Tetrahydrocarbazole compounds, their pharmaceutical compositions and uses

Abstract

The present invention relates to a tetrahydrocarbazole compound, a pharmaceutical composition thereof and uses thereof. The tetrahydrocarbazole compound is a compound represented by formula (I), an optical isomer thereof or a pharmaceutically acceptable salt thereof. On the one hand, the provided tetrahydrocarbazole compound can co-inhibit OCT4 at the transcriptional and functional levels by targeting OCT4 and JAK to promote CSC differentiation. On the other hand, it inhibits the differentiated tumor cells by inhibiting the JAK / STAT pathway. The compound provided by the present invention acts on two drug targets, OCT4 and JAK / STAT, in tumor cells, and synergistically inhibits tumor proliferation and metastasis through dual-target action, and can be used to prepare drugs for treating and / or preventing cancer.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a tetrahydrocarbazole compound, its pharmaceutical composition and uses. Background Art

[0002] Cancer Stem Cells (CSCs) are a small subset of tumor cells, accounting for only a small part of the tumor tissue (usually 1% or less), and having several key characteristics similar to normal stem cells. The first characteristic is self-renewal. CSCs always maintain their ability to continuously divide and differentiate into various types of tumor cells. The second characteristic is the ability to perform asymmetric division, that is, when dividing to produce a daughter CSC with the same characteristics as the parental CSC, it also produces a differentiated daughter cell. These daughter cells rapidly proliferate to occupy most of the tumor volume. Eventually, CSCs and their differentiated daughter cells together constitute a tumor tissue with the ability of self-renewal and rapid proliferation (Clevers H. The cancer stem cell: premises, promises and challenges. Nat Med 2011, 17: 313 - 319). This means that the contribution of CSCs to the long-term survival of tumors is much greater than that of their differentiated tumor cells.

[0003] In recent years, the transformation between Adult Stem Cells (ASCs) and CSCs has also received extensive attention. Mutations in normal ASCs disrupt their self-renewal and induce cell transformation into CSCs. These mutated CSCs have stronger proliferation ability, anti-apoptosis ability and immune escape ability, etc., which are typical malignant tumor characteristics and an important cause of tumor drug resistance and recurrence.

[0004] CSCs are reported to have extensive metastatic potential and can regenerate tumors and form disseminated metastatic tumors (Wang YJ, Herlyn M. The emerging roles of OCT4 in tumor-initiating cells. Am J Physiol Cell Physiol 2015, 309: C709 - 718). Currently, almost all drug treatments and radiotherapy methods can only kill or inhibit rapidly proliferating differentiated tumor cells, but cannot effectively eliminate or inhibit CSCs. Since CSCs and differentiated tumor cells can transform into each other under specific conditions, only combination therapies that simultaneously target and inhibit or kill CSCs and differentiated tumor cells and completely block the bidirectional transformation between the two are likely to eradicate tumors.

[0005] So far, no drug specifically targeting CSCs has been marketed, and a series of candidate drugs (such as Notch and Wnt inhibitors) that can promote the differentiation of CSCs or inhibit their proliferation are in various stages of clinical trials (Maucort C et al. Differentiation of cancer stem cells by using synthetic small molecules: toward new therapeutic strategies against therapy resistance. ChemMedChem. 2021, 16: 14 - 29). CSCs are resistant to traditional radiotherapy, chemotherapy, and molecular targeted therapy drugs, making specific targeting of CSCs an important strategy for cancer treatment and anti-tumor drug resistance research.

[0006] The core stemness transcription factor OCT4 is a member of the POU transcription factor family. Its encoding gene, POU5F1, has multiple transcription start sites and can transcribe different mRNA subtypes, thus translating into multiple OCT4 subtype proteins. As a classical transcription factor, OCT4A (usually simply referred to as OCT4) can specifically recognize and bind to the octamer motif (ATGC(A / T)AAT) in the promoter regions of numerous target genes through its POU domain. The OCT4 protein, in cooperation with interacting proteins such as SOX2, regulates the transcription of hundreds of downstream target genes, which can generally be divided into two categories: one is the stemness factors such as the NANOG gene whose transcription is positively activated by OCT4 / SOX2, and the other is the specific genes of each germ layer differentiation whose transcription is negatively inhibited by OCT4 / SOX2. Therefore, OCT4 / SOX2 maintains the self-renewal and pluripotency of pluripotent stem cells (PSCs) by simultaneously promoting the expression of stemness genes and shutting down the expression of germ layer genes, and is at the center of the PSC homeostasis regulatory network (Jerabek S et al. OCT4: dynamic DNA binding pioneers stem cell pluripotency. Biochim Biophys Acta 2014, 1839: 138 - 154).

[0007] The expression of OCT4 is turned off in normal differentiated tissues, but it is expressed at low levels to varying degrees in various solid tumor tissues and their cell lines, such as liver cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, cervical cancer, ovarian cancer, head and neck tumors, etc. Moreover, under the stimulation of some growth factors and hypoxia conditions, its expression will be significantly upregulated, which further suggests the plasticity of OCT4 expression in these cells. The expression of OCT4 in tumor tissues and tumor cells shows heterogeneity: compared with most differentiated tumor cells, OCT4 is mainly expressed in a small fraction of CSCs, and thus has gradually been regarded as a biomarker of CSCs in recent years (Wang YJ, Herlyn M. The emerging roles of OCT4 in tumor-initiating cells. Am J Physiol Cell Physiol 2015, 309: C709-718).

[0008] Studies in the applicant's laboratory have shown that after knocking out the OCT4 gene with CRISPR / Cas9 in tumor cells such as liver cancer and cervical cancer, the proliferation and metastasis of CSCs will be significantly inhibited, and their differentiation and apoptosis will be promoted, thus confirming that OCT4 is a potential drug target against tumor cells, especially against CSCs (Zhou Y et al. Endogenous authentic OCT4A proteins directly regulate FOS / AP-1 transcription in somatic cancer cells. Cell Death Dis. 2018, 9: 585; Ye C et al. Multiple novel hepatocellular carcinoma signature genes are commonly controlled by the master pluripotency factor OCT4. Cell Oncol (Dordr). 2020, 43: 279-295). Although OCT4 plays a key role in the processes of self-renewal and survival of CSCs, tumor metastasis, and tumor drug resistance, there are almost no reports on small molecule compounds that can specifically target and inhibit OCT4.

[0009] It is known that compounds such as retinoic acid can promote PSC differentiation by inhibiting OCT4 gene transcription. However, these compounds have multiple action targets and mechanisms and do not specifically target and inhibit OCT4. The only small molecule compound KRIBB53 reported so far that can specifically target OCT4 protein downregulates the level of intracellular OCT4 protein by promoting the ubiquitination and degradation of OCT4 protein, but the specific action site and mechanism are still unclear. Moreover, the IC 50 of KRIBB53 for inhibiting the proliferation of two types of PSCs, NCCIT and TERA1, is 10 - 20 μM, and its drug efficacy is weak (Jung J et al. KRIBB53 binds to OCT4 and enhances its degradation through the proteasome, causing apoptotic cell death of OCT4-positive testicular germ cell tumors. Carcinogenesis. 2018, 39: 838 - 849).

[0010] In addition, due to reasons such as the relatively low average expression level of OCT4 in tumor cells, limited drug action effect, and the negative feedback compensation mechanism of tumor signaling pathways, it is difficult to completely inhibit the role of OCT4 in tumor cells by solely targeting the expression and transcription factor function of OCT4. In the regulatory pathway of OCT4, the JAK / STAT pathway can not only directly regulate the transcription of OCT4 and NANOG to maintain the pluripotency of CSCs, but also, because it is highly activated in various tumors and participates in the malignant transformation of tumor cells, it has become an important therapeutic target for differentiating tumor cells. Currently, the commonly used molecular targeting drugs for the JAK / STAT pathway in clinical practice are mainly used to treat autoimmune diseases and myelofibrosis, and their effect of targeted tumor treatment remains to be determined. Therefore, developing JAK / STAT inhibitors with tumor inhibitory effects has good application prospects. Summary of the Invention

[0011] In view of all or part of the deficiencies of the above-mentioned prior art, the present invention provides a tetrahydrocarbazole compound, its pharmaceutical composition and uses. The provided tetrahydrocarbazole compound can, on the one hand, co-inhibit OCT4 at the transcriptional and functional levels by targeting OCT4 and JAK to promote CSC differentiation, and on the other hand, inhibit differentiated tumor cells by inhibiting the JAK / STAT pathway. The compound provided by the present invention acts on two drug targets, OCT4 and JAK / STAT, in tumor cells, and synergistically inhibits tumor proliferation and metastasis through dual-target action, and can be used to prepare drugs for treating and / or preventing cancer.

[0012] In the first aspect of the present invention, there is provided a compound represented by formula (I), its optical isomers or its pharmaceutically acceptable salts:

[0013]

[0014] Wherein,

[0015] is a single bond or a double bond;

[0016] R2 is an unsubstituted or one or more R c substituted group selected from the group consisting of: phenyl, 5-7 membered heteroaryl, 8-12 membered fused heteroaryl;

[0017] M1, M2, M3, M4 are each independently selected from the group consisting of: CH, CD, N; and when M1, M2, M3 or M4 is CH, the hydrogen atom on the CH can be optionally substituted by R a substituted arbitrarily;

[0018] M5 is selected from the group consisting of: CH2, CHD, CD2, NH, S, S(=O), S(=O)2, S(=O)(=NH); and when M5 is CH2 or NH, the hydrogen atom on the CH2 or NH can be optionally substituted by R b substituted arbitrarily;

[0019] The said R a is selected from the group consisting of: hydrogen, deuterium, halogen, hydroxyl, carboxyl, amino, cyano, sulfonyl, C(O)NH2, C1-5 alkyl, C 2-5 alkenyl, C 2-5 alkynyl, C 1-5 alkoxy, C 1-5 haloalkyl, C 2-5 haloalkenyl, C2-5 haloalkynyl, C 1.5 haloalkoxy, C 2-5 ester group, C 1-5 carbonyl, C(O)NH(C 1-5 alkyl), C 3-8 saturated or partially unsaturated carbocyclic group, 3-10 membered saturated or partially unsaturated heterocyclic group, C 6-10 aryl, 5-12 membered heteroaryl; and the said alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkenyl, haloalkynyl, haloalkoxy, ester group, carbonyl, carbocyclic group, heterocyclic group, aryl, heteroaryl are each independently optionally substituted by one or more substituents selected from the group consisting of: halogen, deuterium, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C 1-3 alkyl, C 1-3 amino group;

[0020] The said R bSelected from the group: hydrogen, deuterium, halogen, hydroxyl, carboxyl, cyano, sulfonyl, C 1-5 alkyl, C 2-5 alkenyl, C 2-5 alkynyl, C 1.5 alkoxy, C 1-5 haloalkyl, C 2-5 haloalkenyl, C 2-5 haloalkynyl, C 1-5 haloalkoxy, C 2.5 ester group, C 1.5 carbonyl, C 3.8 saturated or partially unsaturated carbocyclic group, 3- to 10-membered saturated or partially unsaturated heterocyclic group, C 6.10 aryl, 5- to 12-membered heteroaryl; and the alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkenyl, haloalkynyl, haloalkoxy, ester group, carbonyl, carbocyclic group, heterocyclic group, aryl, heteroaryl are each independently optionally substituted with one or more substituents selected from the group: halogen, deuterium, hydroxyl, carboxyl, nitro, cyano, sulfonyl, C 1-3 alkyl, C 1-3 amino group;

[0021] The said R c is selected from the group: hydrogen, deuterium, halogen, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C 1-5 alkyl, C 2.5 alkenyl, C 2-5 alkynyl, C 1-5 alkoxy, C 1-5 haloalkyl, C 2.5 haloalkenyl, C 2-5 haloalkynyl, C 1-5 haloalkoxy, C 1-5 amino group, C 2-5 ester group, C 1.5 carbonyl, C 3-8 saturated or partially unsaturated carbocyclic group, 3- to 10-membered saturated or partially unsaturated heterocyclic group, C 6-10 aryl, 5- to 12-membered heteroaryl; and the alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkenyl, haloalkynyl, amino group, ester group, carbonyl, carbocyclic group, heterocyclic group, aryl, heteroaryl are each independently optionally substituted with one or more substituents selected from the group: halogen, deuterium, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C 1-3 alkyl;

[0022] n is 0, 1, 2 or 3;

[0023] x is 0, 1, 2 or 3;

[0024] y is 0, 1 or 2.

[0025] Preferably, the compound does not include a compound selected from the following group:

[0026] In some embodiments, the compound has a structure represented by formula (II) or formula (III):

[0027]

[0028] wherein, R2, M1, M5, R a , R b , n, x, and y are defined as described in claim 1.

[0029] In another preferred example, the compound has a structure selected from the following group:

[0030]

[0031] In another preferred example, the compound has a structure selected from the following group:

[0032]

[0033] In some embodiments, the R2 is an unsubstituted or one or more R c -substituted phenyl, or an unsubstituted or one or more R c -substituted 5- to 10-membered heteroaryl;

[0034] The R c is selected from the following group: hydrogen, halogen, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C 1-5 alkyl, C 2.5 alkenyl, C 2-5 alkynyl, C 1.5 alkoxy, C 1-5 haloalkyl, C 2-5 haloalkenyl, C 2-5 haloalkynyl, C 1-5 haloalkoxy, C 1.5 amino, C 2-5 ester group.

[0035] In another preferred example, the R2 is selected from the following group: phenyl,

[0036] In some embodiments, the R b is selected from the following group: hydrogen, halogen, hydroxyl, carboxyl, cyano, sulfonyl, C 1-4 alkyl, C 1-4 alkoxy, C 1.4 haloalkyl, C 1-4Halogenated alkoxy, C 2-4 Ester group, C 1-4 Carbonyl group; and, the alkyl group, alkoxy group, halogenated alkyl group, halogenated alkoxy group, ester group, and carbonyl group are each independently optionally substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, carboxyl, nitro, cyano, sulfonyl, C 1-3 Alkyl group, C 1-3 Amino group.

