reaction without purification.

[0338] Synthesis of compound LH-2-110 (ethyl 3-((5,6-dimethoxy-2-(5-(pentylcarbamoyl)thiophene-2-)pyridin-3-yl)amino)-3-oxopropanoate): Refer to the synthesis method of compound LH-2-50 in step 1 of Example 17 for details. Using compound LH-2-108 as raw material, purification by silica gel column chromatography (PE / EA=2 / 1) gave compound LH-2-110 (375 mg, 73%) as a yellow oil.

[0339] Compound LH-2-110. 1 H NMR (400MHz, DMSO) δ10.04(s,1H),8.45(t,J=5.4Hz,1H),7.76–7.60(m,2H),7.23(s,1H),4.17(q,J=7.1Hz,2H),3.94(s,3H),3 .80(s,3H),3.54(s,2H),3.27–3.19(m,2H),1.57–1.47(m,2H),1.32–1.28(m,3H),1.24(t,J=7.1Hz,4H),0.88(t,J=6.8Hz,3H). 13 C NMR (101MHz, DMSO) δ168.04,165.62,161.52,151.34,145.94,143.48,140.28,134.28,128.7 2,126.39,124.39,119.59,61.21,56.41,53.74,43.45,29.35,29.15,22.35,14.54,14.40..

[0340] 2. The preparation of compounds LH-2-133 and LH-2-130 refers to the synthesis of compound LH-2-110 in step 1 of this example.

[0341] Among them, the structural formula of compound LH-2-133 is The NMR characterization results are: 1 HNMR(400MHz,DMSO)δ9.97(s,1H),8.45(s,1H),7.78–7.59(m,2H),7.24(s,1H),3.94(s,3H),3.80(s ,3H),3.69–3.45(m,8H),3.29–3.15(m,2H),1.55–1.45(m,2H),1.38–1.26(m,6H),0.92–0.80(m,5H). 13 C NMR (101MHz, DMSO) δ167.00,166.18,161.54,151.21,145.94,143.43,140.16,134.22,128.7 6,126.60,124.66,119.53,66.56,56.38,53.71,46.73,42.21,29.34,29.14,22.34,14.40.

[0342] Among them, the structural formula of compound LH-2-130 is The NMR characterization results are: 1 H NMR(400MHz,DMSO)δ9.73(s,1H),7.54(s,1H),7.40(s,1H),7.21(s,1H),3.93(s, 3H), 3.80 (s, 3H), 3.68–3.61 (m, 8H), 2.40 (q, J = 7.3Hz, 2H), 1.12 (t, J = 7.3Hz, 3H). 13 C NMR (101MHz, DMSO) δ173.34,162.91,151.28,144.98,143.55,137.03,134 .52,130.09,125.52,125.06,120.23,66.59,56.40,53.66,29.28,9.81.

[0343] 3. Synthesis of compound LH-2-112

[0344]

[0345] Synthesis of compound LH-2-112 (3-((5,6-dimethoxy-2-(5-(pentylcarbamoyl)thiophene-2-)pyridine-3-)amino)-3-oxopropanoic acid): Refer to the synthesis method of compound LH-159 in step 1 of Example 14 for details. Using compound LH-2-110 as the starting material, the ester group was hydrolyzed to a carboxyl group under alkaline conditions, and finally purified by silica gel column chromatography (DCM / MeOH=10 / 1) to give compound LH-2-112 (229 mg, 77%) as a yellow oil.

[0346] Compound LH-2-112. 1 H NMR(400MHz,DMSO)δ10.43(s,1H),8.51(s,1H),7.87–7.65(m,2H),7.30(s,1H),3.94(s,3H), 3.80(s,3H),3.22(d,J=5.9Hz,2H),1.58–1.39(m,2H),1.37–1.07(m,6H),0.91–0.81(m,3H). 13 C NMR(101MHz,DMSO)δ169.97,161.56,151.01,146.14,143.39,143.34,140.13,133.77 ,128.92,126.37,124.75,119.24,56.35,53.69,43.95,29.35,29.15,22.35,14.41.

[0347] 4. Synthesis of compound LH-2-113

[0348]

[0349] Synthesis of compound LH-2-113 (5-(5,6-dimethoxy-3-(3-morpholinyl-3-oxypropionamide)pyridine-2-)-N-pentylthiophene-2-carboxamide): Refer to the synthesis method of compound LH-61 in step 1 of Example 13 for details. Use compound LH-2-112 as raw material and purify by silica gel column chromatography (DCM / MeOH=10 / 1) to obtain yellow oily compound LH-2-113 (32 mg, 41%).

[0350] Compound LH-2-113. 1H NMR(400MHz,DMSO)δ9.97(s,1H),8.45(s,1H),7.78–7.59(m,2H),7.24(s,1H),3.94(s,3H),3.80(s, 3H),3.69–3.45(m,8H),3.29–3.15(m,2H),1.55–1.45(m,2H),1.38–1.26(m,6H),0.92–0.80(m,5H). 13 C NMR (101MHz, DMSO) δ167.00,166.18,161.54,151.21,145.94,143.43,140.16,134.22,128.7 6,126.60,124.66,119.53,66.56,56.38,53.71,46.73,42.21,29.34,29.14,22.34,14.40.

[0351] 5. The preparation of compounds LH-2-109 and LH-2-114 refers to the synthesis of compound LH-2-113 in step 4 of this example.

[0352] Among them, the structural formula of compound LH-2-109 is The NMR characterization results are: 1 HNMR (400MHz, DMSO) δ9.97 (s, 1H), 8.44 (s, 1H), 7.72 (d, J = 16.2Hz, 2H), 7.27 (s, 1H), 3.94 (s, 3H), 3.80 (s, 3H), 3. 58(s,2H),3.31–3.17(m,2H),3.04(s,3H),2.89(s,3H),1.63–1.40(m,2H),1.40–1.25(m,2H),0.95–0.82(m,3H). 13 C NMR (101MHz, DMSO) δ167.56,167.08,161.59,151.12,146.03,143.40,140.11,134.04,12 8.85,126.62,124.73,119.40,56.35,53.68,42.43,37.86,35.39,31.78,20.10,14.17..

[0353] Among them, the structural formula of compound LH-2-114 is The NMR characterization results are: 1HNMR (400MHz, DMSO) δ9.97 (s, 1H), 8.44 (s, 1H), 7.72 (d, J = 16.2Hz, 2H), 7.26 (s, 1H), 3.94 (s, 3H), 3.80 (s, 3H), 3. 57(s,2H),3.29–3.16(m,2H),3.04(s,3H),2.89(s,3H),1.56–1.45(m,2H),1.33–1.27(m,4H),0.89–0.83(m,3H). 13 C NMR (101MHz, DMSO) δ167.55,167.07,161.56,151.11,146.02,143.39,140.12,134.05,128.8 5,126.63,124.74,119.42,56.36,53.69,42.43,37.87,35.40,29.34,29.14,22.35,14.41.

[0354] Example 20: Synthesis of compounds LH-84, LH-133 to LH-134, LH-138 to LH-144, LH-145A, LH-145B, LH-147, LH-151, LH-152, LH-154 to LH-156

[0355] 1. Synthesis of compound LH-84

[0356]

[0357] Synthesis of intermediate compound LH-70 (3-methoxy-4-nitro-1,1'-biphenyl): Pd(PPh3)4 (462 mg, 0.40 mmol) was added to a mixture of 4-chloro-2-methoxy-1-nitrobenzene (1.50 g, 8.0 mmol), phenylboronic acid (1.17 g, 9.6 mmol), potassium carbonate (3.87 g, 28 mmol), DMF (14 mL) and H2O (14 mL) at room temperature. Under argon protection, the mixture was stirred at 100°C for 16 hours. After the reaction was completed, the mixture was extracted with water and ethyl acetate, and the product was dissolved in the organic layer. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EtOAc = 9 / 1) to obtain yellow solid compound LH-70 (1.25 g, 68%). Compound LH-70. 1H NMR (400MHz, CDCl3) δ7.55(d,J=7.5Hz,2H),7.45–7.36(m,2H),7.29(d,J=7.3Hz,1H),7.05(d,J=8.3Hz,2H),6.80(d,J=7.7Hz,1H),3.93(s,3H).

[0358] Synthesis of intermediate compound LH-76 (1-methoxy-[1,1'-biphenyl]-4-amine): Compound LH-70 (700 mg, 3.0 mmol) was dissolved in MeOH (5 mL). 70 mg of Pd / C was added portionwise to the solution, and the mixture was stirred at room temperature for 3 h. Extraction was performed with water and ethyl acetate, and the organic phase was washed with saturated brine. The organic layer was then dried over anhydrous sodium sulfate and concentrated. Purification was performed by silica gel column chromatography (PE / EtOAc = 1 / 1) to obtain compound LH-76 (468 mg, 78%) as a brown oil. 1 H NMR (400MHz, DMSO) δ7.58 (d, J=7.5Hz, 2H), 7.44–7.33 (m, 2H), 7.27–7.17 (m, 1H), 7.08 (d,J=1.7Hz,1H),7.05–6.99(m,1H),6.71(d,J=8.0Hz,1H),4.85(s,2H),3.85(s,3H).

[0359] Synthesis of intermediate compound LH-82 (4-bromo-3-methoxy-1,1'-biphenyl):

[0360] First, compound LH-76 (400 mg, 2.0 mmol) was dissolved in a mixture of 48% hydrobromic acid (5.0 mL), H2O (5.0 mL) and acetonitrile (5.0 mL), and the mixture solution was cooled to below 0°C in an ice-water bath; sodium nitrite (345 mg, 3.0 mmol) was dissolved in H2O (5.0 mL), and the sodium nitrite aqueous solution was added dropwise to the mixture using a dropping funnel and stirred in an ice-water bath to uniformly dissolve it.

[0361] Then, cuprous bromide (573 mg, 2.0 mmol) was dissolved in 48% hydrobromic acid (5.0 mL) to prepare a solution of cuprous bromide and hydrobromic acid. The solution was added dropwise to the sodium nitrite solution and stirred vigorously in an ice-water bath for 1 h. The color of the solution became significantly darker.

[0362] The mixture was heated to 60°C and stirred for 1 hour until nitrogen evolution ceased. After the reaction was complete, the mixture was extracted with water and EtOAc, and the product was dissolved in the organic layer. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield compound LH-82 as a yellow oil (237 mg, 45%). Intermediate compound LH-82 was used directly in the next reaction without further purification.