[0037] In some embodiments, the R a Is selected from the group consisting of: hydrogen, halogen, hydroxyl, carboxyl, cyano, C 1-4 Alkyl group, C 2-4 Alkenyl group, C 2-4 Alkynyl group, C 1-4 Alkoxy group, C 2-4 Ester group, C 1-4 Carbonyl group, 3-8 membered saturated or partially unsaturated heterocyclic group, C 6-10 Aryl group, 5-12 membered heteroaryl group; and, the alkyl group, alkenyl group, alkynyl group, alkoxy group, ester group, carbonyl group, heterocyclic group, aryl group, and heteroaryl group are each independently optionally substituted by one or more substituents selected from the group consisting of: halogen, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C 1-3 Alkyl group, C 1-3 Amino group.

[0038] In a second aspect of the present invention, there is provided a compound represented by formula (IA), its optical isomer or its pharmaceutically acceptable salt:

[0039]

[0040] Wherein,

[0041] R1 is a hydroxyl group and is connected to the carbon atom at position 1 by a single bond, or, R1 is an oxygen atom or -N(CH2) n OH and is connected to the carbon atom at position 1 by a double bond, wherein, n is 1 or 2;

[0042] The carbon atom at position 2 and the carbon atom at position 3 are connected by a single bond or a double bond; R2 is selected from any one of an unsubstituted or substituted monocyclic group or monocyclic heterocyclic group, an unsubstituted or substituted fused ring group or fused heterocyclic group;

[0043] X, Y, Z are each independently selected from hydrogen, halogen, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C 1～5 Alkyl group, C 1～5 Alkoxy group, C 1～5 Halogenated alkyl group, C 1～5 Halogenated alkoxy group.

[0044] In some embodiments, in the compound represented by Formula I, R1 is an oxygen atom and is connected to the carbon atom at the 1-position by a carbon-oxygen double bond; a carbon-carbon double bond is formed between the carbon atom at the 2-position and the carbon atom at the 3-position, including the compound represented by the following Formula (IIA):

[0045]

[0046] In Formula IIA:

[0047] R2 is selected from any one of an unsubstituted or substituted monocyclic group or monocyclic heterocyclic group, an unsubstituted or substituted fused ring group or fused heterocyclic group; the substituents on the monocyclic group, monocyclic heterocyclic group, fused ring group or fused heterocyclic group are selected from halogen, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C 1～5 alkyl, C 1～5 alkoxy, C 1～5 haloalkyl, C 1～5 haloalkoxy, one or more of them.

[0048] In some embodiments, in the compound represented by Formula IIA, R2 is a benzene ring and at least one hydrogen atom on the benzene ring is substituted by a hydroxyl group.

[0049] In some embodiments, the compound is selected from the following group:

[0050]

[0051]

[0052]

[0053] In the second aspect of the present invention, there is provided a pharmaceutical composition comprising (1) the compound as described in the first aspect of the present invention, its optical isomers or its pharmaceutically acceptable salts; and optionally (2) a pharmaceutically acceptable carrier, excipient or other active drug.

[0054] In some embodiments, the pharmaceutical composition further comprises a second therapeutic component, and the second therapeutic component is a DNA methyltransferase inhibitor; preferably, the DNA methyltransferase inhibitor is SGI-1027.

[0055] In the third aspect of the present invention, there is provided the use of the compound as described in the first aspect of the present invention, its optical isomers or its pharmaceutically acceptable salts, or the pharmaceutical composition as described in the second aspect of the present invention in the preparation of a drug for treating and / or preventing cancer.

[0056] In some embodiments, the cancer is selected from the following group: liver cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, cervical cancer, ovarian cancer, head and neck tumors.

[0057] In some embodiments, the treatment and / or prevention of cancer comprises: using the compound represented by formula I according to any one of claims 1-9, its optical isomer or its pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 7 in combination with a DNA methyltransferase inhibitor.

[0058] In some embodiments, the DNA methyltransferase inhibitor is SGI-1027.

[0059] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is a schematic diagram of the synthesis method of some compounds provided by the present invention.

[0061] Figure 2 shows the inhibitory effect of some compounds on OCT4 protein in HeLa cells.

[0062] Figure 3 shows the inhibitory effect of compounds KZT-A5 and AH057 on the transcription of the OCT4 target gene NANOG in HeLa cells.

[0063] Figure 4 shows the inhibitory effect of compounds KZT-A5 and AH057 on the JAK1 / 2-STAT3 signaling pathway in HeLa cells.

[0064] Figure 5 shows the inhibitory effect of compounds KZT-A5 and AH057 on the formation of tumor microspheres by tumor cells.

[0065] Figure 6 shows the effects of compounds KZT-A5 and AH057 on the cell viability of human tumor cell lines from different tissue sources. Among them, Figure 6 A shows the effect of AH057 on the cell viability of human tumor cell lines from different tissue sources, Figure 6 B shows the effect of KZT-A5 on the cell viability of human tumor cell lines from different tissue sources.

[0066] Figure 7 is the in vitro proliferation inhibition effect diagram of compound KZT-A5 alone, SGI-1027 alone, and KZT-A5+SGI-1027 on HeLa cells. Among them, Figure 7 A, Figure 7 B and Figure 7 C are photos of 3 technical replicates.

[0067] Figure 8 It is the mean statistical result of the number of in vitro proliferating clones inhibited by compound KZT-A5 alone, SGI-1027 alone, and KZT-A5 + SGI-1027 in HeLa cells. Detailed implementation manners

[0068] Terms

[0069] As used herein, the term "alkyl" includes straight-chain or branched-chain alkyls. For example, C 1-6 alkyl represents a straight-chain or branched-chain alkyl having 1-6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert-butyl, cyclobutyl, n-pentyl, isopentyl, neopentyl, and the like.

[0070] As used herein, the term "alkenyl" includes straight-chain or branched-chain alkenyls. For example, C 2-6 alkenyl refers to a straight-chain or branched-chain alkenyl having 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups.

[0071] As used herein, the term "alkynyl" includes straight-chain or branched-chain alkynyls. For example, C 2-6 alkynyl refers to a straight-chain or branched-chain alkynyl having 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, or similar groups.

[0072] As used herein, the term "cycloalkyl" refers to a cyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, C3-C 10 alkenyl refers to a cyclic saturated aliphatic hydrocarbon group having 3-10 carbon atoms. It can be a monocyclic group, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. "Cycloalkyl" can also be in a bicyclic form, such as a bridged ring or a spiro ring form.

[0073] As used herein, the term "alkylamino" refers to an amino group substituted by an alkyl group. For example, "C 1-6 alkylamino" refers to an amino group substituted by C 1-6 alkyl, which can be mono-substituted or di-substituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, ditert-butylamino, and the like.

[0074] As used herein, the term "alkoxy" refers to a group having an alkyl-oxy structure. For example, "C 1-8"Alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 8 carbon atoms, including methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, sec-butoxy, pentyloxy, neopentyloxy, and the like.

[0075] As used herein, the term "haloalkyl" represents an alkyl group in which one or more hydrogen atoms are replaced by halogen, wherein the definition of alkyl is as described above.

[0076] As used herein, the term "haloalkoxy" represents an alkoxy group in which one or more hydrogen atoms are replaced by halogen, wherein the definition of alkoxy is as described above.

[0077] As used herein, the term "carbonyl" represents a group containing a specific number of carbon atoms and having an alkyl-C(=O)- or alkyl-C(=O)-alkyl- structure.

[0078] As used herein, the term "heterocyclic group" or "heterocycloalkyl" refers to a saturated or partially saturated cyclic group having a specific number of ring atoms (such as 3 to 10 ring atoms), and in which 1 to 3 atoms are heteroatoms selected from N, S, and O. It can be monocyclic, bicyclic or polycyclic, such as in the form of bridged or spiro rings. Specific examples can be oxetanyl, azetidinyl, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, and pyrrolidinyl, etc.

[0079] As used herein, the terms "aryl", "aromatic ring" or "aromatic cycle" refer to a cyclic group having aromaticity. For example, the term "C6-C10 aryl" refers to an aryl group having 6 to 10 carbon atoms, such as phenyl or naphthyl and similar groups. "Aryl" can also be a fused aryl.

[0080] As used herein, the term "heteroaryl" or "heteroaromatic ring" refers to a cyclic aromatic group in which 1-3 atoms are heteroatoms selected from the group consisting of N, S, and O; for example, the term "5-12 membered heteroaryl" refers to a heteroaryl having 5-12 atoms. It can be monocyclic or in the form of a fused ring. Specific examples can be furyl, thienyl, pyrrolyl, thiazolyl, thiadiazolyl, oxazolyl, dioxazolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, 1,4-dioxacyclohexadienyl, 2H-1,2-azinyl, 4H-1,2-azinyl, 6H-1,2-azinyl, 4H-1,3-azinyl, 6H-1,3-azinyl, 4H-1,4-azinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, oxepinyl, thiepinyl, azepinyl, 1,3-diazepinyl, azocine, benzofuryl, benzisofuryl, benzothienyl, indolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, pyridopyrazolyl, pyridopyrrolyl, pyrimidinopyrazolyl, pyrimidinopyrrolyl, pyridazinopyrazolyl, pyridazinopyrrolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenazinyl, pteridinyl, purinyl, naphthyridinyl, etc.

[0081] As used herein, "halogen" or "halo atom" refers to F, Cl, Br, and I. More preferably, the halogen or halo atom is selected from F, Cl, and Br. "Halogenated" means substituted with an atom selected from F, Cl, Br, and I.

[0082] Unless otherwise specified, the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, the R and S configurations containing an asymmetric center, the (Z) and (E) isomers of a double bond, etc. Therefore, individual stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformational isomers) are within the scope of the present invention.

[0083] As used herein, the term "tautomer" means that structural isomers with different energies can cross a low energy barrier and thus interconvert. For example, proton tautomers (i.e., prototropy) include interconversion through proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion through some reorganization of bonding electrons.

[0084] As used herein, the term "solvate" refers to a complex formed by the coordination of a compound of the present invention with solvent molecules in a specific ratio.

[0085] As used herein, the term "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

[0086] After extensive and in - depth research, the inventors unexpectedly discovered a tetrahydrocarbazole compound. A series of bioactivity tests were then conducted on it, and it was found that it could co - inhibit OCT4 at the transcriptional and functional levels by targeting OCT4 and JAK to promote CSC differentiation. On the other hand, it could inhibit differentiated tumor cells by inhibiting the JAK / STAT pathway. Based on this, the present invention was completed.

[0087] The main advantages of the present invention are as follows:

[0088] 1. The tetrahydrocarbazole compound of the present invention can co - inhibit OCT4 at the gene transcription and functional levels by targeting OCT4 and JAK to promote CSC differentiation, and at the same time inhibit differentiated tumor cells by inhibiting the JAK / STAT pathway, thereby specifically targeting and clearing CSCs and differentiated tumor cells, overcoming drug resistance to existing anti - tumor drugs, and efficiently inhibiting tumor proliferation, invasion and metastasis.

[0089] 2. The tetrahydrocarbazole compound of the present invention has good safety, low toxicity and side effects, and the prepared drug can be taken orally.

[0090] 3. The tetrahydrocarbazole compound of the present invention can be used in combination with other targeted drugs or chemotherapeutic drugs, etc., to achieve the goal of synergistic enhancement, and has the potential to greatly improve the survival period of cancer patients and even cure certain types of cancer.

[0091] 4. The tetrahydrocarbazole compound involved in the present invention is a first - class OCT4 inhibitor and also the first dual - targeted inhibitor of OCT4 and JAK1 / 2.

[0092] The solution of the present invention will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0093] Examples

[0094] Example 1: Synthesis of Compound 1

[0095]

[0096] To a 25 mL single-necked flask, KZT (200 mg, 1.0 mmol), 1-1 (1.5 mmol), concentrated hydrochloric acid (10 mL), and methanol (5 mL) were added successively. The mixture was refluxed for 10 hours until KZT disappeared. The reaction solution was cooled to room temperature and poured into crushed ice. A solid was precipitated, filtered, and the filter cake was washed with pure water. The dried crude product was purified by silica gel column chromatography (petroleum ether∶ethyl acetate∶methanol = 100∶10∶1) to obtain a yellowish-green solid compound 1, with a yield of 53.6% and m.p. = 191 - 193 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 9.39 (s, 1H), 7.57 (s, 1H), 7.46 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.17 (dd, J = 8.5, 1.4 Hz, 1H), 7.11 (d, J = 1.8 Hz, 1H), 7.03 (dd, J = 8.4, 1.7 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 4.09 (q, J = 7.0 Hz, 2H), 3.23 (t, J = 5.7 Hz, 2H), 3.01 (t, J = 6.3 Hz, 2H), 2.40 (s, 3H), 1.37 (t, J = 7.0 Hz, 3H); 13 C NMR (101 MHz, DMSO-d6) δ 180.24, 148.08, 147.05, 137.59, 135.11, 134.52, 132.77, 129.04, 128.87, 127.40, 126.69, 125.86, 123.91, 120.80, 116.05, 115.95, 113.03, 64.43, 27.72, 21.55, 20.61, 15.22. HRMS (ESI) calcd for C 22 H 20 NO3 - (M-H) - 346.1449, found 346.1455.

[0097] Example 2: Synthesis of Compound 2

[0098]

[0099] To a 25 mL single-necked flask, KZT (200 mg, 1.0 mmol), 2-1 (1.5 mmol), concentrated hydrochloric acid (10 mL), and methanol (5 mL) were added successively. The mixture was refluxed for 10 hours until KZT disappeared. The reaction solution was cooled to room temperature and poured into crushed ice. A solid was precipitated, filtered, and the filter cake was washed with pure water. The dried crude product was purified by silica gel column chromatography (petroleum ether∶ethyl acetate∶methanol = 100∶10∶1) to obtain a brown gum 2, yield: 43.7%. 1 H NMR (400 MHz, CDCl3) δ 9.53 (s, 1H), 7.76 (s, 1H), 7.43 (s, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.72 (s, 2H), 3.93 (s, 6H), 3.29 (t, J = 5.9 Hz, 2H), 3.06 (t, J = 6.2 Hz, 2H), 2.45 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 176.72, 145.48, 144.40, 135.13, 132.61, 132.08, 130.15, 126.38, 126.30, 124.91, 124.10, 123.01, 121.24, 119.34, 112.50, 112.26, 110.14, 55.66, 27.15, 21.00, 20.06. HRMS (ESI) calcd for C 22 H 20 NO4 - (M-H) - 362.1398, found 362.1405。

[0100] Example 3: Synthesis of Compound 3

[0101]

[0102] To a 25 mL single-necked flask, KZT (200 mg, 1.0 mmol), 3-1 (1.5 mmol), concentrated hydrochloric acid (10 mL), and methanol (5 mL) were added successively. The mixture was refluxed for 10 hours until KZT disappeared. The reaction solution was cooled to room temperature and poured into crushed ice. A solid was precipitated, filtered, and the filter cake was washed with pure water. The dried crude product was purified by silica gel column chromatography (petroleum ether∶ethyl acetate∶methanol = 100∶10∶1) to obtain a brown oil 3, yield: 43.5%. 11H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 7.47 (d, J = 6.8 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 8.6 Hz, 1H), 6.99 (s, 1H), 6.91 - 6.77 (m, 2H), 3.20 (d, J = 6.4 Hz, 2H), 3.00 (t, J = 6.3 Hz, 2H), 2.39 (s, 3H). 13 13C NMR (101 MHz, DMSO-d6) δ 172.87, 145.69, 137.55, 135.12, 134.08, 132.84, 128.91, 128.74, 126.46, 126.46, 125.86, 122.91, 120.80, 117.64, 117.48, 116.20, 113.00, 27.73, 21.57, 20.62. HRMS (ESI) calcd for C 20 H 16 NO3 - (M - H) - 318.1136, found 3118.1140。

[0103] Example 4: Synthesis of Compound 4

[0104]

[0105] To a 25 mL single-necked flask, add KZT (200 mg, 1.0 mmol), 4-1 (1.5 mmol), concentrated hydrochloric acid (10 mL), and methanol (5 mL) in sequence. Reflux the reaction for 10 hours until KZT disappears. Cool the reaction solution to room temperature and pour it into crushed ice. A solid precipitates. Filter, wash the filter cake with pure water, and purify the dried crude product by silica gel column chromatography (petroleum ether∶ethyl acetate∶methanol = 100∶10∶1) to obtain a brown solid 4, yield: 52.7%, m.p. = 195 - 197 °C. 1 1H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 8.67 (s, 1H), 7.49 (s, 1H), 7.43 (s, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.17 - 7.12 (m, 3H), 3.20 (t, J = 5.6 Hz, 2H), 2.97 (t, J = 6.3 Hz, 2H), 2.38 (s, 3H), 2.21 (s, 6H). 1313C NMR (101 MHz, DMSO-d6) δ 180.35, 154.51, 137.59, 135.05, 134.32, 132.82, 130.91, 129.02, 128.83, 127.07, 126.61, 125.88, 124.80, 120.80, 113.04, 27.76, 21.56, 20.64, 17.15. HRMS (ESI) calcd for C 22 H 20 N02 - (M - H) - 330.1500, found 330.1505.

[0106] Example 5: Synthesis of Compound 5

[0107]

[0108] To a 25 mL single-necked flask, add KZT (200 mg, 1.0 mmol), 5-1 (1.5 mmol), concentrated hydrochloric acid (10 mL), and methanol (5 mL) successively. Reflux the reaction for 10 hours until KZT disappears. Cool the reaction solution to room temperature and pour it into crushed ice. A solid precipitates. Filter it, wash the filter cake with pure water, and purify the dried crude product by silica gel column chromatography (petroleum ether∶ethyl acetate∶methanol = 100∶10∶1) to obtain yellow solid 5, yield: 45.7%, m.p. = 169 - 171 °C. 1 1H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 9.46 (s, 1H), 7.57 (s, 1H), 7.46~7.42 (m, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 8.5, 1.3 Hz, 1H), 7.11 (d, J = 1.8 Hz, 1H), 7.02 (dd, J = 8.3, 1.6 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.82 (s, 3H), 3.27~3.16 (m, 2H), 2.99 (t, J = 6.3 Hz, 2H), 2.38 (s, 3H). 13 13C NMR (101 MHz, DMSO-d6) δ 180.18, 147.94, 147.86, 137.59, 135.07, 134.54, 132.81, 128.94, 128.79, 127.41, 126.56, 125.87, 123.80, 120.80, 115.96, 114.82, 113.00, 55.68, 27.25, 21.10, 20.16. HRMS (ESI) calcd for C 21 H18 NO3 - (M-H) - 332.1292, found 332.1296.

[0109] Example 6: Synthesis of Compound 8

[0110]

[0111] Under a hydrogen atmosphere at room temperature, palladium on carbon (25 mg, 0.180 mmol) was added to a solution of Compound 8-1 (300 mg, 0.900 mmol) in ethyl acetate (1 mL). The reaction was stirred at room temperature overnight and then filtered. The filter cake was washed with ethyl acetate (2 × 10 mL). The filtrate was concentrated and separated by a reverse-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain white solid Compound 8 (54.8 mg, 17.85%). ESI / MS (m / z): 366.1 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6): δ 11.46 (s, 1H), 8.72 (s, 1H), 7.40 (s, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.16 - 7.08 (m, 1H), 6.82 (d, J = 1.6 Hz, 1H), 6.70 (d, J = 7.9 Hz, 1H), 6.63 (dd, J = 8.0, 1.7 Hz, 1H), 3.75 (s, 3H), 3.19 (dd, J = 13.7, 3.9 Hz, 1H), 2.99 (dt, J = 16.4, 4.4 Hz, 1H), 2.85 - 2.71 (m, 2H), 2.59 - 2.52 (m, 1H), 2.37 (s, 3H), 2.15 - 2.01 (m, 1H), 1.78 (m, 1H).

[0112] Example 7: Synthesis of Compound 10

[0113]

[0114] At room temperature, concentrated sulfuric acid (0.2 mL, 3.752 mmol) was added to a mixture of Compound KZT (150 mg, 0.753 mmol) and 10-1 (95 mg, 0.904 mmol) in methanol (2 mL). The reaction mixture was stirred at 60 °C overnight and then the pH was adjusted to 8 with sodium hydroxide. After concentration, the crude product was separated by a reverse-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain yellow solid Compound 10 (49.3 mg, 22.79%). ESI / MS (m / z): 288.2 [M+H] + .1 1H NMR (400 MHz, DMSO-d6): δ 11.66 (s, 1H), 7.62 (s, 1H), 7.52 (d, J = 7.3 Hz, 2H), 7.49 (s, 1H), 7.46 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.17 (dd, J = 8.5, 1.3 Hz, 1H), 3.23 - 3.14 (m, 2H), 3.00 (t, J = 6.2 Hz, 2H), 2.38 (s, 3H).

[0115] Example 8: Synthesis of Compound 11

[0116]

[0117] At room temperature, concentrated sulfuric acid (0.2 mL, 3.752 mmol) was added to a mixture of compound KZT (150 mg, 0.753 mmol) and 11-1 (135 mg, 0.904 mmol) in methanol (2 mL). The reaction mixture was stirred at 60 °C overnight and then the pH was adjusted to 8 with sodium hydroxide. After concentration, the crude product was separated by a reverse-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain yellow solid compound 11 (8.9 mg, 3.57%). ESI / MS (m / z): 332.1 [M+H] + ESI / MS (m / z): 332.1 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 7.57 (d, J = 2.1 Hz, 1H), 7.52 - 7.46 (m, 2H), 7.45 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.16 (dd, J = 8.5, 1.7 Hz, 1H), 7.05 - 6.95 (m, 2H), 4.08 (q, J = 7.0 Hz, 2H), 3.20 (td, J = 6.4, 1.8 Hz, 2H), 3.00 (t, J = 6.3 Hz, 2H), 2.38 (s, 3H), 1.35 (t, J = 7.0 Hz, 3H).

[0118] Example 9: Synthesis of Compound 12

[0119]

[0120] To a 25 mL single-necked flask, KZT (200 mg, 1.0 mmol), 12-1 (1.5 mmol), concentrated hydrochloric acid (10 mL), and methanol (5 mL) were added successively. The mixture was refluxed for 10 hours until KZT disappeared. The reaction solution was cooled to room temperature and poured into crushed ice. A solid was precipitated, filtered, and the filter cake was washed with pure water and dried. The crude product was purified by silica gel column chromatography (petroleum ether∶ethyl acetate∶methanol = 100∶10∶1) to obtain a yellow-green solid compound 12, with a yield of 46.7% and m.p. = 200 - 202 °C. 1 H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H), 7.60 (s, 1H), 7.47 (s, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.21~7.15 (m, 1H), 7.11 (d, J = 7.8 Hz, 1H), 7.08 (s, 1H), 6.98 (dd, J = 8.2, 2.4 Hz, 1H), 3.82 (s, 3H), 3.24~3.16 (m, 2H), 3.02 (t, J = 6.3 Hz, 2H), 2.40 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 180.08, 148.14, 138.09, 135.47, 134.84, 132.61, 128.64, 128.99, 127.81, 126.86, 124.87, 122.60, 120.60, 118.17, 115.26, 114.42, 113.02, 55.66, 27.21, 21.08, 20.06. HRMS (ESI) calcd for C 21 H 18 NO2 - (M-H) - 316.1343, found 316.1346.

[0121] Example 10: Synthesis of Compound 13

[0122]

[0123] At room temperature, potassium hydroxide (211 mg, 3.765 mmol) was added to a methanol (3 mL) solution of compound KZT (150 mg, 0.753 mmol) and 13-1 (132 mg, 0.904 mmol). After the reaction mixture was stirred at 65 °C overnight, the pH was adjusted to 8 with 2 M hydrochloric acid. After concentration, the crude product was separated by a reverse-phase preparative column (mobile phase A: water (10 mmol / L + 0.1% ammonia water), mobile phase B: acetonitrile) to obtain the yellow solid compound 13 (5.0 mg, 2.03%). ESI / MS (m / z): 328.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d6): δ 11.65 (s, 1H), 11.61 (s, 1H), 8.84 (s, 1H), 8.83 (d, J = 7.2 Hz, 1H), 8.74 (d, J = 6.9 Hz, 1H), 8.11 (s, 1H), 7.89 (s, 1H), 7.71 (d, J = 9.1 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.48 (s, 1H), 7.47 - 7.45 (m, 1H), 7.45 - 7.40 (m, 1H), 7.40 - 7.36 (m, 1H), 7.34 (s, 1H), 7.32 (s, 1H), 7.30 (s, 1H), 7.19 - 7.13 (m, 2H), 7.12 - 7.06 (m, 2H), 3.31 (s, 2H), 3.18 (t, J = 5.6 Hz, 2H), 3.10 (d, J = 6.4 Hz, 2H), 3.07 (d, J = 6.1 Hz, 2H), 2.40 (s, 3H), 2.39 (s, 3H).

[0124] Example 11: Synthesis of Compound 14

[0125]

[0126] At room temperature, concentrated sulfuric acid (0.4 mL, 7.504 mmol) was added to a methanol (4 mL) solution of compound KZT (409 mg, 2.053 mmol)) and 14-1 (300 mg, 2.053 mmol). After the reaction mixture was stirred at 65 °C overnight, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate. After concentration, the crude product was separated by a reverse-phase preparative column (mobile phase A: water (10 mmol / L), mobile phase B: acetonitrile) to obtain the brown solid compound 14 (6.3 mg, 0.94%). ESI / MS (m / z): 328.0 [M+H] + .

[0127] Example 12: Synthesis of Compound 15

[0128]

[0129] At room temperature, potassium hydroxide (437 mg, 7.805 mmol) was added to a methanol (4 mL) solution of compound KZT (311 mg, 1.561 mmol) and 15-1 (150 mg, 1.561 mmol). The reaction mixture was stirred at 65 °C overnight, then the pH was adjusted to 8 with 2 M hydrochloric acid. After concentration of the reaction mixture, it was separated by a reversed-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate + 0.1% ammonia water), mobile phase B: acetonitrile) to obtain the yellow solid compound 15 (32.3 mg, 7.41%). ESI / MS (m / z): 278.1 [M-H] - 。 1 1H NMR (400 MHz, DMSO-d6): δ 13.21 (s, 1H), 11.61 (s, 1H), 7.82 (s, 1H), 7.52 (s, 1H), 7.47 (s, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.17 (dd, J = 8.5, 1.3 Hz, 1H), 6.66 (s, 1H), 3.53 - 3.37 (m, 2H), 3.04 (t, J = 6.2 Hz, 2H), 2.39 (s, 3H).