[0363] Synthesis of compound LH-84 (5-(3-methoxy-[1,1'-biphenyl]-4-yl)thiophene-2-carboxylic acid): Catalyst Pd(PPh3)4 (35 mg, 0.03 mmol) was added to a mixture of compound LH-82 (160 mg, 0.55 mmol), 5-carboxythiophene-2-boronic acid (86 mg, 0.5 mmol), sodium carbonate Na2CO3 (106 mg, 1.0 mmol), MeCN (3.0 mL) and H2O (3.0 mL) at room temperature, and then the mixture solution was stirred at 80 °C for 4 h under argon protection.

[0364] After the reaction is completed, the mixture is extracted with water and EtOAc. At this time, the aqueous phase is alkaline and the product is a carboxylic acid derivative, so the product will dissolve in the aqueous phase. The organic layer is then discarded and the pH of the aqueous phase is adjusted to 2-3 with a 2M HCl aqueous solution to reduce the solubility of the carboxylic acid derivative in the aqueous phase. A light yellow precipitate is precipitated in the solution. It is then extracted with pure EtOAc and the product is dissolved in the organic layer. Finally, the organic phase is washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. Purification by silica gel column chromatography (DCM / MeOH=10 / 1) gives compound LH-84 (45 mg, 26%) as a yellow solid.

[0365] Compound LH-84. 1 H NMR (400MHz, MeOD) δ7.85(d,J=8.0Hz,1H),7.74(s,1H),7.69(d,J=7.3Hz,2H),7.61(s,1H),7.50–7.42(m,2H),7.41–7.25(m,3H),4.06(s,3H). 13 C NMR(101MHz,DMSO)δ163.73,156.17,145.01,142.17,139.78,134.00,133.1 7,129.42,128.72,128.43,127.26,126.02,120.93,119.81,110.96,56.37..

[0366] 2. Synthesis of compound LH-133

[0367]

[0368] Synthesis of compound LH-133 ((5-(3-methoxy-[1,1'-biphenyl]-4-)thiophene-2-)(4-(pyridine-2-)piperazine-1-)methanone): HATU (171 mg, 0.45 mmol), compound LH-84 (60 mg, 0.2 mmol), 1-(pyridine-2-)piperazine (163 mg, 1.0 mmol) and DIPEA (58 mg, 0.45 mol) were added to DMF (1.0 mL), and the mixture was stirred at room temperature for 12 h. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA=3 / 1) to give compound LH-133 (85 mg, 93%) as a white solid.

[0369] Compound LH-133. 1 H NMR (400MHz, DMSO) δ8.14(s,1H),7.91(d,J=7.3Hz,1H),7.78(d,J=6.4Hz,2H),7.67(s,1H),7.61–7.54(m,1H),7.53–7 .45(m,3H),7.45–7.29(m,3H),6.85(d,J=8.1Hz,1H),6.68(s,1H),4.05(s,3H),3.86–3.73(m,4H),3.66–3.55(m,4H). 13 CNMR(101MHz,DMSO)δ163.10,159.11,156.01,148.06,141.76,139.86,138.13,137.07,137.02,129.44, 129.41,128.58,128.35,127.24,125.20,121.07,119.79,113.76,110.95,107.66,56.35,45.03,39.98..

[0370] 3. The preparation of compounds LH-134, LH-138 to LH-144, LH-145A, LH-145B, LH-147, LH-151, LH-152, and LH-154 to LH-156 followed the synthesis of compound LH-133 in step 1 of this example. The structures and NMR characterization results of compounds LH-134, LH-138 to LH-144, LH-145A, LH-145B, LH-147, LH-151, LH-152, and LH-154 to LH-156 are shown in Table 7.

[0371] Table 7: Structural formula and NMR characterization results of each compound

[0372]

[0373]

[0374]

[0375] Example 21: Synthesis of Compound LH-187

[0376]

[0377] Synthesis of intermediate compound LH-125 (5-(2-methoxy-4-nitrophenyl)thiophene-2-carboxylic acid methyl ester): Pd(PPh3)4 (231 mg, 0.2 mmol) was added to a mixture of 5-bromothiophene-2-carboxylic acid methyl ester (884 mg, 4.0 mmol), (2-methoxy-4-nitrophenyl)boric acid (866 mg, 4.4 mmol), sodium bicarbonate (1008 mg, 12.0 mmol), DME (16 mL), and H2O (12 mL). Under argon protection, the mixture was stirred at 100°C for 18 h. After the reaction was completed, the mixture was extracted with water and EtOAc and concentrated, and recrystallized from dichloromethane and petroleum ether to obtain yellow solid compound LH-125 (395 mg, 27%). The intermediate compound LH-125 was used directly in the next reaction without purification by column chromatography.

[0378] Synthesis of intermediate compound LH-136 (methyl 5-(4-amino-2-methoxyphenyl)thiophene-2-carboxylate): Referring to the synthesis steps for compound LH-76 in Example 20, compound LH-125 was used as the starting material and hydrogen reduction was performed to obtain compound LH-136 (270 mg, 60%) as a yellow solid. Intermediate compound LH-136 was used directly in the next reaction without purification.

[0379] Synthesis of intermediate compound LH-178 (methyl 5-(4-((tert-butoxycarbonyl)amino)-2-methoxyphenyl)thiophene-2-carboxylate): Compound LH-136 (200 mg, 0.68 mmol), (Boc)2O (1000 mg, 4.6 mmol), and TEA (0.36 mL, 5.0 mmol) were dissolved in THF (10 mL) and stirred at room temperature for 12 h. The mixture was then extracted with water and EtOAc, and the product was dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 5 / 1) to obtain compound LH-178 (203 mg, 66%) as a yellow solid. Compound LH-178. 1H NMR (400MHz, CDCl3) δ7.74(d,J=4.0Hz,1H),7.58(d,J=8.4Hz,1H),7.39(d,J=3.8 Hz,2H),6.83–6.70(m,1H),6.60(s,1H),3.97(s,3H),3.89(s,3H),1.53(s,9H).

[0380] Synthesis of intermediate compound LH-181 (5-(4-((tert-Butyloxycarbonyl)amino)-2-methoxyphenyl)thiophene-2-carboxylic acid): Referring to the synthesis steps for compound LH-159 in Example 14, compound LH-178 was used as the starting material. The ester group was hydrolyzed to a carboxyl group to obtain compound LH-181 (90 mg, 87%) as a yellow solid. Intermediate compound LH-181 was used directly in the next step without purification.

[0381] Synthesis of intermediate compound LH-182 (tert-butyl(3-methoxy-4-(5-(4-(pyridine-2-)piperazine-1-carbonyl)thiophene-2-)phenyl)carbamate): Compound LH-181 was used as the starting material through an amide condensation reaction to obtain compound LH-182 (91 mg, 62%) as a yellow solid. Intermediate compound LH-182 was used directly in the next reaction without purification by column chromatography.

[0382] Synthesis of intermediate compound LH-183 ((5-(4-amino-2-methoxyphenyl)thiophene-2-)(4-(pyridine-2-)piperazine-1-)methanone): Compound LH-182 (90 mg, 0.18 mmol) and trifluoroacetic acid (5 mL) were dissolved in DCM (10 mL) and stirred at room temperature for 12 h. After completion of the reaction, the solvent was evaporated under vacuum to afford compound LH-183 as a yellow oil (52 mg, 73%). Intermediate compound LH-183 was used directly in the next reaction without purification.

[0383] Synthesis of compound LH-187 ((5-(2-methoxy-4-(1H-tetrazol-1-yl)phenyl)thiophene-2-yl)(4-(pyridin-2-yl)piperazine-1-)methanone): Compound LH-183 (50 mg, 0.1 mmol), NaN3 (13 mg, 0.2 mmol) and trimethyl orthoformate (30 mg, 0.3 mmol) were dissolved in AcOH (5 mL) and slowly stirred at 80°C for 4 h. The mixture was then extracted with water and EtOAc, and the product was dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (DCM / MeOH=5 / 1) to give compound LH-187 (37 mg, 73%) as a yellow solid.

[0384] Compound LH-187. 1 H NMR (400MHz, DMSO) δ10.21(s,1H),8.22–8.03(m,2H),7.80–7.69(m,2H),7.67–7.55(m,2H),7.51(d,J=4 .0Hz,1H),6.87(d,J=8.6Hz,1H),6.72–6.64(m,1H),4.08(s,3H),3.90–3.74(m,4H),3.69–3.56(m,4H).

[0385] Example 22: Synthesis of Compound LH-189

[0386]

[0387] Synthesis of intermediate compound LH-174 (methyl 5-(4-cyanophenyl)thiophene-2-carboxylate): See the synthesis procedure for compound LH-2-163 in Step 1 of Example 14 for details, to obtain yellow solid compound LH-174 (371 mg, 74%). Compound LH-174. 1 H NMR (400MHz, CDCl3) δ7.82 (d, J = 3.9 Hz, 1H), 7.78–7.70 (m, 4H), 7.41 (d, J = 4.0 Hz, 1H), 3.95 (s, 3H).

[0388] Synthesis of intermediate compound LH-185 (5-(4-cyanophenyl)thiophene-2-carboxylic acid): Referring to the synthesis procedure for compound LH-159 in Step 1 of Example 14, compound LH-174 was used as the starting material to obtain compound LH-185 as a yellow solid (187 mg, 91%). Intermediate compound LH-185 was used directly in the next reaction without purification.

[0389] Synthesis of intermediate compound LH-186 (4-(5-(4-(pyridine-2-)piperazine-1-carbonyl)thiophene-2-)benzonitrile): Refer to the synthesis procedure for compound LH-61 in Step 1 of Example 13. Using compound LH-186 as the starting material, a yellow solid compound LH-186 (256 mg, 80%) was obtained. Compound LH-186. 1 H NMR (400MHz, CDCl3) δ8.32–8.17(m,1H),7.77–7.69(m,4H),7.62–7.52(m,1H) ,7.40–7.34(m,2H),6.77–6.69(m,2H),4.02–3.90(m,4H),3.76–3.61(m,4H).

[0390] Synthesis of compound LH-189 ((5-(4-(1H-tetrazolyl-5-)phenyl)thiophene-2-)(4-(pyridine-2-)piperazine-1-)methanone): Compound LH-186 (149 mg, 0.4 mmol) was dissolved in anhydrous DMF (5 mL), and then NaN3 (104 mg, 1.6 mmol) and NH4Cl (86 mg, 1.6 mmol) were added and stirred at 100°C for 24 h. The mixture was then extracted with water and EtOAc, and the product was dissolved in the organic phase. After completion of the reaction, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (DCM / MeOH=5 / 1) to give compound LH-189 (77 mg, 47%) as a yellow solid.