[0130] Example 13: Synthesis of Compound 16

[0131]

[0132] At room temperature, potassium hydroxide (42.5 mg, 0.759 mmol) was added to a 1,4-dioxane (2 mL) mixture of KZT (37.0 mg, 0.253 mmol) and 16-1 (60.5 mg, 0.303 mmol). The reaction was heated to 100 °C and reacted for 3 hours. The reaction was quenched by adding water, and the resulting mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (methanol∶dichloromethane = 1∶20) to obtain the yellow solid compound 16 (2.0 mg, 2.41%). ESI / MS (m / z) 327.9 [M+H] + 。

[0133] Example 14: Synthesis of Compound 17

[0134]

[0135] Under zero-degree conditions, an aqueous solution (20 mL) of sodium nitrite (1.25 g, 18.162 mmol) was slowly added to an aqueous solution (83 mL) of compound 17-1 (1 g, 9.081 mmol) and concentrated hydrochloric acid (2.70 mL, 88.903 mmol). The reaction solution was stirred at 0 °C for 30 minutes, and then a solution of compound 17-2 (1.15 g, 9.081 mmol) and sodium acetate (1.64 g, 19.978 mmol) in a methanol and water mixture (80 mL, 1:1) was slowly added to the reaction solution. The reaction solution was continuously stirred at 0 °C for 0.5 hours, and then the pH was adjusted to 8 with saturated aqueous sodium bicarbonate solution. After concentration, the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 5:1) to obtain the yellow solid compound 17-3 (400 mg, 20.09%). ESI / MS (m / z): 220.1 [M+H] + 。

[0136] At room temperature, acetic acid (12 mL, 209.359 mmol) and concentrated sulfuric acid (1.20 mL, 22.508 mmol) were added to a methanol (3 mL) solution of compound 17-3. The reaction solution was refluxed and stirred at 65 °C for 2 hours. The pH was adjusted to 8 with saturated aqueous sodium bicarbonate solution. After concentration, the crude product was separated by silica gel column chromatography (dichloromethane:methanol = 5:1) to obtain the yellow solid compound 17-4 (300 mg, 43.42%). ESI / MS (m / z): 203.1 [M+H] + 。

[0137] At room temperature, pyridine (587 mg, 7.420 mmol) and trifluoromethanesulfonic anhydride (935 mg, 4.452 mmol) were added to a dichloromethane (10 mL) mixture of compound 17-4 (300 mg, 1.484 mmol) and the mixture was stirred for 2 hours. After concentration, the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain the yellow solid compound 17-5 (210 mg, 42.35%). ESI / MS (m / z): 334.8 [M+H] + 。

[0138] Under a nitrogen atmosphere, at 0 °C, trimethylaluminum (226 mg, 3.143 mmol) was slowly added to a mixture of compound 17-5 (150 mg, 0.449 mmol) and Pd(PPh3)4 (52 mg, 0.045 mmol) in tetrahydrofuran (5 mL). The reaction solution was refluxed at 80 °C for 1 hour. After concentration of the reaction solution, the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain the yellow solid compound 17-6 (70 mg, 77.90%). ESI / MS (m / z): 200.9 [M+H] + 。

[0139] At room temperature, concentrated sulfuric acid (0.1 mL) was added to a mixture of compound 17-6 (60 mg, 0.300 mmol) and 17-7 (50 mg, 0.330 mmol) in methanol (1 mL). The reaction mixture was stirred and refluxed at 65 °C overnight. The pH was adjusted to 8 with saturated aqueous sodium bicarbonate, and after concentration, the crude product was separated by reverse-phase preparative column chromatography to give the yellow solid compound 17 (43.8 mg, 42.54%). ESI / MS (m / z): 335.0 [M+H] + 。 1 H NMR (400 MHz, DMSO-d6): δ 11.86 (s, 1H), 9.54 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.63 (s, 1H), 7.20 (dd, J = 8.4, 3.8 Hz, 1H), 7.15 (s, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 3.84 (s, 3H), 3.26 (t, J = 6.0 Hz, 2H), 3.07 (t, J = 6.2 Hz, 2H), 2.57 (s, 3H).

[0140] Example 15: Synthesis of Compound 18

[0141]

[0142] At room temperature, concentrated hydrochloric acid (1 mL) was added to a solution of compound 18-1 (1 g, 7.237 mmol) and 18-2 (0.97 g, 8.684 mmol) in acetic acid (10 mL). The reaction mixture was stirred at 65 °C for 2 hours and then concentrated directly. The residue was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 8∶1) to give the light brown solid compound 18-3 (110 mg, 7.06%). ESI / MS (m / z): 216.2 [M+H] + 。 1 H NMR (400 MHz, DMSO-d6): δ 11.44 (s, 1H), 7.30 (d, J = 8.8 Hz, 1H), 7.10 (s, 1H), 6.97 (d, J = 8.8 Hz, 1H), 3.78 (s, 3H), 2.92 (s, 2H), 2.54 (s, 2H), 2.14 (s, 2H).

[0143] At room temperature, concentrated sulfuric acid (0.3 mL, 5.628 mmol) was added to a methanol (3 mL) solution of compound 18-3 (110 mg, 0.511 mmol) and 18-4 (93 mg, 0.613 mmol). After the reaction mixture was stirred at 65 °C overnight, the pH was adjusted to 8 using saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate (3 × 20 mL), and the organic phase was washed with saturated brine (30 mL). After drying over anhydrous sodium sulfate and concentration, the crude product was separated by reverse-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain the yellow solid compound 18 (5.9 mg, 3.30%). ESI / MS (m / z): 350.0 [M+H] + . 1 H NMR (400 MHz, DMSO-d6): δ 11.58 (s, 1H), 9.44 (s, 1H), 7.56 (s, 1H), 7.32 (d, J = 8.9 Hz, 1H), 7.11 (t, J = 2.4 Hz, 2H), 7.03 (dd, J = 8.3, 1.5 Hz, 1H), 6.98 (dd, J = 9.0, 2.5 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H), 3.22 (t, J = 5.8 Hz, 2H), 3.00 (t, J = 6.3 Hz, 2H).

[0144] Example 16: Synthesis of Compound 19

[0145]

[0146] At room temperature, acetic acid AcOH (15 mL) and concentrated hydrochloric acid (3 mL) were added to a methanol (60 mL) solution of compound 19-1 (5 g, 22.371 mmol) and 19-2 (3.01 g, 26.845 mmol). After the reaction mixture was stirred at 65 °C overnight, the pH was adjusted to 8 using saturated aqueous sodium bicarbonate. After concentration, the mixture was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 5∶1) to obtain the light yellow compound 19-3 (700 mg, 11.85%). ESI / MS (m / z): 263.8 [M+H] + .

[0147] At room temperature, concentrated sulfuric acid (1.5 mL, 28.143 mmol) was added to a mixture of compound 19-3 (300 mg, 1.136 mmol) and 19-4 (259 mg, 1.704 mmol) in MeOH (15 mL). The reaction mixture was stirred overnight at 80 °C. After the reaction mixture was cooled to room temperature, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate and concentrated to obtain a crude product, which was separated by reverse-phase preparative column chromatography to give the yellow solid compound 19 (66.8 mg, 14.80%). ESI / MS (m / z): 398.0 [M+H] + 。 1 HNMR (400 MHz, DMSO-d6): δ 11.94 (s, 1H), 9.48 (s, 1H), 7.93 (s, 1H), 7.59 (s, 1H), 7.43 (dd, J = 8.8, 1.9 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.12 (d, J = 1.7 Hz, 1H), 7.04 (dd, J = 8.2, 1.6 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.82 (s, 3H), 3.28 - 3.19 (m, 2H), 3.02 (t, J = 6.3 Hz, 2H).

[0148] Example 17: Synthesis of Compound 20

[0149]

[0150] At room temperature, concentrated hydrochloric acid (2.4 mL, 78.991 mmol) was added to a solution of 20-1 (2.5 g, 15.020 mmol) and 20-2 (2.02 g, 18.024 mmol) in acetic acid (11.8 mL). The reaction mixture was refluxed at 120 °C for 1 hour. After the reaction mixture was concentrated, it was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to give the yellow solid compound 20-3 (400 mg, 12.67%). ESI / MS (m / z): 211.1 [M+H] + 。

[0151] At room temperature, concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a methanol (5 mL) solution of 20-3 (200 mg, 0.951 mmol) and 20-4 (173.69 mg, 1.141 mmol). The reaction mixture was stirred at 65 °C overnight. The pH of the reaction mixture was adjusted to 8 with saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate (3 × 20 mL), and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by preparative reverse-phase column chromatography (mobile phase A: 0.05% ammonia water, mobile phase B: acetonitrile) to obtain a yellow solid compound 20 (15.6 mg, 4.65%). ESI / MS (m / z): 345.0 [M+H] + 。 1 H NMR (400 MHz, DMSO-d6): δ 12.28 (s, 1H), 8.34 (s, 1H), 7.64 (dd, J = 8.6, 1.4 Hz, 1H), 7.61 (s, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.15 - 7.10 (m, 1H), 7.09 - 6.99 (m, 1H), 6.87 (d, J = 8.2 Hz, 1H), 3.82 (s, 3H), 3.28 - 3.25 (m, 2H), 3.07 (t, J = 6.8 Hz, 2H).

[0152] Example 18: Synthesis of Compound 21

[0153]

[0154] At room temperature, concentrated sulfuric acid (1 mL, 18.762 mmol) was added to an acetic acid (10 mL) solution of compound 21-1 (920 mg, 4.540 mmol) and 21-2 (610 mg, 5.448 mmol). The reaction mixture was stirred at 65 °C for 2 hours and then the pH was adjusted to 8 with saturated aqueous sodium bicarbonate. After extraction with ethyl acetate (3 × 50 mL), it was dried over anhydrous sodium sulfate and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 2∶1) to obtain a brown solid compound 21-3 (480 mg, 43.46%). ESI / MS (m / z): 244.2 [M+H] + 。

[0155] At room temperature, concentrated sulfuric acid (0.4 mL, 7.505 mmol) was added to a methanol (4 mL) solution of compound 21-3 (150 mg, 0.617 mmol) and 21-4 (112 mg, 0.740 mmol). After the reaction mixture was stirred at 80 °C overnight, the pH was adjusted to 8 using saturated aqueous sodium bicarbonate. After concentration, the crude product was separated by reversed-phase preparative column (mobile phase A: water (0.05% formic acid), mobile phase B: acetonitrile) to obtain the yellow solid compound 21 (20.04 mg, 8.35%). ESI / MS (m / z): 378.2 [M+H] + 。 1 H NMR (400 MHz, DMSO-d6): δ 12.16 (s, 1H), 9.50 (s, 1H), 8.4 - 8.31 (m, 1H), 7.92 (dd, J = 8.7, 1.6 Hz, 1H), 7.60 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 1.8 Hz, 1H), 7.05 (dd, J = 8.2, 1.6 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.26 (t, J = 5.2 Hz, 2H), 3.10 (t, J = 6.3 Hz, 2H).

[0156] Example 19: Synthesis of Compound 22

[0157]

[0158] Under a nitrogen atmosphere, sodium hydride NaH (29.9 mg, 1.250 mmol) was added to a DMF (5 mL) solution of compound 22-1 (300 mg, 1.136 mmol) and 22-2 (162 mg, 1.136 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 4 hours. The reaction was quenched with water, extracted with ethyl acetate (3 × 20 mL), washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 5∶1) to obtain the white solid compound 22-3 (300 mg, 68.73%). ESI / MS (m / z): 384.0 [M+H] + 。

[0159] Under a nitrogen atmosphere, at room temperature, to a solution of compound 22-3 (300 mg, 0.781 mmol), Cs2CO3 (763 mg, 2.343 mmol) and 22-4 (236.9 mg, 2.343 mmol) in 1,4-dioxane (8 mL) was added the catalyst Pd-PEPPSI-IHeptCl3-chloropyridine (76 mg, 0.078 mmol). The reaction mixture was stirred at 100 °C overnight under a nitrogen atmosphere. After cooling, the crude product obtained by concentration was separated by silica gel column chromatography (dichloromethane∶methanol = 1∶1) to give a light yellow solid compound 22-5 (150 mg, 47.50%). ESI / MS (m / z): 405.2 [M+H] + 。

[0160] At room temperature, concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a solution of compound 22-5 (150 mg, 0.371 mmol) and 22-6 (68 mg, 0.445 mmol) in methanol (5 mL). The reaction mixture was stirred at 65 °C overnight and then the pH was adjusted to 8 using saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate (3×20 mL), washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by reverse-phase preparative column (mobile phase A: water (0.05% formic acid), mobile phase B: acetonitrile) to give a yellow solid compound 22 (25.3 mg, 15.99%). ESI / MS (m / z): 419.1 [M+H]+. 1 1H NMR (400 MHz, DMSO-d6): δ 11.46 (s, 1H), 9.43 (s, 1H), 7.55 (s, 1H), 7.28 (d, J = 9.0 Hz, 1H), 7.15 (dd, J = 9.1, 2.2 Hz, 1H), 7.10 (d, J = 1.5 Hz, 1H), 7.03 (s, 1H), 7.02 - 6.99 (m, 1H), 6.8δ (d, J = 8.1 Hz, 1H), 4.66 (d, J = 3.8 Hz, 1H), 3.82 (s, 3H), 3.59 (dq, J = 8.9, 4.4 Hz, 1H), 3.47 - 3.36 (m, 2H), 3.21 (t, J = 5.6 Hz, 2H), 2.98 (t, J = 6.1 Hz, 2H), 2.81 - 2.71 (m, 2H), 1.91 - 1.79 (m, 2H), 1.55 (q, J = 10.9, 9.2 Hz, 2H).

[0161] Example 20: Synthesis of compound 23

[0162]

[0163] Under a nitrogen atmosphere, at room temperature, into a solution of compound 23-1 (200 mg, 0.520 mmol), cesium carbonate (508 mg, 1.560 mmol), catalyst EPhosPdG4 (47 mg, 0.052 mmol), ligand EPhos (28 mg, 0.052 mmol) and 23-2 (116 mg, 0.624 mmol) in 1,4-dioxane (5 mL). Under a nitrogen atmosphere, after the reaction mixture was stirred at 100 °C for 4 hours, it was quenched with water. Extracted with ethyl acetate (3 × 10 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to obtain white solid compound 23-3 (182 mg, 71.42%). ESI / MS (m / z): 490.3 [M+H] + 。

[0164] At room temperature, concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a mixture of compound 23-3 (150 mg, 0.286 mmol) and 23-4 (56 mg, 0.367 mmol) in methanol (5 mL). The reaction mixture was stirred at 65 °C overnight and then the pH was adjusted to 7 using saturated aqueous sodium bicarbonate. Extracted with ethyl acetate (3 × 20 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by reversed-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain light yellow solid compound 23 (10.1 mg, 8.17%). ESI / MS (m / z): 404.0 [M+H] + 。 1 1H NMR (400 MHz, DMSO-d6): δ 11.47 (s, 1H), 7.55 (s, 1H), 7.29 (d, J = 9.0 Hz, 1H), 7.15 (dd, J = 9.0, 2.0 Hz, 1H), 7.10 (d, J = 1.5 Hz, 1H), 7.03 (d, J = 1.6 Hz, 1H), 7.01 (s, 1H), 6.86 (d, J = 8.1 Hz, 1H), 3.82 (s, 3H), 3.21 (t, J = 5.8 Hz, 2H), 2.98 (t, J = 4.6 Hz, 6H), 2.86 (s, 4H).

[0165] Example 21: Synthesis of Compound 24

[0166]

[0167] Under a nitrogen atmosphere, at room temperature, potassium acetate (371 mg, 3.786 mmol) was added to a solution of compound 24-1 (500 mg, 1.893 mmol), 24-2 (734 mg, 3.786 mmol), and the catalyst Pd(dppf)Cl2CH2Cl2 (231 mg, 0.284 mmol) in 1,4-dioxane (10 mL) and water (2 mL). Under a nitrogen atmosphere, the reaction mixture was stirred at 100 °C overnight, quenched with water, extracted with ethyl acetate (3 × 20 mL), and washed with saturated brine (20 mL). Dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to obtain the brown solid compound 24-3 (100 mg, 21.02%). ESI / MS (m / z): 252.2 [M+H] + .

[0168] At room temperature, concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a solution of compound 24-3 (100 mg, 0.398 mmol) and 24-4 (72 mg, 0.478 mmol) in methanol (5 mL). The reaction mixture was stirred at 65 °C overnight, adjusted to pH 8 with saturated aqueous sodium bicarbonate, concentrated, and the crude product was separated by reversed-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain the brown compound 24 (12.2 mg, 7.80%). ESI / MS (m / z): 386.1 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6): δ 12.86 (s, 1H), 11.69 (s, 1H), 9.41 (s, 1H), 8.04 (s, 2H), 7.90 (s, 1H), 7.61 (dd, J = 8.7, 1.6 Hz, 1H), 7.59 (s, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.13 (d, J = 1.6 Hz, 1H), 7.04 (dd, J = 8.2, 1.5 Hz, 1H), 6.88 (d, J = 8.1 Hz, 1H), 3.83 (s, 3H), 3.26 (t, J = 5.7 Hz, 2H), 3.07 (t, J = 6.2 Hz, 2H).

[0169] Example 22: Synthesis of Compound 25

[0170]

[0171] Under a nitrogen atmosphere at room temperature, triethylamine (228.6 mg, 2.259 mmol) and 25-2 (222 mg, 2.259 mmol) were added to a mixture of compound 25-1 (300 mg, 0.753 mmol), Pd(dppf)Cl2·CH2Cl2 (123 mg, 0.151 mmol) and copper(I) iodide (14 mg, 0.075 mmol) in DMF (5 mL). The reaction mixture was stirred at 80 °C for 2 h. The reaction was quenched with water (1 mL), and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic layer was washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to give the yellow solid compound 25-3 (150 mg, 47.92%). ESI / MS (m / z): 416.0 [M+H] + 。

[0172] Potassium carbonate (100 mg, 0.722 mmol) was added to a mixture of compound 25-3 (150 mg, 0.361 mmol) in DMF (5 mL) at room temperature. After stirring the reaction mixture at 60 °C for 2 h, the reaction was quenched by adding water (1 mL). The mixture was extracted with ethyl acetate (3 × 10 mL). The organic layer was washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by reverse-phase preparative column chromatography to give the yellow solid compound 25 (20.3 mg, 16.38%). ESI / MS (m / z): 344.0 [M+H] + 。 1 1H NMR (400 MHz, DMSO-d6): δ 11.93 (s, 1H), 7.88 (d, J = 1.2 Hz, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.38 (dd, J = 8.6, 1.4 Hz, 1H), 7.12 (d, J = 1.7 Hz, 1H), 7.04 (dd, J = 8.3, 1.5 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 4.03 (s, 1H), 3.82 (s, 3H), 3.24 (t, J = 5.8 Hz, 2H), 3.04 (t, J = 6.3 Hz, 2H).

[0173] Example 23: Synthesis of Compound 26

[0174]

[0175] At room temperature, lithium hydroxide (3 mg, 0.130 mmol) was added to a methanol (1 mL) solution of compound 26-1 (10 mg, 0.026 mmol). After the reaction mixture was stirred at 65 °C overnight, the pH was adjusted to 8 with 2 M hydrochloric acid. The concentrated crude product was separated by reverse-phase preparative column (mobile phase A: water (0.05% formic acid), mobile phase B: acetonitrile) to obtain the brown solid compound 26 (2 mg, 20.77%). ESI / MS (m / z): 364.0 [M+H] + 。

[0176] Example 24: Synthesis of Compound 27

[0177]

[0178] At room temperature, concentrated hydrochloric acid (0.3 mL, 9.874 mmol) was added to an aqueous (12 mL) solution of compound 27-1 (300 mg, 1.845 mmol) and 27-2 (248 mg, 2.214 mmol). The reaction mixture was stirred at 120 °C overnight. The resulting solid was collected and washed with water (30 mL) to obtain the brown solid compound 27-3 (180 mg, 48.01%). ESI / MS (m / z): 204.0 [M+H] + 。 1 HNMR (400 MHz, DMSO-d6): δ 11.69 (s, 1H), 7.45 (dd, J = 9.5, 2.2 Hz, 1H), 7.40 (dd, J = 9.0, 4.5 Hz, 1H), 7.17 (td, J = 9.2, 2.3 Hz, 1H), 2.92 (t, J = 5.9 Hz, 2H), 2.62 - 2.53 (m, 2H), 2.15 (p, J = 6.0 Hz, 2H).

[0179] At room temperature, concentrated sulfuric acid (0.3 mL, 5.628 mmol) was added to a methanol (3 mL) mixed solution of compound 27-3 (180 mg, 0.886 mmol) and 27-4 (594 mg, 5.302 mmol). After the reaction mixture was stirred at 65 °C overnight, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate. The concentrated crude product was separated by reverse-phase preparative column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain the yellow solid compound 27 (20.4 mg, 6.83%). ESI / MS (m / z): 338.0 [M+H] + 。 11H NMR (400 MHz, DMSO-d6): δ 11.83 (s, 1H), 9.48 (s, 1H), 7.59 (s, 1H), 7.48 (dd, J = 9.5, 2.5 Hz, 1H), 7.42 (dd, J = 9.0, 4.5 Hz, 1H), 7.19 (td, J = 9.2, 2.6 Hz, 1H), 7.12 (d, J = 1.8 Hz, 1H), 7.04 (dd, J = 8.3, 1.7 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.82 (s, 3H), 3.27 - 3.20 (m, 2H), 3.01 (t, J = 6.3 Hz, 2H).

[0180] Example 25: Synthesis of Compound 28

[0181]

[0182] At room temperature, triethylamine (0.19 mL, 1.347 mmol) was added to a solution of Compound 28-1 (150 mg, 0.449 mmol) and catalyst Pd(dppf)Cl2·CH2Cl2 (73 mg, 0.090 mmol) in methanol (20 mL). The reaction mixture was stirred at 80 °C overnight under a carbon monoxide gas atmosphere. After concentration, the residue was purified by silica gel column chromatography (dichloromethane∶methanol = 20∶1) to afford a light yellow solid, Compound 28-2 (70 mg, 63.87%). ESI / MS (m / z): 245.0 [M+H] + 。 1 1H NMR (400 MHz, DMSO-d6): δ 12.19 (s, 1H), 8.02 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 8.7 Hz, 1H), 3.91 (s, 3H), 3.05 (t, J = 5.7 Hz, 2H), 2.66 (t, J = 6.2 Hz, 2H), 2.21 (p, J = 5.8 Hz, 2H).

[0183] At room temperature, concentrated sulfuric acid (0.3 mL) was added to a mixture of Compound 28-2 (50 mg, 0.205 mmol) and 28-3 (37 mg, 0.246 mmol) in methanol (3 mL). The reaction mixture was stirred at 65 °C overnight and then adjusted to pH 8 with saturated aqueous sodium bicarbonate. After concentration, the crude product was purified by reversed-phase preparative column chromatography (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to afford a yellow solid, Compound 28 (12.5 mg, 16.14%). ESI / MS (m / z): 379.0 [M+H] + 。 11H NMR (400 MHz, DMSO-d6): δ 12.07 (s, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.64 (s, 1H), 7.15 (s, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 3.91 (s, 3H), 3.83 (s, 3H), 3.31 (s, 2H), 3.13 (d, J = 6.3 Hz, 2H).

[0184] Example 26: Synthesis of Compound 29

[0185]

[0186] At room temperature, triethylamine (397 mg, 3.927 mmol) and HATU (746.42 mg, 1.963 mmol) were added to a solution of Compound 29-1 (300 mg, 1.309 mmol) and ammonium bicarbonate (310 mg, 3.927 mmol) in DMF (10 mL). The reaction mixture was stirred at room temperature for 2 hours, quenched with saturated aqueous ammonium chloride solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to obtain a gray solid, Compound 29-2 (150 mg, 50.22%). ESI / MS (m / z): 226.9 [M-H] - . 1 1H NMR (400 MHz, DMSO-d6): δ 11.82 (s, 1H), 8.31 (s, 1H), 7.92 (s, 1H), 7.84 (s, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.20 (s, 1H), 2.99 (t, J = 5.9 Hz, 2H), 2.64 - 2.55 (m, 2H), 2.19 (q, J = 6.0 Hz, 2H).

[0187] At room temperature, concentrated sulfuric acid (0.3 mL, 5.628 mmol) was added to a solution of Compound 29-2 (150 mg, 0.657 mmol) and Compound 29-3 (120 mg, 0.788 mmol) in methanol (3 mL). The reaction mixture was stirred at 65 °C overnight and then the pH was adjusted to 8 using saturated aqueous sodium bicarbonate solution. After concentration, the crude product was separated by reversed-phase preparative column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain a brown solid, Compound 29 (8.5 mg, 3.57%). ESI / MS (m / z): 361.0 [M-H] - . 11H NMR (400 MHz, DMSO-d6): δ 11.97 (s, 1H), 9.50 (s, 1H), 8.33 (s, 1H), 7.95 (s, 1H), 7.88 (dd, J = 8.7, 1.4 Hz, 1H), 7.60 (s, 1H), 7.44 (d, J = 8.7 Hz, 1H), 7.22 (s, 1H), 7.14 (d, J = 1.8 Hz, 1H), 7.05 (dd, J = 8.3, 1.6 Hz, 1H), 6.88 (d, J = 8.1 Hz, 1H), 3.83 (s, 3H), 3.28 (t, J = 5.7 Hz, 2H), 3.08 (t, J = 6.3 Hz, 2H).

[0188] Example 27: Synthesis of Compound 30

[0189]

[0190] To a solution of Compound 30-1 (300 mg, 1.309 mmol) and 30-2 (106 mg, 1.571 mmol) in DMF (3 mL) at room temperature was added DIEA (1.14 mL, 6.545 mmol) and HATU (597 mg, 1.571 mmol), and the reaction mixture was stirred at room temperature for 2 h. After quenching with water, the mixture was extracted with ethyl acetate (3 × 10 mL). After concentration, the crude product was separated by silica gel column chromatography (dichloromethane∶methanol = 10∶1) to give a brown compound 30-3 (180 mg, 56.77%). ESI / MS (m / z): 243.0 [M + H] + 。 1 1H NMR (400 MHz, DMSO-d6): δ 11.84 (s, 1H), 8.39 (d, J = 4.4 Hz, 1H), 8.25 (s, 1H), 7.83 (dd, J = 8.7, 1.4 Hz, 1H), 7.43 (d, J = 8.7 Hz, 1H), 2.99 (t, J = 5.9 Hz, 2H), 2.82 (d, J = 4.5 Hz, 3H), 2.66 - 2.55 (m, 2H), 2.18 (p, J = 5.9 Hz, 2H).

[0191] At room temperature, concentrated sulfuric acid (0.3 mL, 5.629 mmol) was added to a methanol (3 mL) solution of compound 30-3 (180 mg, 0.743 mmol) and 30-4 (135 mg, 0.892 mmol). After the reaction mixture was stirred at 80 °C overnight, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate. After concentration, the crude product was separated by reversed-phase preparative column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain the yellow solid compound 30 (11.3 mg, 4.04%). ESI / MS (m / z): 377.1 [M+H] + 。 1 1H NMR (400 MHz, DMSO-d6): δ 11.96 (s, 1H), 9.47 (s, 1H), 8.39 (d, J = 4.6 Hz, 1H), 8.25 (s, 1H), 7.83 (d, J = 10.4 Hz, 1H), 7.59 (s, 1H), 7.42 (s, 1H), 7.13 (s, 1H), 7.05 (d, J = 10.1 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.83 (s, 3H), 3.27 (t, J = 6.4 Hz, 2H), 3.07 (t, J = 6.3 Hz, 2H), 2.80 (d, J = 4.5 Hz, 3H).