[0391] Compound LH-189. 1 H NMR (400MHz, DMSO) δ8.20–8.10(m,1H),8.04(d,J=8.4Hz,2H),7.75(d,J=8.4Hz,2H),7.61–7.54(m,2H ),7.53–7.48(m,1H),6.87(d,J=8.6Hz,1H),6.72–6.65(m,1H),3.88–3.75(m,4H),3.67–3.58(m,4H). 13 C NMR (101MHz, DMSO) δ162.46,159.07,156.76,148.04,145.96,138.13,137.4 5,134.73,131.16,127.94,126.75,126.62,124.87,113.76,107.66,44.98..

[0392] Example 23: Synthesis of Compound LH-2-11

[0393]

[0394] Synthesis of intermediate compound LH-2-9 (5-(4-cyanophenyl)-N-methyl-N-pentylthiophene-2-carboxamide): Referring to the synthesis procedure for compound LH-61 in Step 1 of Example 13, using compound LH-185 as the starting material, a yellow solid compound LH-2-9 (145 mg, 57%) was obtained. Intermediate compound LH-2-9 was used directly in the next reaction without purification.

[0395] Synthesis of compound LH-2-11 (5-(4-(1H-tetrazol-5-yl)phenyl)-N-methyl-N-pentylthiophene-2-carboxamide): Refer to the synthesis steps of compound LH-189 in step 1 of Example 22 for details. Using LH-2-9 as raw material, a white solid compound LH-2-11 (56 mg, 34%) was obtained.

[0396] Compound LH-2-11. 1 H NMR (400MHz, DMSO) δ8.08(d,J=8.2Hz,2H),7.82(d,J=8.1Hz,2H),7.58(d,J=3.7Hz,1H) ,7.48(s,1H),3.14(s,3H),1.68–1.48(m,2H),1.46–0.96(m,6H),0.87(t,J=6.9Hz,3H). 13 C NMR (101MHz, DMSO) δ172.46,158.38,146.25,133.50,129.37,127.44,126.47,124.38,28.82,22.33,21.55,14.38.

[0397] Example 24: Synthesis of Compound LH-2-42

[0398]

[0399] Synthesis of intermediate compound LH-2-25 (2-((5-bromo-2-iodopyridin-3-yl)oxy)acetonitrile): To acetone (20 mL) was added KCO (276 mg, 2.0 mmol), 5-bromo-2-iodopyridin-3-ol (600 mg, 2.0 mmol), and 2-bromoacetonitrile (240 mg, 20.0 mmol), and the mixture was stirred at room temperature for 12 h. The mixture was then diluted with water and extracted with ethyl acetate. The mixture was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 3 / 1) to obtain compound LH-2-25 (640 mg, 94%) as a white solid. Compound LH-2-25.1 H NMR (400MHz, CDCl3) δ8.26 (s, 1H), 7.31 (s, 1H), 4.87 (s, 2H).

[0400] Synthesis of intermediate compound LH-2-30 (5-(5-bromo-3-(cyanomethoxy)pyridin-2-yl)thiophene-2-carboxylic acid): Pd(PPh3)4 (139 mg, 0.12 mmol) was added to a mixture of compound LH-2-25 (600 mg, 1.8 mmol), 5-boronic acid-thiophene-2-carboxylic acid (276 mg, 1.6 mmol), sodium carbonate (678 mg, 6.4 mmol), MeCN (20 mL), and H2O (10 mL). The resulting mixture was stirred at 90°C for 12 h under argon protection. After completion of the reaction, the mixture was extracted with water and EtOAc, and the product was dissolved in the aqueous phase. The aqueous phase was then adjusted to pH 2-3 with 2M HCl aqueous solution, extracted with EtOAc, and the product was dissolved in the organic layer. The product was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a yellow solid (320 mg, 59%). The intermediate compound LH-2-30 was directly used in the next reaction without purification.

[0401] Synthesis of intermediate compound LH-2-32 (methyl 5-(5-bromo-3-(cyanomethoxy)pyridin-2-yl)thiophene-2-carboxylate): Compound LH-2-30 (300 mg, 0.88 mmol) was dissolved in THF (10 mL), and potassium carbonate (533 mg, 4.0 mmol) and dimethyl sulfate (300 mg, 2.4 mmol) were added. The mixture was stirred at room temperature for 24 hours. After completion of the reaction, water was added portionwise to the reaction mixture, and the mixture was extracted with EtOAc. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE / EtOAc = 1 / 1) to obtain compound LH-2-32 (220 mg, 71%) as a yellow solid. 1 H NMR (400MHz, DMSO) δ8.36(d,J=1.7Hz,1H),8.08(d,J=4.1Hz,1H),7.82(d,J=4.1Hz,1H),7.76(d,J=1.7Hz,1H),4.84(s,2H),3.84(s,3H).

[0402] Synthesis of intermediate compound LH-2-37 (methyl 5-(5-(3-(tert-butyl)phenyl)-3-(cyanomethoxy)pyridin-2-yl)thiophene-2-carboxylate): To a mixture of compound LH-2-32 (130 mg, 0.36 mmol), 560132-24-3 (142 mg, 0.8 mmol), potassium carbonate (354 mg, 2.6 mmol), PhMe (5.0 mL), dioxane (5.0 mL) and H2O (2.0 mL) was added Pd(PPh3)4 (70 mg, 0.06 mmol). Under argon protection, the resulting mixture was stirred at 100°C for 12 h. The mixture was extracted with water and EtOAc, and the product was dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give compound LH-2-37 (98 mg, 69%) as a yellow solid. The intermediate compound LH-2-37 was used directly in the next reaction without purification.

[0403] Synthesis of intermediate compound LH-2-40 (5-(5-(3-(tert-butyl)phenyl)-3-(carboxymethyloxy)pyridin-2-yl)thiophene-2-carboxylic acid): Compound LH-2-37 (90 mg, 0.25 mmol) and LiOH (50 mg, 2 mmol) were dissolved in 1,4-dioxane (20 mL) and H2O (10 mL) and stirred at 100°C for 2 h. The cyano group was hydrolyzed to a carboxyl group under alkaline conditions. After completion of the reaction, the mixture was adjusted to pH 2-3 with 2M HCl solution. Water was added portionwise to the reaction mixture, and extraction was performed with EtOAc. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound LH-2-40 (53 mg, 52%) as a yellow solid. Intermediate compound LH-2-40 was used directly in the next reaction without purification.

[0404] Synthesis of compound LH-2-42 (5-(5-(3-(tert-butyl)phenyl)-3-(2-oxo-2-(pentylamino)ethoxy)pyridin-2-yl)-N-pentylthiophene-2-carboxamide): HATU (171 mg, 0.45 mmol), compound LH-2-40 (40 mg, 0.1 mmol), n-pentylamine (26 mg, 0.3 mmol), and DIPEA (150 mg, 1.5 mol) were added to DMF (2.0 mL), and the mixture was stirred at room temperature for 12 h. After completion of the reaction, the mixture was extracted with water and ethyl acetate, and the organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. Purification by silica gel column chromatography (PE / EA = 1 / 1) gave compound LH-2-42 (36 mg, 66%) as a yellow oil.

[0405] Compound LH-2-42. 1H NMR (400MHz, DMSO) δ8.56(s,2H),8.23(s,1H),8.06(s,1H),7.74(d,J=13.4Hz,2H),7.59(d,J=25.6Hz,2H),7.46(d,J=10 .5Hz,2H),4.91(s,2H),3.26–3.20(m,2H),3.18–3.11(m,2H),1.40–1.32(m,13H),1.25–1.22(m,8H),0.91–0.84(m,6H). 13 C NMR (101MHz, DMSO) δ167.35,161.57,152.10,151.49,145.65,140.99,140.09,139.67,136.66,129.33,128.88,128.78,125 .84,124.67,124.28,119.04,67.84,38.81,35.10,31.59,29.70,29.50,29.37,29.18,28.99,22.38,22.26,14.43,14.31.

[0406] Example 25: Synthesis of Compound LH-2-45

[0407]

[0408] Synthesis of intermediate compound LH-2-36 (methyl 5-(5-(4-(tert-butyl)phenyl)-3-(cyanomethoxy)pyridin-2-yl)thiophene-2-carboxylate): To a mixture of compound LH-2-32 (130 mg, 0.36 mmol), 4-tert-butylphenylboronic acid (123324-71-0, 142 mg, 0.8 mmol), potassium carbonate (354 mg, 2.6 mmol), PhMe (5 mL), 1,4-dioxane (5 mL), and H2O (2 mL) was added Pd(PPh3)4 (70 mg, 0.06 mmol). The mixture was stirred at 100°C for 12 h under argon protection. The mixture was extracted with water and EtOAc, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA=1 / 1) to obtain yellow solid compound LH-2-36 (103 mg, 73%). 1H NMR (400MHz, DMSO) δ8.57 (s, 1H), 8.11 (d, J = 3.5Hz, 1H), 7.92 (s, 1H), 7.83 ( d,J=3.5Hz,1H),7.73–7.67(m,4H),4.89(s,2H),3.85(s,3H),1.33(s,9H).

[0409] Synthesis of intermediate compound LH-2-44 (5-(5-(4-(tert-butyl)phenyl)-3-(carboxymethyloxy)pyridin-2-yl)thiophene-2-carboxylic acid): Compound LH-2-37 (90 mg, 0.25 mmol) and LiOH (50 mg, 2 mmol) were dissolved in 1,4-dioxane (20 mL) and H2O (10 mL) and stirred at 100°C for 2 h. The cyano group was hydrolyzed to a carboxyl group under alkaline conditions. After completion of the reaction, the mixture was adjusted to pH 2-3 with 2M HCl solution. Water was added to the reaction mixture in portions and extracted with EtOAc. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound LH-2-44 (60 mg, 59%) as a yellow solid. Intermediate compound LH-2-44 was used directly in the next reaction without purification.

[0410] Synthesis of compound LH-2-45 (5-(5-(4-(tert-butyl)phenyl)-3-(2-oxo-2-(pentylamino)ethoxy)pyridin-2-yl)-N-pentylthiophene-2-carboxamide): HATU (171 mg, 0.45 mmol), compound LH-2-40 (40 mg, 0.1 mmol), n-pentylamine (26 mg, 0.3 mmol), and DIPEA (150 mg, 1.5 mol) were added to DMF (2.0 mL), and the mixture was stirred at room temperature for 12 h. The mixture was then extracted with water and ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 1 / 1) to obtain compound LH-2-45 (43 mg, 78%) as a yellow oil.