[0192] Example 28: Synthesis of Compound 31

[0193]

[0194] At room temperature, concentrated hydrochloric acid (5 mL) was added to an aqueous (100 mL) solution of compound 31-1 (1.2 g, 5.945 mmol) and 31-2 (1 g, 8.918 mmol). The reaction mixture was refluxed at 120 °C overnight. After cooling to room temperature, the resulting solid was collected by filtration, washed with ice water, and purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to obtain the yellow solid compound 31-3 (120 mg, 8.23%). ESI-MS (m / z): 245.95 [M+H] + 。

[0195] At room temperature, concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a methanol (5 mL) solution of compound 31-3 (120 mg, 0.489 mmol) and 31-4 (89 mg, 0.587 mmol). The reaction mixture was stirred at 65 °C overnight, and the pH was adjusted to 8 with saturated aqueous sodium carbonate. After concentration, the crude product was separated by reversed-phase preparative column to obtain the yellow solid compound 31 (74.0 mg, 38.31%). ESI / MS (m / z): 380.0 [M+H] + 。 11H NMR (400 MHz, DMSO-d6): δ 11.49 (s, 1H), 9.42 (s, 1H), 7.54 (s, 1H), 7.10 (s, 2H), 7.01 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.85 (s, 1H), 3.82 (s, 6H), 3.79 (s, 3H), 3.19 (t, J = 6.4 Hz, 2H), 2.98 (t, J = 6.3 Hz, 2H).

[0196] Example 29: Synthesis of Compound 32

[0197]

[0198] At room temperature, concentrated hydrochloric acid (0.5 mL, 16.456 mmol) was added to a solution of Compound 32-1 (300 mg, 1.695 mmol) and 32-2 (228 mg, 2.034 mmol) in water (20 mL). The reaction mixture was stirred at 120 °C overnight. The resulting solid was collected and washed with water (30 mL) to obtain brown solid Compound 32-3 (170 mg, 39.48%). ESI / MS (m / z): 255.1 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6): δ 12.17 (s, 1H), 7.79 (s, 1H), 7.47 (s, 1H), 2.94 (d, J = 11.7 Hz, 2H), 2.64 - 2.54 (m, 2H), 2.15 (p, J = 5.9 Hz, 2H).

[0199] At room temperature, concentrated sulfuric acid (0.3 mL, 5.629 mmol) was added to a solution of Compound 32-3 (170 mg, 0.669 mmol) and 32-4 (122 mg, 0.803 mmol) in methanol (3 mL). The reaction mixture was stirred at 65 °C overnight and then the pH was adjusted to 8 using saturated aqueous sodium bicarbonate. After concentration, the crude product was separated by a reverse-phase preparative column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain yellow solid Compound 32 (27 mg, 10.40%). ESI / MS (m / z): 388.1 [M+H] + . 11H NMR (400 MHz, DMSO-d6): δ 12.30 (s, 1H), 9.51 (s, 1H), 7.81 (s, 1H), 7.62 (s, 1H), 7.50 (d, J = 1.6 Hz, 1H), 7.13 (s, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 8.1 Hz, 1H), 3.83 (s, 3H), 3.24 (t, J = 5.8 Hz, 2H), 3.02 (t, J = 6.2 Hz, 2H).

[0200] Example 30: Synthesis of Compound 33

[0201]

[0202] At room temperature, concentrated hydrochloric acid (2.40 mL, 78.989 mmol) was added to a solution of Compound 33-1 (1.2 g, 6.833 mmol) and 33-2 (0.92 g, 8.200 mmol) in water (100 mL). The reaction mixture was stirred at 120 °C overnight. After concentration, the crude product was purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to obtain yellow solid Compound 33-3 (400 mg, 27.07%). ESI / MS (m / z): 217.1 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H), 7.73 (d, J = 8.9 Hz, 1H), 6.79 (d, J = 8.9 Hz, 1H), 3.91 (s, 3H), 2.94 (t, J = 6.0 Hz, 2H), 2.63 - 2.54 (m, 2H), 2.14 (p, J = 6.2 Hz, 2H).

[0203] At room temperature, concentrated sulfuric acid (0.40 mL, 7.511 mmol) was added to a solution of Compound 33-3 (400 mg, 1.850 mmol) and 33-4 (309 mg, 2.035 mmol) in methanol (5 mL). The reaction mixture was stirred at 65 °C overnight and then the pH was adjusted to 8 with saturated aqueous sodium bicarbonate. After concentration, the crude product was separated by preparative reverse-phase column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain yellow solid Compound 33 (110 mg, 16.43%). ESI / MS (m / z): 351.0 [M+H] + . 11H NMR (400 MHz, DMSO-d6): δ 11.87 (s, 1H), 9.48 (s, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.61 (s, 1H), 7.13 (s, 1H), 7.03 (s, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 8.9 Hz, 1H), 3.91 (s, 3H), 3.84 (s, 3H), 3.23 (t, J = 5.7 Hz, 2H), 3.01 (t, J = 6.2 Hz, 2H).

[0204] Example 31: Synthesis of Compound 34

[0205]

[0206] Sodium ethoxide (1 g, 15.848 mmol) was added to a solution of compound 34-1 (1 g, 7.924 mmol) and ethyl formate (880 mg, 11.886 mmol) in 1,4-dioxane (10 mL) at 0 °C. After the reaction mixture was stirred at room temperature for 1 hour, the pH was adjusted to 2 with 2 M hydrochloric acid, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain a brown oily liquid 34-2 (600 mg, 49.10%). ESI / MS (m / z): 153.2 [M-H] - .

[0207] A solution of sodium nitrite (643 mg, 9.332 mmol) in water (10 mL) was added to a solution of p-toluidine (500 mg, 4.666 mmol) and concentrated hydrochloric acid (3.7 mL, 123.416 mmol) in water (20 mL) at 0 °C. A mixed solution of compound 34-2 (719 mg, 4.666 mmol) and sodium acetate (842 mg, 10.265 mmol) in methanol and water (40 mL, 1:1) was further added to this reaction mixture at 0 °C. The reaction mixture was continuously stirred at room temperature for 2 hours. The resulting solid was collected and washed with water (30 mL) to obtain a yellow solid compound 34-3 (255 mg, 22.37%). ESI-MS (m / z): 245.0 [M+H] + .

[0208] At room temperature, concentrated sulfuric acid MeOH (0.1 mL) was added to a methanol (1 mL) solution of compound 34-3 (250 mg, 1.023 mmol) and acetic acid (0.1 mL). The reaction mixture was stirred at 65 °C for 24 hours, and then the pH was adjusted to 8 using saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate (2 × 10 mL), and after concentration, the crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 3∶1) to obtain yellow solid compound 34-4 (88 mg, 37.84%). ESI / MS (m / z): 228.3 [M+H] + 。

[0209] At room temperature, concentrated sulfuric acid (0.2 mL, 3.752 mmol) was added to a mixed methanol (2 mL) solution of compound 34-4 (80 mg, 0.352 mmol) and 34-5 (64 mg, 0.422 mmol). The reaction mixture was stirred at 65 °C overnight, and then the pH was adjusted to 8 using saturated aqueous sodium bicarbonate. After concentration, the crude product was separated by a reverse-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain yellow solid compound 34 (28 mg, 22.01%). ESI / MS (m / z): 362.1 [M+H] + 。 1 H NMR (400 MHz, DMSO-d6): δ 11.60 (s, 1H), 9.47 (s, 1H), 7.66 (s, 1H), 7.61 (s, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.16 - 7.11 (m, 1H), 7.11 (d, J = 1.6 Hz, 1H), 7.04 (dd, J = 8.2, 1.5 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.82 (s, 3H), 3.07 (s, 2H), 2.39 (s, 3H), 1.47 (s, 6H).

[0210] Example 32: Synthesis of compound 35

[0211]

[0212] At 0 °C, sodium hydride (1.71 g, 71.328 mmol) was added to a toluene (23 mL) solution of compound 35-1 (2 g, 23.776 mmol) and ethyl formate (6.28 g, 71.328 mmol). The reaction mixture was stirred at room temperature for 2 hours, and then the pH was adjusted to 2 using 2 M hydrochloric acid. The mixture was extracted with ethyl acetate (3 × 50 mL), and after concentration, a colorless oily liquid 35-2 (1.5 g, 56.26%) was obtained. ESI / MS (m / z): 113.1 [M+H] + 。

[0213] Aqueous solution (20 mL) of p-toluidine (477 mg, 4.459 mmol) and concentrated hydrochloric acid (1 mL) was added with aqueous solution (10 mL) of sodium nitrite (615 mg, 8.918 mmol) at 0 °C. A mixed solution of compound 35-2 (500 mg, 4.459 mmol) and sodium acetate (804 mg, 9.810 mmol) in methanol and water (40 mL, 1:1) was added to the above mixture at 0 °C. The reaction solution was stirred at 0 °C for 1 hour. The resulting solid was collected and washed with water (3 × 50 mL) to obtain yellow solid compound 35-3 (800 mg, 88.70%). ESI / MS (m / z): 203.0 [M+H] + 。

[0214] Concentrated sulfuric acid (0.4 mL, 7.504 mmol) was added to a solution of compound 35-3 (400 mg, 1.978 mmol) in acetonitrile (10 mL) at room temperature. The reaction solution was stirred at 65 °C for 2 hours and then the pH was adjusted to 7 using saturated aqueous sodium bicarbonate solution. The mixture was extracted with ethyl acetate (3 × 30 mL), and the crude product after concentration was separated by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain yellow solid compound 35-4 (150 mg, 40.95%). ESI / MS (m / z): 186.2 [M+H] + 。

[0215] Concentrated sulfuric acid (0.4 mL, 7.504 mmol) was added to a mixed solution of compound 35-4 (150 mg, 0.810 mmol) and 35-5 (147 mg, 0.972 mmol) in methanol at room temperature. The reaction solution was stirred at 65 °C overnight and then the pH was adjusted to 8 using saturated aqueous sodium bicarbonate solution. The crude product after concentration was separated by reversed-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain yellow solid compound 35 (22 mg, 8.51%). ESI / MS (m / z): 320.0 [M+H] + 。 1 H NMR (400 MHz, DMSO-d6): δ 11.72 (s, 1H), 9.62 (s, 1H), 7.57 (s, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.32 (s, 1H), 7.30 (s, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 3.99 (s, 2H), 3.89 (s, 3H), 2.41 (s, 3H).

[0216] Example 33: Synthesis of Compound 36

[0217]

[0218] At 0 °C, sodium methoxide (0.47 g, 8.607 mmol) was added to a mixture of compound 36-1 (1 g, 8.607 mmol) and ethyl formate (0.64 g, 8.607 mmol) in dioxane (10 mL). After the reaction mixture was stirred at room temperature for 2 h, the pH was adjusted to 7 with 2 M hydrochloric acid, and the mixture was extracted with ethyl acetate (3 × 30 mL), washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 5∶1) to obtain the light brown solid compound 36-2 (490 mg, 39.48%). ESI / MS (m / z): 145.0 [M+H] + 。

[0219] At 0 °C, a solution of sodium nitrite (431 mg, 6.242 mmol) in water (10 mL) was added to a solution of p-toluidine (334 mg, 3.121 mmol) and concentrated hydrochloric acid (1.5 mL) in water (10 mL). Compound 36-2 (450 mg, 3.121 mmol) and sodium acetate (563 mg, 6.242 mmol) were added to the reaction mixture in a water and methanol solution (20 mL, 1∶1). The reaction mixture was stirred at room temperature for 2 h. The resulting solid was collected and washed with water (3 × 100 mL) to obtain the yellow solid compound 36-3 (180 mg, 24.61%). ESI / MS (m / z): 235.0 [M+H] + 。

[0220] At room temperature, concentrated sulfuric acid (0.2 mL, 3.005 mmol) was added to a solution of compound 36-3 (160 mg, 0.683 mmol) and acetic acid (0.1 mL) in methanol (1 mL). The reaction mixture was stirred at 65 °C overnight. After concentration, the reaction mixture was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 5∶1) to obtain the yellow solid compound 36-4 (100 mg, 67.40%). ESI-MS (m / z): 218.2 [M+H] + 。

[0221] At room temperature, concentrated sulfuric acid (0.1 mL, 1.876 mmol) was added to a mixture of compound 36-4 (80 mg, 0.390 mmol) and 36-5 (71 mg, 0.468 mmol) in methanol (1 mL). The reaction mixture was stirred at 65 °C overnight, and then the pH was adjusted to 8 with saturated aqueous sodium bicarbonate. After concentration, the crude product was separated by a reversed-phase preparative column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain the yellow solid compound 36 (8.1 mg, 5.91%). ESI / MS (m / z): 352.0 [M+H] + 。1 1H NMR (400 MHz, DMSO-d6): δ 11.85 (s, 1H), 7.58 (s, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.31 (s, 1H), 7.22 - 7.16 (m, 1H), 7.11 (d, J = 1.5 Hz, 1H), 7.06 - 7.01 (m, 1H), 6.89 (d, J = 8.1 Hz, 1H), 4.39 (s, 2H), 3.83 (s, 3H), 2.38 (s, 3H).

[0222] Example 34: Synthesis of Compound 37

[0223]

[0224] At 0 °C, sodium ethoxide (1.21 g, 17.830 mmol) was added to a mixed solution of compound 37-1 (1 g, 8.915 mmol) and ethyl formate (1.32 g, 17.830 mmol) in dioxane (10 mL). After stirring the reaction mixture at 0 °C for 1 hour, the pH was adjusted to 7 with 2 M hydrochloric acid. The mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 10∶1) to obtain light brown solid oily liquid 37-2 (700 mg, 56.01%). ESI / MS (m / z): 141.1 [M + H] + 。

[0225] At 0 °C, a solution of sodium nitrite (590 mg, 8.560 mmol) in water (5 mL) was added to a solution of p-toluidine (458 mg, 4.280 mmol) and concentrated hydrochloric acid (1.50 mL, 48.382 mmol) in water (20 mL). While maintaining 0 °C, a mixed solution of compound 37-2 (600 mg, 4.280 mmol) and sodium acetate (772 mg, 9.416 mmol) in methanol and water (40 mL, 1∶1) was added to this mixture. The reaction mixture was stirred at room temperature for 2 hours. The formed solid was collected, washed with water (3 × 50 mL), and separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 2∶1) to obtain brown solid compound 37-3 (300 mg, 30.43%). ESI / MS (m / z): 231.0 [M + H] + 。

[0226] At room temperature, concentrated sulfuric acid (0.5 mL, 10.944 mmol) was added to a methanol (5 mL) solution of compound 37-3 (280 mg, 1.216 mmol) and acetic acid (1 mL, 24.320 mmol). After the reaction mixture was stirred at 65 °C overnight, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate, and then extracted with ethyl acetate, washed with saturated brine, and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to obtain the brown solid compound 37-4 (135 mg, 52.06%). ESI-MS (m / z): 214.2 [M+H] + 。 1 1H NMR (400 MHz, DMSO-d6): δ 11.19 (s, 1H), 7.43 (s, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.14 - 7.05 (m, 1H), 3.12 - 2.96 (m, 2H), 2.79 - 2.68 (m, 2H), 2.37 (s, 3H), 1.96 (p, J = 6.1 Hz, 2H), 1.86 (p, J = 5.7, 5.0 Hz, 2H).

[0227] At room temperature, concentrated sulfuric acid (0.3 mL, 5.628 mmol) was added to a methanol (3 mL) mixture of compound 37-4 (100 mg, 0.469 mmol) and 37-5 (85 mg, 0.563 mmol). After the reaction mixture was stirred at 65 °C overnight, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate, and then concentrated. The crude product was separated by a reverse-phase preparative column (mobile phase A: water (10 mmol / L ammonium bicarbonate + 0.1% ammonia water), mobile phase B: acetonitrile) to obtain the yellow solid compound 37 (32.6 mg, 19.81%). ESI / MS (m / z): 348.1 [M+H] + 。 1 1H NMR (400 MHz, DMSO-d6): δ 11.27 (s, 1H), 9.38 (s, 1H), 7.51 (s, 1H), 7.44 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 8.4, 1.2 Hz, 1H), 7.07 (d, J = 1.7 Hz, 1H), 6.98 (dd, J = 8.2, 1.6 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.83 (s, 3H), 3.11 (t, J = 6.3 Hz, 2H), 2.90 - 2.81 (m, 2H), 2.39 (s, 3H), 2.12 (dt, J = 11.9, 6.2 Hz, 2H).

[0228] Example 35: Synthesis of Compound 38

[0229]

[0230] At room temperature, potassium hydroxide (89.8 mg, 1.600 mmol) was added to a mixture of compound 38-1 (78.5 mg, 0.400 mmol) and KZT (159.4 mg, 0.800 mmol) in 1,4-dioxane (3 mL). The reaction was heated to 100 °C and stirred for 2 hours. The reaction mixture was cooled to room temperature, quenched with water, and the resulting mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate∶petroleum ether = 1∶5) to give yellow solid 38 (21.8 mg, 14.44%). ESI / MS (m / z) 377.9 [M+H] + 。 1 H NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H), 7.57 (s, 1H), 7.45 (s, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.17 (dd, J = 8.4 Hz, J = 1.3 Hz, 1H), 6.83 (s, 2H), 3.82 (s, 6H), 3.71 (s, 3H), 3.23 (t, J = 4.8 Hz, 2H), 3.01 (t, J = 6.2 Hz, 2H), 2.38 (s, 3H).

[0231] Example 36: Synthesis of Compound 39

[0232]

[0233] At room temperature, potassium hydroxide (168.6 mg, 3.011 mmol) was added to a mixture of KZT (200.0 mg, 1.004 mmol) and compound 39-1 (149.8 mg, 1.004 mmol) in 1,4-dioxane (4 mL). The reaction was heated to 100 °C and reacted for 6 hours. The reaction was quenched with water, and the resulting mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (methanol∶dichloromethane = 1∶20) to give orange solid compound 39 (20.87 mg, 6.29%). ESI / MS (m / z) 330.9 [M+H] + 。 1HNMR (400 MHz, CDCl3) δ 8.77 (s, 1H), 7.78 (s, 1H), 7.49 - 7.43 (m, 3H), 7.34 (d, J = 8.5 Hz, 1H), 7.22 (dd, J = 8.5, 1.4 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 3.34 (t, J = 6.4 Hz, 2H), 3.09 - 3.03 (m, 8H), 2.48 (s, 3H).

[0234] Example 37: Synthesis of Compound 40

[0235]

[0236] Under room temperature conditions, potassium hydroxide (168.6 mg, 3.011 mmol) was added to a mixture of KZT (200.0 mg, 1.004 mmol) and 40-1 (175.9 mg, 1.004 mmol) in 1,4-dioxane (4 mL). The reaction was heated to 100 °C and reacted for 6 hours. The reaction was quenched by adding water, and the resulting mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (methanol∶dichloromethane = 1∶20) to obtain the orange solid compound 40 (30.79 mg, 8.60%). ESI / MS (m / z) 357.0 [M + H] + . 1 HNMR (400 MHz, CDCl3) δ 8.88 (s, 1H), 7.79 (s, 1H), 7.47 - 7.45 (m, 3H), 7.34 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.4, 1.4 Hz, 1H), 6.62 (d, J = 8.8 Hz, 2H), 3.40 - 3.33 (m, 6H), 3.06 (t, J = 6.4 Hz, 2H), 2.48 (s, 3H), 2.09 - 2.02 (m, 4H).

[0237] Example 38: Synthesis of Compound 41

[0238]

[0239] Under room temperature conditions, potassium hydroxide (168.6 mg, 3.011 mmol) was added to a mixture of KZT (200.0 mg, 1.004 mmol) and 41-1 (205.1 mg, 1.004 mmol) in 1,4-dioxane (4 mL). The reaction was heated to 100 °C and reacted for 6 hours. Water was added to quench the reaction, and the resulting mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (methanol∶dichloromethane = 1∶20) to obtain a dark yellow solid compound 41 (120 mg, 31.0%). ESI / MS (m / z) 385.9 [M+H] + 。 1 H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 7.77 (s, 1H), 7.45 (d, J = 8.5 Hz, 3H), 7.35 (d, J = 8.4 Hz, 1H), 7.23 (dd, J = 8.5, 1.4 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 3.40 - 3.26 (m, 6H), 3.06 (t, J = 6.4 Hz, 2H), 2.67 - 2.54 (m, 4H), 2.48 (s, 3H), 2.39 (s, 3H).

[0240] Biological Test Example 1 Verification of the Mechanism of Action of the Compound

[0241] 1. Verification of the dual-target inhibition mechanism of the compound:

[0242] (1) Inhibitory effect of the compound on OCT4 protein in cancer stem cells (CSC)

[0243] Test compounds: KZT, EKZT, KZT-A1 (Compound 10), KZT-A3 (Compound 11), KZT-A5 (Compound 5), KZT-A6 (Compound 2), KZT-A7 (Compound 12)

[0244] Table 1: Test Compounds Inhibiting OCT4 Protein and Their Structural Formulas

[0245]

[0246]

[0247] 1. Test procedure

[0248] 1) Cell source: The HeLa cell line 3A11 with endogenous high expression of OCT4 was constructed by the inventor's laboratory (Zhou Y et al. Endogenous authentic OCT4A proteins directly regulate FOS / AP-1 transcription in somatic cancer cells. Cell Death Dis. 2018, 9: 585).

[0249] 2) Cell culture and subculture: The above cells were cultured adherently in 6 cm culture dishes (#430166, Corning) or T75 culture flasks (#3276, Corning) containing complete medium [DMEM high-glucose basal medium (#SH30243.01B, HyClone) supplemented with 10% FBS (#1101-500, Shanghai Pufei) and Penicillin-Streptomycin double antibody (#SV30010, HyClone)]. The culture dishes (flasks) were placed in a cell culture incubator (#3111, ThermoFisher Scientific) at 37°C, 5% CO2, and saturated humidity. For subculture, first aspirate the medium, wash twice with PBS phosphate buffer solution (#GNM-10944, Hangzhou Jinuo), then add an appropriate amount of 0.25% trypsin-0.02% EDTA (#25200-072, Gibco), shake the culture dish (flask) to evenly cover the cells, and observe under a phase contrast microscope. When most cells retract and become round and detach easily with gentle shaking, quickly add complete medium twice the volume of trypsin to terminate, and gently pipette the cells into single cells. Transfer the cell suspension to a centrifuge tube of appropriate size, centrifuge at 800 rpm for 5 min. Discard the supernatant, resuspend the cell pellet with fresh complete medium, pipette into single cells again, and subculture and inoculate into new culture dishes (flasks) at a ratio of 1:3 - 1:6 and supplement with complete medium. Place in a cell culture incubator at 37°C, 5% CO2 for culture.

[0250] 3) Cell drug treatment: Digest and count the above HeLa cells, and inoculate each well of a 96-well cell culture plate (#3988, Corning) at a density of 5000 cells / 200 μl culture medium, and place in a cell culture incubator at 37°C, 5% CO2 for 24 h to allow the cells to adhere well. Then replace the original culture medium with complete culture medium containing various test compounds at gradient dilutions (such as 100 μM, 10 μM, etc.) (set 3 replicate wells for each concentration of each compound) and DMSO (#D5879, Sigma-Aldrich) solvent control, and continue to culture for 48 - 72 h.

[0251] 4) Detection sample preparation: Collect HeLa cells after being treated with the above compound for 48 - 72 h. After centrifugation, aspirate the supernatant culture medium, wash twice with ice-cold PBS, and dry the liquid. Add 200 μl of cell lysis buffer (#P0013, Beyotime), shake vigorously for 30 s, let stand on ice for 5 min, and repeat 3 times. Centrifuge the cell lysate sample at 13000 rpm at 4 °C for 6 min, take the supernatant and mix it with 4×Laemmli loading buffer (#161 - 0747, Bio-Rad) in a volume ratio of 3:1 to form a cell lysate protein sample, and denature it in a metal bath at 100 °C for 6 min. The sample is used for the following Western blot detection.

[0252] 5) Detection of OCT4 protein biomarker: Install a 4 - 15% precast gradient gel (456 - 8084, Bio-Rad) into the electrophoresis tank, and add sufficient 1×SDS-PAGE electrophoresis buffer. Use a 20 μl pipette to add 20 μl of the above cell lysate protein sample. Cover the lid of the electrophoresis tank and turn on the power supply. First, electrophorese at 80 V for about 30 min. When the bromophenol blue in the sample forms a thin line at the boundary between the stacking gel and the separating gel, adjust the voltage to 120 V, and adjust the electrophoresis duration according to the sizes of the target protein and the internal reference protein bands. Pour the pre-cooled 1×transfer buffer into a container of appropriate size, and assemble a "sandwich" structure of foam pad - filter paper - gel - PVDF membrane - filter paper - foam pad according to the instructions, and place it in the electrophoresis tank. Add ice cubes and ice-bath the entire electrophoresis tank, connect the power supply for transfer, 250 mA, 2 h. Place the PVDF membrane (IPVH00010, Millipore) in 5% non-fat milk prepared with 1×TBST and block it at room temperature for 1 h. Then, incubate with different primary antibodies (anti-OCT4A, #2890S, CST; anti-GAPDH[HRP], #A00191 - 40, GenScript) and secondary antibodies (anti-Mouse IgG HRP-linked antibody, #7076, CST; anti-Rabbit IgG HRP-linked antibody, #7074, CST) in sequence (incubate at room temperature for 1 h each) and wash the membrane (wash the membrane 3 times with 1×TBST, 5 min each time). Finally, place the PVDF membrane in the middle of the plastic film, add ECL and react on the membrane for 3 min, cover with another layer of plastic film, and expose it in a full-automatic chemiluminescence / fluorescence image analysis system (5200-Multi, Tianneng).

[0253] 2. Test results

[0254] Figure 2 It is the inhibitory effect of the test compound on OCT4 protein in HeLa cells. As Figure 2As shown, each test compound had inhibitory effects on OCT4 protein to varying degrees, among which KZT-A5 had the most obvious effect on downregulating OCT4 protein. Moreover, compared with the administration concentration of KZT-A5 at 10 μM, the inhibitory effect of KZT-A5 at the administration concentration of 100 μM on OCT4 protein was more obvious, indicating that KZT-A5 could inhibit the expression of OCT4 protein in HeLa cells in a dose-dependent manner.

[0255] (2) Inhibitory effect of the compound on the transcription of the OCT4 target gene NANOG in cancer stem cells (CSCs)

[0256] Test compounds: KZT-A5 (Compound 5), AH057 (the structural formula of AH057 is shown in Formula A below, and this compound will be referred to as AH057 hereinafter)

[0257]

[0258] 1. Experimental procedures

[0259] 1) Cell source: The HeLa cell line 3A11 with endogenous high expression of OCT4 was prepared by the inventors' laboratory.

[0260] 2) Cell culture and subculture: The methods were the same as those in "Inhibitory effect of the compound on OCT4 protein in cancer stem cells (CSCs)" above.

[0261] 3) Cell drug treatment: The methods were the same as those in "Inhibitory effect of the compound on OCT4 protein in cancer stem cells (CSCs)" above. The cervical cancer cells HeLa-3A11 were treated with DMSO (NC, blank control), 20 μM AH057, and 20 μM KZT-A5 for 3, 6, and 12 hours respectively. After extracting RNA from the cell samples, the mRNA levels of the indicated genes were detected by fluorescence quantitative PCR (qRT-PCR).

[0262] 2. Experimental results

[0263] Figure 3 The inhibitory effects of KZT-A5 and the control compound (AH057) on the transcription of the OCT4 target gene NANOG in HeLa cells are shown, reflecting the inhibitory effects on OCT4 protein. As Figure 3As shown, after treatment with 20 μM KZT-A5 for different times, the mRNA levels of OCT4 and NANOG were downregulated to varying degrees. This result suggests that KZT-A5 can inhibit the binding of OCT4 protein to the promoter regions of target genes such as NANOG by competitively disrupting the formation of the OCT4 / SOX2 / DNA complex, thereby inhibiting its transcription. The effect of KZT-A5 on inhibiting the transcription of the NANOG gene is basically equivalent to that of AH057, suggesting that KZT-A5 may have the same action target and mechanism as AH057, that is, by competitively disrupting the formation of the OCT4 / SOX2 / DNA complex, inhibiting the binding of OCT4 protein to target genes, and then promoting the differentiation of PSC and CSC. It should be noted that after 6 hours of treatment, the downregulation effect of the mRNA level of NANOG was the most obvious, and when the cells were treated for 6 hours and 12 hours, the ability of KZT-A5 to downregulate the mRNA level of NANOG was better than that of AH057.