[0411] Compound LH-2-45. 1H NMR(400MHz,DMSO)δ8.54(d,J=1.7Hz,1H),8.53–8.46(m,1H),8.21–8.15(m ,1H),8.04(d,J=4.0Hz,1H),7.74(d,J=4.1Hz,1H),7.70(d,J=8.4Hz,3H),7. 64(d,J=1.6Hz,1H),7.54(d,J=8.5Hz,2H),4.88(s,2H),3.26–3.22(m,2H),3 .16–3.12(m,2H),1.35–1.31(m,13H),1.25–1.23(m,8H),0.88–0.86(m,6H). 13 C NMR (101MHz, DMSO) δ167.31,161.60,151.56,145.63,140.93,139.78,139.57,136.03,134.00,128.86,128.67 ,127.04,126.38,34.87,34.84,31.48,30.19,29.35,29.22,29.17,29.08,29.00,22.36,22.28,14.40,14.28..

[0412] Example 26: Synthesis of Compound LH-2-58

[0413]

[0414] Synthesis of intermediate compound LH-2-20 (2-((5-bromo-2-iodopyridin-3-yl)oxy)acetonitrile): K2CO3 (276 mg, 2.0 mmol), 6-bromo-3-hydroxy-2-iodopyridine (CAS: 188057-35-4; brand: Bidler; 600 mg, 2.0 mmol), and 2-bromoacetonitrile (240 mg, 20.0 mmol) were added to acetone (20 mL) and stirred at room temperature for 12 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 1 / 1) to obtain compound LH-2-20 (550 mg, 71%) as a white solid. Compound LH-2-20. 1 H NMR (400MHz, CDCl3) δ7.45 (d, J = 8.4Hz, 1H), 7.10 (d, J = 8.5Hz, 1H), 4.85 (s, 2H).

[0415] Synthesis of intermediate compound LH-2-24 (5-(5-bromo-3-methoxypyridine-2-)thiophene-2-carboxylic acid): Pd(PPh3)4 (70 mg, 0.06 mmol) was added to a mixture of compound LH-2-20 (370 mg, 1.1 mmol), 5-boronic acid-thiophene-2-carboxylic acid (172 mg, 1.0 mmol), sodium carbonate (424 mg, 4.0 mmol), MeCN (20 mL) and H2O (10 mL). Under argon protection, the resulting mixture was stirred at 80°C for 24 h. The mixture was then extracted with water and EtOAc, and the product was dissolved in the aqueous phase. The aqueous phase was adjusted to pH = 2-3 with 2M HCl aqueous solution and extracted with EtOAc. The product was dissolved in the organic layer. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give compound LH-2-24 (220 mg, 58%) as a yellow solid. The intermediate compound LH-2-24 was used directly in the next reaction without purification.

[0416] Synthesis of intermediate compound LH-2-28 (methyl 5-(6-bromo-3-(2-methoxy-2-oxyethoxy)pyridin-2-yl)thiophene-2-carboxylate): Compound LH-2-24 (220 mg, 0.65 mmol) and concentrated sulfuric acid (5.0 mL) were dissolved in MeOH (40 mL) and stirred at 65°C for 12 h. The cyano group was hydrolyzed under acidic conditions. The mixture was extracted with water and EtOAc, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE / EA = 2 / 1) to obtain compound LH-2-28 (188 mg, 75%) as a white solid. 1 H NMR (400MHz, CDCl3) δ8.13(d,J=4.1Hz,1H),7.82(d,J=4.1Hz,1H),7.35(d,J =8.6Hz,1H),7.08(d,J=8.6Hz,1H),4.82(s,2H),3.93(s,3H),3.87(s,3H).

[0417] Synthesis of intermediate compound LH-2-53 (methyl 5-(3-(2-methoxy-2-oxyethoxy)-6-phenylpyridin-2-yl)thiophene-2-carboxylate): To a mixture of compound LH-2-28 (200 mg, 0.5 mmol), phenylboronic acid (142 mg, 0.8 mmol), potassium carbonate (354 mg, 2.6 mmol), PhMe (5.0 mL), 1,4-dioxane (5.0 mL) and H2O (2.0 mL) was added Pd(PPh3)4 (70 mg, 0.06 mmol). Under argon protection, the mixture was stirred at 100°C for 12 h. The mixture was extracted with water and EtOAc, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain yellow solid compound LH-2-53 (155 mg, 81%). Intermediate compound LH-2-53 was used directly in the next reaction without purification.

[0418] Synthesis of intermediate compound LH-2-57 (5-(3-(carboxymethyloxy)-6-phenylpyridin-2-yl)thiophene-2-carboxylic acid): Compound LH-2-53 (150 mg, 0.38 mmol) and LiOH (50 mg, 2 mmol) were dissolved in 1,4-dioxane (20 mL) and H2O (10 mL), and the mixture was stirred at 100°C for 2 h. The cyano group was hydrolyzed to a carboxyl group under alkaline conditions. The mixture was adjusted to pH 2-3 with 2M HCl solution, extracted with water and EtOAc, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound LH-2-57 (60 mg, 59%) as a yellow solid. Intermediate compound LH-2-57 was used directly in the next reaction without purification.

[0419] Synthesis of compound LH-2-58 (N-(tert-butyl)-5-(3-(2-(tert-butylamino)-2-oxoethoxy)-6-phenylpyridin-2-yl)thiophene-2-carboxamide): HATU (171 mg, 0.45 mmol), compound LH-2-57 (100 mg, 0.25 mmol), tert-butylamine (73 mg, 1.0 mmol), and DIPEA (150 mg, 1.5 mol) were added to DMF (2.0 mL), and the mixture was stirred at room temperature for 12 h. The mixture was then extracted with water and ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 1 / 1) to obtain compound LH-2-58 (49 mg, 43%) as a yellow oil.

[0420] Compound LH-2-58. 1H NMR (400MHz, DMSO) δ8.12(d,J=7.6Hz,2H),8.07(d,J=3.8Hz,1H),7.94(d,J=8.7Hz,1H),7.87–7.79 (m,2H),7.75(s,1H),7.57–7.47(m,3H),7.46–7.38(m,1H),4.72(s,2H),1.40(s,9H),1.33(s,9H). 13 C NMR (101MHz, DMSO) δ166.53,161.60,150.81,148.04,145.80,142.40,140.06,138.16,129 .25,129.08,129.03,128.71,126.45,122.16,120.47,68.14,51.59,50.97,29.11,28.91.

[0421] Example 27: Synthesis of Compound LH-2-39

[0422]

[0423] Synthesis of the intermediate compound LH-2-21 (dimethyl 2-(5,5-dimethyl-1,3,2-dioxanaphthene-2-yl) terephthalate): dimethyl o-bromobenzoate (CAS No.: 18643-86-2; brand: Bidex; 1365 mg, 5.0 mmol), neopentyl glycol diborate (CAS No.: 201733-56-4; brand: Bidex; (1694 mg, 7.5 mmol), KOAc (1470 mg, 15.0 mm To a mixture of 1% daptomycin (1% daptomycin) and DMSO (40 mL) was added PdCl(dppf) (70 mg, 0.06 mmol). The resulting mixture was stirred at 80°C for 5 h under argon protection. The mixture was then extracted with water and EtOAc, and the product was dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 5 / 1) to obtain compound LH-2-21 (1.2 g, 80%) as a clear oil.

[0424] Compound LH-2-21. 1 H NMR (400MHz, CDCl3) δ8.18(d,J=1.4Hz,1H),8.08–8.01(m,1H),7.98–7.93(m,1H),3.93(d,J=5.7Hz,6H),3.80(s,4H),1.12(s,6H).

[0425] Synthesis of compound LH-2-39 (dimethyl 2-(5-(methoxycarbonyl)thiophen-2-yl)terephthalate): Pd(PPh3)4 (100 mg, 0.09 mmol) was added to a mixture of compound LH-2-39 (900 mg, 3.0 mmol), methyl 5-bromothiophene-2-carboxylate (CAS No. 62224-19-5; brand: Bidler; 800 mg, 4.0 mmol), K3PO4 (1.0 mg, 9.0 mmol), and 1,4-dioxane (20 mL). The resulting mixture was stirred at 90°C for 12 h under argon. The mixture was then extracted with water and EtOAc, and the product was dissolved in the organic phase. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (PE / EA=5 / 1) to obtain compound LH-2-39 (616 mg, 66%) as a transparent oil.

[0426] Compound LH-2-39. 1 H NMR (400MHz, CDCl3) δ8.14(s,1H),8.10(d,J=8.1Hz,1H),7.83(d,J=8.1Hz,1H),7 .75(d,J=3.8Hz,1H),7.04(d,J=3.8Hz,1H),3.93(d,J=19.6Hz,6H),3.77(s,3H).

[0427] Example 28: Synthesis of Compound LH-3-26

[0428]

[0429] Synthesis of intermediate compound LH-3-17 (2-iodo-5-isopropylphenol): To AcOH (40 mL) were added KIO (856 mg, 4.0 mmol), iodine (2030 mg, 8.0 mmol), 3-isopropylphenol (2720 mg, 20.0 mmol), and H O (10 mL), and the mixture was stirred at room temperature for 48 h. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated sodium thiosulfate solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-17 (4.2 g, 80%) as a yellow solid. Compound LH-3-17. 1 H NMR (400MHz, CDCl3) δ7.54(d,J=8.1Hz,1H),6.89(s,1H),6.58(d,J=8.2Hz,1H),5.31(s,1H),2.84(dt,J=13.7,6.8Hz,1H),1.22(d,J=6.9Hz,6H).

[0430] Synthesis of intermediate compound LH-3-21 (1-iodo-4-isopropyl-2-methoxybenzene): KCO (950 mg, 6.9 mmol), compound LH-3-17 (524 mg, 20.0 mmol), and MeI (0.12 mL, 2.0 mmol) were added to EtOH (40 mL), and the mixture was stirred at reflux for 12 h. The mixture was then diluted with aqueous NaOH, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-21 (497 mg, 90%) as a yellow oil. Compound LH-3-21. 1 H NMR (400MHz, CDCl3) δ7.66(d,J=8.0Hz,1H),6.70(s,1H),6.61(d,J=8.0Hz,1H),3.88(s,3H),2.94–2.78(m,1H),1.25(d,J=6.9Hz,6H).

[0431] Synthesis of intermediate compound LH-3-24 (5-(4-isopropyl-2-methoxyphenyl)thiophene-2-carboxylic acid): Refer to the synthesis method for compound LH-2-157 in Step 1 of Example 14. Using compound LH-3-21 as the starting material, yellow solid compound LH-3-24 (231 mg, 58%) was obtained. Compound LH-3-24. 1 H NMR (400MHz, MeOD) δ7.70(d,J=3.6Hz,1H),7.67(d,J=8.0Hz,1H),7.51(d,J=3.5Hz,1H),6.98(s, 1H),6.92(d,J=8.0Hz,1H),3.97(s,3H),3.31(s,3H),3.00–2.82(m,1H),1.29(d,J=6.8Hz,6H).

[0432] Synthesis of intermediate compound LH-3-25 (tert-butyl 4-(5-(4-isopropyl-2-methoxyphenyl)thiophene-2-carbonyl)piperazine-1-carboxylate): A yellow solid compound LH-3-25 (107 mg, 83%) was obtained by similar synthetic procedures to compound LH-61. Compound LH-3-25. 1H NMR (400MHz, CDCl3) δ7.57(d,J=8.0Hz,1H),7.38(d,J=3.7Hz,1H),7.26(s,1H),6.88(d,J=8.0Hz,1H),6.84(s, 1H),3.94(s,3H),3.82–3.68(m,4H),3.56–3.45(m,4H),2.99–2.84(m,1H),1.48(s,9H),1.28(d,J=6.8Hz,6H).

[0433] Synthesis of compound LH-3-26 ((5-(4-isopropyl-2-methoxyphenyl)thiophen-2-yl)(piperazin-1-yl)methanone): Compound LH-3-25 (89 mg, 0.2 mmol) was dissolved in 4 M HCl / dioxane solution (10.0 mL), and the mixture was stirred at room temperature for 12 h. The mixture was then diluted with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (DCM / MeOH = 10 / 1) to obtain compound LH-3-26 (41 mg, 60%) as a white solid.

[0434] Compound LH-3-26. 1 H NMR (400MHz, MeOD) δ7.65(d,J=8.0Hz,1H),7.49(d,J=3.1Hz,1H),7.40(d,J=2.8Hz,1H),6.98(s,1H ),6.91(d,J=8.0Hz,1H),3.95(s,7H),3.25–3.11(m,4H),3.00–2.86(m,1H),1.28(d,J=6.7Hz,7H).

[0435] Example 29: Synthesis of Compound LH-3-30

[0436]

[0437] Synthesis of intermediate compound LH-3-20 (5-(tert-butyl)-2-iodophenol): To AcOH (40 mL) were added KIO (856 mg, 4.0 mmol), iodine (2030 mg, 8.0 mmol), 3-tert-butylphenol (3000 mg, 20.0 mmol), and H O (10 mL), and the mixture was stirred at room temperature for 48 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated sodium thiosulfate solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-20 (4.3 g, 78%) as a yellow solid. Compound LH-3-20.1H NMR (400 MHz, CDCl3) δ7.55 (d, J=8.4 Hz, 1H), 7.05 (s, 1H), 6.73 (d, J=8.4 Hz, 1H), 5.31 (s, 1H), 1.29 (s, 9H).

[0438] Synthesis of intermediate compound LH-3-22 (4-(tert-butyl)-1-iodo-2-methoxybenzene): KCO (950 mg, 69 mmol), compound LH-3-20 (552 mg, 2.0 mmol), and MeI (0.12 mL, 2.0 mmol) were added to EtOH (40 mL), and the mixture was stirred at reflux for 12 h. The mixture was then diluted with aqueous NaOH, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-22 (477 mg, 89%) as a yellow oil. Compound LH-3-22. 1 H NMR (400MHz, CDCl3) δ7.67 (d, J = 8.2 Hz, 1H), 6.86 (s, 1H), 6.76 (d, J = 8.2 Hz, 1H), 3.89 (s, 3H), 1.32 (s, 9H).

[0439] Synthesis of intermediate compound LH-3-27 (5-(4-(tert-butyl)-2-methoxyphenyl)thiophene-2-carboxylic acid): Refer to the synthesis method for compound LH-2-157 in Step 1 of Example 14. Using compound LH-3-22 as the starting material, yellow solid compound LH-3-27 (267 mg, 63%) was obtained. Compound LH-3-27. 1 H NMR (400MHz, MeOD) δ7.71–7.62(m,2H),7.49(d,J=3.1Hz,1H),7.11–6.99(m,2H),3.96(s,3H),1.34(s,9H).

[0440] Synthesis of intermediate compound LH-3-29 (tert-butyl 4-(5-(4-(tert-butyl)-2-methoxyphenyl)thiophene-2-carbonyl)piperazine-1-carboxylate): Referring to the synthesis of compound LH-61 in Step 1 of Example 13, a yellow solid (96 mg, 89%) was obtained using compound LH-3-27 as the starting material via an amide condensation reaction. Intermediate compound LH-3-29 was used directly in the next reaction without purification.

[0441] Synthesis of compound LH-3-30 ((5-(4-(tert-butyl)-2-methoxyphenyl)thiophene-2-)(piperazine-1-)methanone): Compound LH-3-29 (90 mg, 0.2 mmol) was dissolved in 4M HCl / dioxane solution (10.0 mL) and stirred at room temperature for 12 h. The mixture was then extracted with water and ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. Purification by silica gel column chromatography (DCM / MeOH = 10 / 1) afforded compound LH-3-30 (47 mg, 67%) as a white solid.

[0442] Compound LH-3-30. 1 H NMR(400MHz,MeOD)δ7.67(d,J=8.1Hz,1H),7.51(d,J=3.1Hz,1H),7.41(d,J=3.2Hz,1H), 7.12(s,1H),7.08(d,J=8.2Hz,1H),4.04–3.87(m,7H),3.26–3.13(m,4H),1.36(s,10H). 13 C NMR(101MHz,MeOD)δ166.18(s),130.83(s),128.58(s),125.15(s),120.4 9(s),119.26(s),110.21(s),56.00(s),45.12(s),35.91(s),31.60(s)..

[0443] Example 30: Synthesis of compounds LH-2-189, LH-2-197 and LH-2-198

[0444] 1. Synthesis of compound LH-2-189

[0445]

[0446] Synthesis of Compound: 5-Methyl-N-(thiophen-2-ylmethyl)-1H-indole-2-carboxamide (LH-2-189): According to the reagents and conditions (a), 10241-97-1 (CAS No.: 10241-97-1; Brand: Bidler; 60 mg, 0.34 mmol), HATU (230 mg, 0.6 mmol), 2-thiophenemethylamine (40 mg, 0.34 mmol), and DIPEA (58 mg, 0.45 mol) were added to DMF (3.0 mL) and stirred at room temperature for 12 h. The mixture was extracted with water and ethyl acetate, concentrated, and purified by silica gel column chromatography (PE / EA = 2 / 1) to afford LH-2-189 (63 mg, 69%) as a white solid.

[0447] Compound LH-2-189. 1 H NMR (400MHz, DMSO) δ11.45(s,1H),9.15–8.90(m,1H),7.40–7.31(m,2H),7. 28(d,J=8.4Hz,1H),7.04–6.89(m,4H),4.61(d,J=6.0Hz,2H),2.32(s,3H). 13 C NMR (101MHz, DMSO) δ161.45,143.13,135.36,131.80,128.75,127.76,127.11,125.91,125.70,125.52,121.25,112.48,102.71,37.77,21.60.

[0448] 2. Synthesis of compound LH-2-197

[0449]

[0450] Synthesis of compound LH-2-197 (N-((2-chloropyridin-4-yl)methyl)-5-methyl-1H-indole-2-carboxamide): Compound 10241-97-1 (CAS No.: 10241-97-1; Brand: Bidex; 60 mg, 0.34 mmol), HATU (230 mg, 0.6 mmol), 2-chloropyridine-4-methylamine (71 mg, 0.5 mmol), and DIPEA (58 mg, 0.45 mol) were added to DMF (3.0 mL) and stirred at room temperature for 12 h. The mixture was then extracted with water and ethyl acetate and concentrated. Purification by silica gel column chromatography (PE / EA = 2 / 1) afforded compound LH-2-197 (75 mg, 73%) as a white solid.

[0451] Compound LH-2-197. 1H NMR (400MHz, DMSO) δ11.51(s,1H),9.20–9.04(m,1H),8.36(d,J=5.1Hz,1H),7.41(d,J=9.1Hz,2H), 7.38–7.29(m,2H),7.11(d,J=1.5Hz,1H),7.02(d,J=8.2Hz,1H),4.54(d,J=6.0Hz,2H),2.36(s,3H). 13 C NMR(101MHz,DMSO)δ162.02,153.47,150.87,150.33,135.48,131.49,128.8 7,127.76,125.88,122.76,122.09,121.29,112.54,102.98,41.49,21.61.

[0452] 3. Synthesis of compound LH-2-198

[0453]

[0454] Synthesis of compound LH-2-198 (5-methoxy-N-(thiophen-2-ylmethyl)-1H-indole-2-carboxamide): Compound 4382-54-1 (CAS No. 4382-54-1; Brand: Bidex; 70 mg, 0.37 mmol), HATU (230 mg, 0.6 mmol), 2-thiophenemethylamine (80 mg, 0.7 mmol), and DIPEA (58 mg, 0.45 mol) were added to DMF (3.0 mL) and stirred at room temperature for 12 h. The mixture was then extracted with water and ethyl acetate, concentrated, and purified by silica gel column chromatography (PE / EA = 2 / 1) to afford compound LH-2-198 (53 mg, 76%) as a white solid.

[0455] Compound LH-2-198. 1 H NMR (400MHz, DMSO) δ11.46(s,1H),9.07(t,J=5.8Hz,1H),7.45–7.37(m,1H),7.31(d,J=8.9Hz,1 H),7.10–7.02(m,3H),7.00–6.95(m,1H),6.87–6.80(m,1H),4.65(d,J=5.9Hz,2H),3.75(s,3H). 13C NMR (101MHz, DMSO) δ161.36,154.20,143.16,132.22,132.13,127.82,127.12,125.89,125.52,115.04,113.58,102.93,102.46,55.71,37.78.

[0456] Example 31: Synthesis of Compounds LH-2-48 and LH-3-5

[0457] 1. Synthesis of compound LH-2-48

[0458]

[0459] Synthesis of intermediate compound LH-2-200 (ethyl 5-phenyl-1H-indole-2-carboxylate): Pd(PPh3)4 (210 mg, 0.18 mmol) was added to a mixture of compound 16732-70-0 (brand: Bidler; 1410 mg, 5.3 mmol), phenylboronic acid (768 mg, 6.3 mmol), potassium carbonate (1600 mg, 11.6 mmol), H2O (20 mL), and 1,4-dioxane (20 mL). The mixture was stirred at 110°C for 3 h under argon. After completion of the reaction, the mixture was extracted with water and EtOAc, and the product was dissolved in the organic phase, which was then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 5 / 1) to yield compound LH-2-200 (117 mg, 72%) as a yellow solid.

[0460] Compound LH-2-200. 1 H NMR (400MHz, CDCl3) δ9.03(s,1H),7.90(s,1H),7.65(d,J=7.6Hz,2H),7.60(d,J=8.6Hz,1H),7 .53–7.43(m,3H),7.38–7.31(m,1H),7.29(s,1H),4.45(q,J=6.9Hz,2H),1.44(t,J=6.9Hz,3H).

[0461] Synthesis of intermediate compound LH-3-1 (5-phenyl-1H-indole-2-carboxylic acid): Compound LH-2-200 (130 mg, 0.5 mmol) and NaOH (80 mg, 2 mmol) were dissolved in a mixture of MeOH (10 mL), THF (10 mL), and H2O (3.0 mL) and stirred at room temperature for 3 h. After completion of the reaction, the mixture was adjusted to pH 2-3 with 2M HCl solution, then diluted with water in portions and extracted with EtOAc. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow solid (103 mg, 88%). Intermediate compound LH-3-1 was used directly in the next reaction without purification.

[0462] Synthesis of compound LH-2-48 (5-phenyl-N-(thiophen-2-ylmethyl)-1H-indole-2-carboxamide): HATU (171 mg, 0.45 mmol), compound LH-3-1 (50 mg, 0.21 mmol), 2-chloropyridine-4-methylamine (71 mg, 0.5 mmol), and DIPEA (58 mg, 0.45 mol) were added to DMF (3.0 mL), and the mixture was stirred at room temperature for 12 h. The mixture was then extracted with water and ethyl acetate and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 3 / 1) to give compound LH-2-48 (56 mg, 70%) as a white solid.

[0463] Compound LH-2-48. 1 H NMR (400MHz, DMSO) δ11.73(s,1H),9.21(t,J=6.0Hz,1H),8.37(d,J=5.1Hz,1H),7.92(s,1H),7.68(d,J=7.2Hz,2H),7 .52(s,2H),7.49–7.41(m,3H),7.38(d,J=5.1Hz,1H),7.34–7.29(m,1H),7.28(d,J=1.9Hz,1H),4.57(d,J=5.9Hz,2H). 13 C NMR(101MHz,DMSO)δ161.86,153.42,150.88,150.37,141.82,136.63,132.80,132.26,12 9.27,128.14,127.18,126.91,123.57,122.78,122.10,120.00,113.26,103.94,41.51..

[0464] 2. The synthesis method of compound LH-3-5 refers to the synthesis of compound LH-2-48 in step 1 of this example.

[0465] Among them, the structural formula of compound LH-3-5 is The NMR characterization results are: 1 HNMR(400MHz,DMSO)δ11.72(s,1H),9.20(s,1H),7.89(s,1H),7.71–7.61(m,2H),7.51(s,2 H),7.47–7.38(m,3H),7.34–7.26(m,1H),7.22(s,1H),7.02(d,J=28.7Hz,2H),4.67(s,2H). 13 C NMR (101MHz, DMSO) δ161.30,143.07,141.85,136.53,132.71,132.58,129.25,128.1 4,127.17,127.13,126.88,125.95,125.56,123.41,119.95,113.21,103.67,37.82.

[0466] Example 32: Synthesis of Compound LH-194

[0467]

[0468] Synthesis of compound LH-194 (2-(5-(4-(pyridine-2-)piperazine-1-carbonyl)thiophene-2-)isonicotinonitrile): To DMF (3.0 mL) were added compound LH-26 (93 mg, 0.4 mmol), HATU (230 mg, 0.6 mmol), 1-(2-pyridyl)piperazine (80 mg, 0.5 mmol), and DIPEA (106 mg, 0.9 mol), and stirred at room temperature for 12 h. The mixture was extracted with water and ethyl acetate, concentrated, and purified by column chromatography to afford compound LH-194 (95 mg, 63%) as a yellow solid.

[0469] Compound LH-194. 1 H NMR (400MHz, DMSO) δ8.81(d,J=5.0Hz,1H),8.58(s,1H),8.15(d,J=3.5Hz,1H),7.99(d,J=3.9Hz,1H),7.80(d,J =5.0Hz,1H),7.63–7.50(m,2H),6.86(d,J=8.6Hz,1H),6.73–6.62(m,1H),3.78(s,4H),3.63(d,J=5.2Hz,4H).

[0470] Example 33: Synthesis of Compound LH-3-7

[0471]

[0472] Synthesis of compound LH-3-7 (5-(3-ethoxy-5-phenylpyridin-2-yl)-N-pentylthiophene-2-carboxamide): To DMF (3.0 mL) were added compound LH-3-6 (50 mg, 0.15 mmol), HATU (230 mg, 0.6 mmol), n-pentylamine (43 mg, 0.5 mmol), and DIPEA (106 mg, 0.9 mol), and the mixture was stirred at room temperature for 12 h. The mixture was extracted with water and ethyl acetate, concentrated, and purified by column chromatography to afford compound LH-3-7 (38 mg, 63%) as a yellow solid.

[0473] Compound LH-3-7. 1 H NMR (400MHz, DMSO) δ8.56–8.44(m,2H),7.93(d,J=3.9Hz,1H),7.86–7.76(m,3H),7.73(d,J=3.9Hz,1H),7.58–7.48(m,2H) ,7.47–7.39(m,1H),4.38(q,J=6.8Hz,2H),3.29–3.17(m,2H),1.57–1.44(m,5H),1.34–1.26(m,4H),0.88(t,J=6.6Hz,3H). 13 C NMR (101MHz, DMSO) δ161.65,151.96,145.43,140.96,139.49,139.37,136.97,136.28,129 .54,128.83,128.73,128.04,127.46,118.54,64.92,29.36,29.18,22.36,15.00,14.40.

[0474] Example 34: Synthesis of Compounds LH-3-48 and LH-3-49

[0475] 1. Synthesis of compound LH-3-48

[0476]

[0477] Synthesis of intermediate compound LH-3-32 (4-bromo-2-methoxy-5-nitroaniline): 4-bromo-2-methoxyaniline (1145 mg, 5.67 mmol) was dissolved in concentrated sulfuric acid (5.0 mL) in an ice bath. After the solid dissolved, KNO3 (573 mg, 5.67 mmol) was added to the solution, and the mixture was stirred in an ice bath for 24 hours. The mixture was then diluted with water, and the pH of the solution was adjusted to pH = 7 with NaOH. The mixture was then extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-32 (938 mg, 67%) as a yellow solid. Compound LH-3-32. 1 H NMR (400MHz, DMSO) δ7.26 (s, 1H), 7.09 (s, 1H), 5.51 (s, 2H), 3.84 (s, 3H).

[0478] Synthesis of intermediate compound LH-3-33 (N,N'-tert-butoxycarbonyl-4-bromo-2-methoxy-5-nitroaniline): 4-Bromo-2-methoxy-5-nitroaniline (compound LH-3-32) (500 mg, 2.0 mmol) was dissolved in THF (10.0 mL). After the solid dissolved, (Boc)2O (880 mg, 4.0 mmol), TEA (1.0 mL), and DMAP (12 mg, 0.1 mmol) were added to the solution, and the mixture was stirred at 60°C for 24 h. The mixture was then diluted with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-33 (733 mg, 82%) as a yellow solid. Compound LH-3-33. 1 H NMR (400MHz, CDCl3) δ7.85(s,1H),7.20(s,1H),3.92(s,3H),1.40(s,18H).

[0479] Synthesis of intermediate compound LH-3-34 (N,N'-tert-butoxycarbonyl-4-phenyl-2-methoxy-5-nitroaniline): Compound LH-3-33 (670 mg, 1.5 mmol), phenylboronic acid (274 mg, 2.25 mmol), XPhos Pd G3 (26 mg, 0.03 mmol), and potassium phosphate (956 mg, 4.5 mmol) were added to THF (5.0 mL) and H2O (8.0 mL). Under argon protection, the mixture was stirred at 40°C for 12 h. The mixture was then diluted with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-34 (567 mg, 85%) as a light yellow solid. Compound LH-3-34. 1 H NMR (400MHz, CDCl3) δ7.90(s,1H),7.52–7.41(m,3H),7.38–7.31(m,2H),6.86(s,1H),3.94(s,3H),1.49(s,18H).

[0480] Synthesis of intermediate compound LH-3-36 (5-methoxy-2-nitro-[1,1'-biphenyl]-4-amine): Compound LH-3-34 (445 mg, 1.0 mmol) was added to 4M HCl / dioxane (10.0 mL) and stirred at room temperature for 12 h. The mixture was then diluted with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-36 (170 mg, 70%) as a yellow oil. Compound LH-3-36. 1 H NMR (400MHz, CDCl3) δ7.47–7.26(m,6H),6.71(s,1H),4.11(s,2H),3.94(s,3H)..

[0481] Synthesis of intermediate compound LH-3-37 (N,N'-tert-butoxycarbonyl-4-bromo-2-methoxy-5-nitroaniline): Compound LH-3-36 (80 mg, 0.33 mmol) and p-TsOH (172 mg, 1.0 mmol) were dissolved in MeCN (5.0 mL), and the mixture was stirred at 0°C for 10 min. NaNO2 (140 mg, 2.0 mmol), KI (332 mg, 2.0 mmol), and H2O (8.0 mL) were then added to the solution, and the mixture was stirred at 0°C for 12 h. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to obtain compound LH-3-37 (90 mg, 76%) as a yellow solid. Compound LH-3-37. 1 H NMR (400MHz, CDCl3) δ8.42(s,1H),7.49–7.39(m,3H),7.33–7.27(m,2H),6.72(s,1H),3.96(s,3H).

[0482] Synthesis of compound LH-3-38 (5-(5-methoxy-2-nitro-[1,1'-biphenyl]-4-yl)thiophene-2-carboxylic acid): Pd(PPh3)4 (35 mg, 0.03 mmol) was added to a mixture of compound LH-3-37 (178 mg, 0.5 mmol), 5-carboxythiophene-2-boronic acid (86 mg, 0.5 mmol), sodium carbonate (106 mg, 1.0 mmol), MeCN (3 mL) and H2O (3 mL). The mixture was stirred at 80°C for 4 h under argon protection. The mixture was extracted with water and EtOAc, and the product was dissolved in the aqueous phase. The pH of the aqueous phase was adjusted to 2-3 with 2M aqueous HCl, extracted with EtOAc, then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. Purification by silica gel column chromatography (DCM / MeOH=10 / 1) gave yellow solid compound LH-3-38 (107 mg, 61%).

[0483] Compound LH-3-38. 1 H NMR (400MHz, DMSO) δ13.16(s,1H),8.49(s,1H),7.86(d,J=4.0Hz,1H),7.75(d,J=4.0Hz,1H),7.57–7.36(m,5H),7.23(s,1H),4.11(s,3H). 13C NMR (101MHz, DMSO) δ163.49,157.84,142.52,142.14,137.69,137.44,135.5 7,133.11,129.13,128.86,128.30,127.75,124.56,121.51,115.57,57.41..

[0484] 2. Synthesis of compound LH-3-48

[0485]

[0486] Synthesis of compound LH-3-48 (tert-butyl 4-(5-(5-methoxy-2-nitro-[1,1'-biphenyl]-4-yl)thiophene-2-carbonyl)piperazine-1-carboxylate): HATU (171 mg, 0.45 mmol), compound LH-3-38 (100 mg, 0.28 mmol), 1-tert-butyloxycarbonylpiperazine (56 mg, 0.3 mmol), and DIPEA (58 mg, 0.45 mol) were added to DMF (1.0 mL), and the mixture was stirred at room temperature for 12 h. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 3 / 1) to obtain compound LH-3-48 (91 mg, 62%) as a white solid.

[0487] Compound LH-3-48. 1 H NMR(400MHz,DMSO)δ8.47(s,1H),7.81(d,J=3.6Hz,1H),7.53–7.39(m,6H),7 .23(s,1H),4.10(s,3H),3.73–3.62(m,4H),3.48–3.41(m,4H),1.43(s,9H). 13 C NMR (101MHz, DMSO) δ162.84,157.73,154.27,142.53,139.04,138.49,137.49,137.34,1 29.40,129.11,128.82,128.30,126.92,124.33,121.70,115.51,79.69,57.37,28.51..

[0488] 3. Synthesis of compound LH-3-49

[0489]

[0490] Synthesis of compound LH-3-49 ((5-(5-methoxy-2-nitro-[1,1'-biphenyl]-4-yl)thiophen-2-yl)(piperazin-1-yl)methanone): Compound LH-3-48 (80 mg, 0.15 mmol) was added to 4M HCl / dioxane (6.0 mL) and stirred at room temperature for 12 h. The mixture was then diluted with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE / EA = 10 / 1) to afford compound LH-3-49 (38 mg, 60%) as a yellow solid.

[0491] Compound LH-3-49. 1 H NMR(400MHz,DMSO)δ8.44(s,1H),7.77(d,J=3.9Hz,1H),7.50–7.45(m,3H),7.4 4–7.40(m,3H),7.20(s,1H),4.08(s,3H),3.66–3.62(m,4H),2.90–2.69(m,4H). 13 C NMR(101MHz,DMSO)δ162.61,157.70,142.47,138.69,138.65,137.49,137.2 9,129.12,128.81,128.29,126.82,124.29,121.71,115.45,57.34,45.70.

[0492] Test Case

[0493] 1. Detection of the affinity level of each compound in Examples 1 to 34 above with GAS41 protein

[0494] Compounds LH-33, LH-60 and LH-61 used the Biacore 8K model SPR device and display interface, and other compounds used the Biacore T200 model SPR device and operation interface. The SPR device measures the equilibrium dissociation constant KD value of the binding of the molecule to the GAS41 protein, thereby evaluating the affinity level of the compound to the protein. The working principle and operation method of the Biacore 8K model SPR device are the same as those of the Biacore T200 model SPR device, and the same CM7 chip is used, with only slight differences in the display interface and image. Among them, the detection results of some compounds are shown in the table below, and the SPR test results of compound LH-145B and compound LH-143 are shown in the figure below. Figure 1 and Figure 2 shown.

[0495] Compound Equilibrium dissociation constant KD value (μM) Compound Equilibrium dissociation constant KD value (μM) LH-57 42 LH-133 78.9 LH-138 54.7 LH-145A 78.9 LH-140 78.2 LH-143 73 LH-98 62.1 LH-156 126 LH-151 68.1 LH-165 20.7 LH-95 88.6 LH-145B 65.3 LH-87 77.7 LH-60 18 LH-134 68.3 LH-33 39 LH-94 93.5 LH-61 85

[0496] 2. CCK8 cytotoxicity assay to test the effects of compounds LH-145B and LH-2-169 on cell viability of T98G, SNB19, and BT-325 cell lines

[0497] The compounds LH-145B and LH-2-169 prepared in the above examples were used to perform CCK8 cytotoxicity experiments on different commercially available glioma cell lines (T98G, SNB19, and BT-325). The specific experimental methods are as follows:

[0498] The cell suspensions of commercially available T98G, SNB19, and BT-325 cell lines were injected into 96-well plates at a volume of 100 μl per well, with 5,000 cells per well. The different cell lines were then pre-incubated for 24 hours at 37°C and 5% CO2. 10 μl of culture medium solution of small molecule compounds with different concentration gradients (0, 25 μM, 50 μM, 100 μM) was then added to each well and incubated for 72 hours. 10 μl of CCK8 reagent was then added to each well, and CCK8 solution was added to the wells without cells as a control, and the culture was continued for 4 hours. Finally, the absorbance data was measured using a microplate reader. The test results are shown in Figure 2. Figure 3 As shown, the concentration gradient of the small molecule compound on the horizontal axis is set to 0, 25 μM, 50 μM, and 100 μM.

[0499] The results showed that both LH-145B and LH-2-169 exhibited inhibitory activity against the aforementioned cell lines. Compound LH-145B has a phenylbithiophene skeleton, while LH-2-169 has a pyridinylbithiophene skeleton. This further demonstrates that compounds containing both pyridinylbithiophene and phenylbithiophene skeletons exhibit tumor cell inhibitory activity in glioma cell lines.

[0500] 3. Proliferation inhibition experiment of compound LH-145B in DIPG tumor cells, U118MG tumor cells and PPC cell lines

[0501] DIPG17 cells are commercially available diffuse intrinsic pontine glioma (DIPG) cells, U118MG is a commercially available glioblastoma cell line, and PPC cells are embryonic pontine progenitor cells (not tumor cells). The three cells were treated with the compound LH-145B prepared in the above example for 48 hours, and the inhibition rate (Cell Inhibition %) of the compound LH-145B in different cells was determined. The results are shown in Figures 4 to 6 ,in Figures 4 to 6The horizontal axis represents the concentration of compound LH-145B, and the vertical axis represents the inhibition rate of cells. The Ki-67% (i.e., cell proliferation inhibition experiment, where Ki-67% represents the cell proliferation index using Ki-67 as a tumor cell proliferation marker) of LH-145B (5.0 μM) in the DIPG17 tumor cell line was determined using bivariate flow cytometry. The results are shown in Figure 7 It was found that the inhibition rate (Cell Inhibition%) of DIPG17 tumor cells, U118MG tumor cells and PPC cells increased with the increase of the concentration of compound LH-145B. According to the fitting curve of concentration gradient and inhibition rate, when the inhibition rate reached 50%, the half-inhibitory concentration (IC) of LH-145B in different cell lines can be obtained according to the corresponding concentration value. 50 Among them, the half-inhibitory concentration IC of compound LH-145B in different cells 50 and 95% confidence intervals, the results are shown in the following table:

[0502]

[0503]

[0504] As shown in the table above, the IC of compound LH-145B in DIPG17 cell line 50 The IC in U118MG cell line is 6.1 μM. 50 The IC in PPC cells is 6.2 μM. 50 It is 11.3μM.

[0505] The results of the cell proliferation inhibition experiment showed that after DIPG17 cells were treated with compound LH-145B at a concentration of 5.0 μM for 48 hours, the Ki-67% of the experimental groups of different tumor cell lines was significantly lower than that of the control group, indicating that the proliferation ability of the cells in the experimental group was significantly inhibited, and compound LH-145B can effectively inhibit the proliferation of DIPG17 tumor cells.

[0506] 4. Cell Titer-Glo assay to test the effect of compound LH-145B on mouse embryonic fibroblast (MEF) cell viability

[0507] ATP is a key indicator of viable cell metabolism. The Cell Titer-Glo cell viability assay is an effective method for measuring viable cell count or cell proliferation. It works by quantitatively detecting ATP and thereby determining cell viability. The Cell Viability% value represents the ratio of the viable cell count in the experimental group to the blank control group, with the default Cell Viability% for the blank control group being 100%. Lower Cell Viability% values ​​in the experimental group indicate lower cell viability and proliferation, indicating a higher inhibitory activity of the compound.

[0508] The Cell Titer-Glo Luminescent Cell Viability Assay was used to test the effect of compound LH-145B on the viability of MEF cells (mouse embryonic fibroblasts, which are derived from normal tissue and are not tumor cells). The specific detection steps are as follows:

[0509] (1) MEF cells were cultured in a 96-well plate with 90 μL DMEM medium per well, and the cells were allowed to reach 60% confluence.

[0510] (2) Ten groups of DMEM culture medium solutions containing compound LH-145B were prepared, with concentrations set to 0, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM and 500 μM.

[0511] (3) Divide the 96-well plate into ten groups, and add ten groups of DMEM solutions of compound LH-145B with different concentrations in sequence. Add 10 μL of solution to each well and add it to the original culture medium to obtain 100 μL, which is equivalent to a 10-fold dilution.

[0512] (4) Culture MEF cells for 72 hours.

[0513] (5) Take out the 96-well plate containing MEF cells, add Cell Titer-Glo reagent to the 96-well plate, and place it in a 37°C incubator in the dark for 10 minutes.

[0514] (6) Use an enzyme-labeled instrument to detect the fluorescence signal value and calculate the corresponding Cell Viability% value.

[0515] Test results see Figure 8 , Figure 8The horizontal axis shows the concentration of compound LH-145B in the culture medium, and the vertical axis shows the Cell Viability% (or Relative Cell Viability%, relative cell activity) of MEF cells after treatment with compound LH-145B. The lower the Cell Viability%, the lower the cell viability and the higher the inhibitory activity of the drug.

[0516] The control group was treated with LH-145B at a concentration of 0, while the experimental groups were treated with concentrations ranging from 10 to 50 μM. The experimental results showed that when LH-145B concentrations did not exceed 30 μM, the Cell Viability% of MEF cells did not decrease, and cell viability was not inhibited. When the concentration of LH-145B was increased to 50 μM, the Cell Viability% remained high at 57.8%, indicating that the inhibitory effect of LH-145B on MEF cell viability was weak and that MEF cells were less sensitive to LH-145B. This further demonstrates that the compounds of the present invention do not significantly inhibit the cell viability of normal cells.

[0517] As mentioned above, a lower concentration (5 μM) of compound LH-145B significantly inhibited the viability of DIPG17 tumor cells, but even a higher concentration (50 μM) of compound LH-145B still had a weaker inhibitory effect on the viability of MEF cells. These results indicate that mouse embryonic fibroblasts (MEF cells) have a low sensitivity to compound LH-145B, while DIPG tumor cells are more sensitive to compound LH-145B. Compared with normal cells, compound LH-145B exhibits a certain degree of selectivity for tumor cells.

[0518] 5. CCK8 cytotoxicity assay to test the effects of compounds LH-145B and LH-2-169 on cell viability of U87-LUC, HCMEC / D3, and M-231 cell lines

[0519] The compounds LH-145B and LH-2-169 prepared in the above examples were used to perform CCK8 cytotoxicity experiments on different commercially available cell lines (U87-LUC, HCMEC / D3 and M-231 cell lines). The specific detection steps are shown in step 2 of this example. The test results are shown in FIG. Figures 9 to 11 As shown, the abscissa in the figure shows the compound concentration gradient set to 0, 6.25μM, 12.5μM, 25μM, 50μM and 100μM, and the ordinate shows the corresponding Cell Viability%. The results showed that compounds LH-145B and LH-2-169 both exhibited inhibitory activity against U87-LUC, HCMEC / D3 and M-231 cell lines.

[0520] 6. Cell spheroidization assay to test the inhibitory activity of compounds LH-143 and LH-145B against DIPG tumor cells

[0521] DIPG cells were used for a cell spheroidization experiment. The experimental group of DIPG cells was treated with a 5.0 μM culture medium solution (0.1% DMSO) of the compound LH-143 or LH-145B. The initial cell number was 1000. The control group of cells (No. 151201) was treated with a culture medium (containing 0.1% DMSO) and cultured at 37°C and 5% CO2 for 14 days. The cell spheroidization effect after treatment with different compounds was detected using an optical microscope. The results are shown in Figure 2. Figure 12 As shown in the figure, compared with the control group, the number of tumor cells in the culture dish was significantly reduced, the cell density was significantly decreased, and the cells did not aggregate or grow in overlapping clusters. Therefore, the results indicate that both compounds LH-143 and LH-145 have the most prominent tumor cell inhibitory activity.

[0522] 7. Detection of blood-brain barrier permeability of compound LH-145B

[0523] 1) Twelve commercially available Balb / c mice were intraperitoneally injected with compound LH-145B. The changes in the drug concentration of compound LH-145B in plasma, brain, and cerebrospinal fluid within 24 hours were detected using mass spectrometry to study the blood-brain barrier permeability of compound LH-145B. The drug concentration of compound LH-145B was 50 mg / kg. Compound LH-145B can be diluted with a solution. The content of compound LH-145B in the solution was 10 mg / 1000 μL. The solution formula was DMSO (5%), PEG400 / 600 (30%), and H2O (65%). The test results are as follows: Figure 13 shown.

[0524] like Figure 13 As shown in Figure A, following administration to Balb / c mice, the plasma concentration of compound LH-145B immediately peaked. Subsequently, as the drug is metabolized in the mice, the plasma concentration decreased over the 24-hour experimental duration. Blood concentrations were measured twice at each time point, and the average was taken. The plasma concentration reached 7650 ng / mL 0.5 hours after administration, decreased to 2785 ng / mL 2 hours later, and remained at only 73.5 ng / mL 24 hours later. This indicates that compound LH-145B is metabolized at a slightly elevated rate in mice, with a half-life of no more than 2 hours.

[0525] like Figure 13 As shown in Figure B, after administration, the concentration of compound LH-145B in brain tissue began to increase significantly, and after 4 hours, the concentration showed a downward trend.

[0526] like Figure 13 As shown in Figure C, after administration, the concentration of compound LH-145B in the cerebrospinal fluid (CSF) decreased, rebounded after 1 hour, decreased after 2 hours, and decreased to below the detection limit after 8 hours.

[0527] 2) After the Balb / c mice were administered the drug in step 1), the concentration changes of compound LH-145B in plasma and brain, as well as the brain / plasma drug concentration ratio are shown in the table below.

[0528]

[0529] As shown in the table above, following administration, the concentration of compound LH-145B in brain tissue initially increased, then decreased after 4 hours. From 4 to 24 hours, brain concentrations continued to decline due to metabolism. Concentrations were measured twice at each time point, and the average value was taken. The values ​​reached 51,500 ng / g at 0.5 hours, rose to 79,000 ng / g at 4 hours, and decreased to 5,165 ng / g at 24 hours. These values ​​are significantly higher, indicating that brain concentrations of compound LH-145B remained high throughout the testing period, demonstrating its strong ability to cross the blood-brain barrier.

[0530] Brain / blood drug concentration ratio (C Brain / C Plasma The α-D-ratio (α-D-ratio) is an important parameter for evaluating brain distribution and can be used to assess a molecule's blood-brain barrier permeability. A value below 0.1 indicates that the molecule cannot freely cross the blood-brain barrier. Experimental results showed that this parameter reached 6.73 at 0.5 hours, 16.4 at 1 hour, 28.0 at 2 hours, and 79.5 at 24 hours, significantly exceeding 0.1. This indicates that compound LH-145B can efficiently cross the blood-brain barrier.

[0531] These experimental results demonstrate that compound LH-145B, due to its high efficiency in crossing the blood-brain barrier, can be administered using conventional routes in the treatment of brain tumors, eliminating the need for less tolerated head injections. Once the drug molecules enter the brain through the blood-brain barrier via the circulatory system, they accumulate in the brain tumor site, where capillaries are typically abundant. This increases drug exposure within the brain and effectively inhibits brain tumor growth.

[0532] 3) After administration to Balb / c mice in step 1), the concentration changes of compound LH-145B in plasma and cerebrospinal fluid, as well as the cerebrospinal fluid / plasma drug concentration ratio are shown in the following table (BLOQ=below LLOQ, below the lower limit of quantification):

[0533]

[0534] As shown in the table above, the concentration of compound LH-145B in the cerebrospinal fluid decreased after administration, rebounded after 1 hour, decreased after 2 hours, and dropped below the detection limit after 8 hours. During the whole process, the concentration of the drug in the cerebrospinal fluid was always lower than 10 ng / mL, which was much lower than the blood concentration. The cerebrospinal fluid can transfer metabolites from brain tissue, and the cerebrospinal fluid / blood concentration ratio (C CSF / C Plasma The drug metabolism ratio (BMR) can, to a certain extent, assess drug metabolism in the brain. Residual LH-145B enters the cerebrospinal fluid (CSF) and is transported out of the brain by the CSF. The CSF / blood drug concentration ratio ranges from 0.0008 to 0.0022. After 8 hours, the CSF drug concentration falls below the detection limit and cannot be calculated. The low level of LH-145B residue in the CSF and the high brain / blood drug concentration ratio mentioned above indicate that LH-145B molecules can accumulate in brain tissue.

[0535] In summary, compound LH-145B has a strong ability to cross the blood-brain barrier and can efficiently cross the blood-brain barrier, which has significant advantages for the treatment of brain diseases. It is conducive to the use of conventional trunk injection administration methods, eliminating the need for high-risk and poorly tolerated head injection administration methods. It also facilitates the enrichment of small molecule drugs in brain tumor tissue and can improve the drug's brain exposure. Therefore, compound LH-145B has great potential for the treatment of brain tumors.

[0536] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0537] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A compound, which is a compound represented by formula (VIII) or a stereoisomer, tautomer, or pharmaceutically acceptable salt of a compound represented by formula (VIII): X2 is C or N; R3 is -C 1-3 alkoxy; R1 is -C(O)-NR 1a R 1a '; R 1a and R 1a ' are each independently selected from H, -C optionally substituted with one or more R7 1-10 Alkyl, optionally substituted with one or more R7-C 1-10 Alkylene-R 1c or a saturated or unsaturated 4-10 membered cycloalkyl group optionally substituted by one or more R7; or N and R 1a With R 1a ' are connected together to form a saturated or unsaturated 4-10 membered heterocyclic alkyl group containing R 1a and R 1a ' 4-10 membered heterocycloalkyl is optionally substituted by one or more R8; R 1c is a saturated or unsaturated 4- to 10-membered cycloalkyl group optionally substituted by one or more R9 or a 4- to 10-membered heterocycloalkyl group optionally substituted by one or more R9; R7 is halogen, -OH, or -C 1-4 Alkyl, -C 1-4 alkoxy; R8 is halogen, -C(O)-R 11 、-C 1-4 Alkyl, -C 1-4 Alkoxy, saturated or unsaturated 4-7 membered cycloalkyl or saturated or unsaturated 4-7 membered heterocycloalkyl; R9 is halogen, -C 1-4 Alkyl, -C 1-4 Alkoxy or -C 1-4 Haloalkyl; R 11 It is H or -OH.

2. The compound according to claim 1, characterized in that R1 is 3. The compound according to claim 1, characterized in that R1 is 4. A compound, which is a compound as shown below or a stereoisomer, tautomer, or pharmaceutically acceptable salt of a compound as shown below:

5. A GAS41 inhibitor, characterized in that The invention comprises the compound according to any one of claims 1 to 4.

6. A pharmaceutical composition, characterized in that The compound comprising any one of claims 1 to 4; Optionally, it further includes pharmaceutically acceptable carriers, excipients, and vehicles.

7. Use of the compound according to any one of claims 1 to 4 in the preparation of a reagent for binding to GAS41.

8. The use according to claim 7, characterized in that The agent is used to bind to GAS41 and inhibit GAS41 activity or expression.

9. Use of the compound according to any one of claims 1 to 4, the GAS41 inhibitor according to claim 5, or the pharmaceutical composition according to claim 6 in the preparation of a medicament for preventing or treating a disease associated with GAS41 overexpression.

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