[0264] (3) Inhibitory effect of the compound on the JAK / STAT signaling pathway

[0265] Test compounds: KZT-A5 (compound 5), AH057

[0266] 1. Experimental procedure

[0267] 1) Cell source: The HeLa cell line 3A11 with endogenous high expression of OCT4 was prepared by the inventors' laboratory.

[0268] 2) Cell culture and passage: The cell culture and passage methods were the same as those in the above-mentioned "Inhibitory effect of the compound on OCT4 protein in cancer stem cells (CSC)".

[0269] 3) Cell treatment with the drug: Digest and count the above-mentioned HeLa cells, and inoculate each well of a 96-well cell culture plate (#3988, Corning) at a density of 5000 cells / 200 μl of culture medium. Place it in a cell culture incubator at 37 °C and 5% CO2 for 24 h to allow the cells to adhere fully. Then replace the original culture medium with complete culture medium containing 1 μM, 10 μM, 100 μM of KZT compound and AH057 (3 replicate wells are set for each concentration of each compound) and DMSO (#D5879, Sigma-Aldrich) solvent control, and continue to culture for 72 h.

[0270] 4) Preparation of test samples: The preparation method of test samples was the same as that in the above-mentioned "Inhibitory effect of the compound on OCT4 protein in cancer stem cells (CSC)".

[0271] 5) Detection of JAK / STAT signaling pathway biomarkers: HeLa cells have been reported to continuously activate JAK1 / 2 through the IL-6 autocrine pathway and further activate STAT3. The method for detecting signaling pathway biomarkers is the same as that in the above "Inhibitory effect of the compound on OCT4 protein in cancer stem cells (CSCs)". The antibodies are: primary antibodies (anti-OCT4A, #2890S, CST; anti-AKT (Pan), #4821, CST; anti-pAKT-S473, #4060, CST; anti-STAT3, #4904, CST; anti-pSTAT3-Y705, #9145, CST; anti-GAPDH[HRP], #A00191-40, GenScript) and secondary antibodies (anti-Mouse IgG HRP-linked antibody, #7076, CST; anti-Rabbit IgG HRP-linked antibody, #7074, CST).

[0272] 2. Test results

[0273] Figure 4 It is the inhibitory effect of KZT-A5 and the control compound (AH057) on the JAK / STAT signaling pathway biomarkers in HeLa cells, and also reflects the inhibitory effect on OCT4 protein. As Figure 4 shown, the activity of STAT3 is inhibited, indicating that KZT-A5 can effectively inhibit the activation of the JAK1 / 2-STAT3 signaling pathway in a dose-dependent manner. By inhibiting the kinase activity of JAK1 / 2, the activation of STAT3 is inhibited, and further the transcription of its target gene OCT4 is inhibited. Since STAT3 can partially mediate the transcription of stemness genes such as OCT4 (Kim SY et al. Role of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells. Cell Signal. 2013, 25: 961-9), KZT-A5 can synergistically block the transcription of stemness genes such as OCT4 and NANOG through different pathways, promote the differentiation of cancer stem cells and inhibit their proliferation.

[0274] The results of the inhibitory effects of the above compounds on OCT4 protein in HeLa cells and the JAK1 / 2-STAT3 signaling pathway indicate that the KZT series of compounds can target both OCT4 protein and JAK1 / 2. On the one hand, it inhibits the transcription of OCT4 and NANOG stemness factors by blocking the formation of the OCT4 / SOX2 / DNA complex. On the other hand, it inhibits the transcriptional activity of STAT3 downstream by inhibiting the kinase activity of JAK1 / 2. It is a candidate drug with a dual-target mechanism that simultaneously inhibits at two links: OCT4 self-transcription and OCT4 transcriptional activity. Such a dual-target inhibition mechanism can simultaneously target and inhibit, and eliminate CSCs and differentiated tumor cells, providing the possibility to completely block the bidirectional transformation between CSCs and differentiated tumor cells, and has good potential application prospects in eradicating tumors.

[0275] Biological Test Example 2 In vitro Pharmacodynamic Test of Compounds

[0276] (1) Inhibitory Effect of Compounds on the Formation of Tumor Spheroids by Tumor Cells

[0277] Test Compounds: KZT-A5 (Compound 5), AH057

[0278] 1. Test Procedure

[0279] 1) Cell Source: Cervical cancer cells HeLa and Caski, liver cancer cells Hep3B and Huh7, all purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0280] 2) Tumor Spheroid Culture: For the specific culture method of tumor spheroids, refer to the literature (Cheng J et al. Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells. Nat Commun. 2015, 6: 7209.). Each type of tumor cell was cultured in a medium supplemented with 2% B27, 10 ng / ml BFGF, 5 ng / ml EGF, and 10 ng / ml LIF to form tumor spheroids rich in CSCs.

[0281] 3) Cell Drug Treatment: The HeLa cell line and Huh7 cell line were seeded in a 6-well plate with the above medium at a density of 200 cells per well, and the Hep3B cell line and Caski cell line were seeded in a 6-well plate with the above medium at a density of 2000 cells per well. Then, KZT-A5 and AH057 with concentrations of 1 μM and 10 μM were added. After 48 hours of treatment, the morphology of the tumor spheroids was observed and photographed.

[0282] 2. Test Results

[0283] Figure 5 Cell morphological characterization after 48-hour treatment of tumor microspheres with KZT-A5 and the control compound (AH057). As Figure 5 shown, the tumor microspheres treated with KZT-A5 were scattered, and the KZT-A5 administration concentration of 10 μM had a better effect on inhibiting the formation of tumor microspheres than the KZT-A5 administration concentration of 1 μM, indicating that KZT-A5 can inhibit the formation of tumor microspheres in a dose-dependent manner, confirming its ability to promote CSC differentiation and / or inhibit CSC proliferation. According to the experimental results of the tumor microsphere system, KZT-A5 can promote the differentiation and apoptosis of CSCs and tumor cells, and inhibit the proliferation, invasion and migration of tumor cells.

[0284] By comparing the effects of AH057 and KZT-A5 on tumor microspheres cultured from Huh7 cells, it can be seen that 1 μM of KZT-A5 had a more obvious inhibitory effect than AH057 at the same concentration, indicating that KZT-A5 has the potential to have better efficacy than AH057 in inhibiting the proliferation and differentiation of hepatocellular carcinoma cells.

[0285] By comparing the effects of AH057 and KZT-A5 on tumor microspheres cultured from HeLa and Caski cells, it can be seen that when the concentration of KZT-A5 reached 10 μM, its inhibitory effect on tumor microspheres cultured from cervical cancer cells was more obvious than that of the control compound AH057.

[0286] (2) Inhibitory effects of the compound on human tumor cell lines from different tissue sources (I)

[0287] Test compound: KZT-A5 (Compound 5)

[0288] 1. Test procedures

[0289] 1) Cell sources: HeLa (human cervical cancer cells), CaSki (human cervical cancer intestinal metastasis cells), Huh7 (human hepatocellular carcinoma cells), HepG2 (human hepatocellular carcinoma cells), Hep3B (human hepatocellular carcinoma cells) were all purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai); LO2 (human normal hepatocytes), HcerEpic (human normal uterine epithelial cells) were from ATCC.

[0290] 2) Cell culture and passage: The cell culture and passage methods were the same as those in the above "Inhibitory effects of the compound on OCT4 protein in cancer stem cells (CSCs)".

[0291] 3) Cell drug treatment: Digest and count each type of the above cells, and inoculate them into each well of a 96-well cell culture plate (#3988, Corning) at a density of 5000 cells / 200 μl culture medium. Place the plate in a cell culture incubator at 37 °C with 5% CO2 for 24 h to allow the cells to adhere well. Then replace the original culture medium with complete culture medium containing gradient-diluted KZT-A5 (set 3 replicate wells for each concentration) and DMSO (#D5879, Sigma-Aldrich) solvent control, and continue culturing for 72 h.

[0292] 4) Pharmacodynamic determination and statistics: First, use an inverted phase contrast microscope (X71, Olympus) to observe the morphology of the cells treated with the above compounds and take pictures for record. Then replace the original culture medium with phenol red-free culture medium containing CCK-8 detection reagent (#E606335, Sangon Biotech), and continue culturing in the incubator for 2 h. Then measure the absorbance value at OD450nm (OD value) on a multi-functional microplate reader (168-1130, Bio-Rad). After the test compound treats the cells, the calculation formulas for cell survival rate or cell growth rate are as follows: Survival rate = OD value of the drug-treated group / OD value of the control group × 100%; The calculation formula for the growth inhibition rate of the compound on cell proliferation is: Inhibition rate = (OD value of the control group - OD value of the drug-treated group) / OD value of the control group × 100%. Further calculate the IC 50 of each compound in SPSS based on the inhibition rate values. 50 The IC

[0293] 2. Test results

[0294] Table 2 Inhibitory activities of KZT-A5 on the in vitro proliferation of multiple human tumor cell lines

[0295]

[0296]

[0297] As can be seen from Table 2, the IC 50 values of KZT-A5 for the Huh7 cell line in human liver cancer cells and the HeLa cell line in human cervical cancer cells are relatively small, both lower than 1 μM, indicating that it has excellent ability to inhibit the proliferation of liver cancer cells and cervical cancer cells. In addition, compounds 8, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 31, 32, 33, 34, 35, 36, 37 and 38 on the HeLa cell line in human cervical cancer cells50 The values are all less than 50 μM.

[0298] (3) Inhibitory effect of the compound on human tumor cell lines from different tissue sources (Part II)

[0299] Test compounds: KZT-A5 (Compound 5), AH057

[0300] 1. Test procedures

[0301] 1) Cell sources: Huh7 (human liver cancer cells), HepG2 (human liver cancer cells), Hep3B (human liver cancer cells), and HeLa (human cervical cancer cells) were all purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai), and LO2 (human normal liver cells) was from ATCC.

[0302] 2) Cell culture and subculture: The methods were the same as those in "Inhibitory effect of the compound on human tumor cell lines from different tissue sources (Part I)" above.

[0303] 3) Cell treatment with the compound: The methods were the same as those in "Inhibitory effect of the compound on human tumor cell lines from different tissue sources (Part I)" above.

[0304] 4) Pharmacodynamic determination and statistics: The methods were the same as those in "Inhibitory effect of the compound on human tumor cell lines from different tissue sources (Part I)" above. The IC 50 values of the compound against each type of tumor cell were the average ± standard deviation of three replicate wells in one experiment.

[0305] 2. Test results

[0306] Figure 6 are the effects of KZT-A5 and AH057 on the activities of different tumor cells. As Figure 6 shown, compared with AH057, KZT-A5 had a more obvious inhibitory effect on the activities of Huh7 and HepG2 cells in liver cancer cells.

[0307] Table 3 In vitro antitumor activities of KZT-A5 and AH057 against human tumor cells

[0308]

[0309] As can be seen from Table 3, using AH057 as a control, the in vitro pharmacodynamic effects of KZT-A5 and AH057 on human liver cancer cells and cervical cancer cells were compared. It was found that the inhibitory effect of KZT-A5 on Hep3B and HeLa cell lines was comparable to that of AH057. Compared with AH057, KZT-A5 had lower IC 50The value is significant with a high degree of significance. This result confirms that KZT-A5 has a significantly better inhibitory effect on the proliferation of liver cancer cells than AH057, and has better in vitro drug efficacy.

[0310] Biological Test Example 3 KZT-A5 and SGI-1027 Synergistically Inhibit the In Vitro Proliferation of HeLa Cells

[0311] Test compound: KZT-A5

[0312] SGI-1027 is an inhibitor of DNA methyltransferase (DNA Methyltransferase Inhibitor II), and its CAS number is 1020149-73-8. SGI-1027 was selected as the compound to be used in combination with KZT-A5 by screening based on the Bliss independence model. The screening process was carried out according to the method in the following literature: Goldoni M, Johansson C. A mathematical approach to study combined effects of toxicants in vitro: evaluation of the Bliss independence criterion and the Loewe additivity model. Toxicol In Vitro. 2007, 21: 759-69.

[0313] 1. Experimental procedure

[0314] 1) Cell source: HeLa cells, purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0315] 2) Cell culture and subculture: The same as the cell culture and subculture methods in the above "Inhibitory Effect of Compounds on OCT4 Protein in Cancer Stem Cells (CSC)".

[0316] 3) Cell drug treatment: Inoculate at a density of 200 cells per well in a 6-well plate of the above medium. Divide into four groups and add DMSO (as the blank control group), 700 nM KZT-A5 (as the KZT-A5 single drug group), 100 nM SGI-1027 (as the SGI-1027 single drug group), and 700 nM KZT-A5 + 100 nM SGI-1027 (as the combined drug administration group). After two weeks, take pictures and count the number of clones in each group respectively.

[0317] 2. Experimental results

[0318] Figure 7The inhibitory effects of the blank control group, the KZT-A5 single-drug group, the SGI-1027 single-drug group, and the combination drug group on the proliferation of HeLa cells. Figure 7 A, Figure 7 B, and Figure 7 C respectively represent the cell proliferation conditions of three technical replicates. As Figure 7 shown, compared with the DMSO control group, the SGI-1027 single drug only slightly inhibited colony formation, the KZT-A5 single drug significantly inhibited colony formation, while the combination of the two drugs, KZT-A5 and SGI-1027, almost completely inhibited colony formation.

[0319] Figure 8 The statistical result of the average number of colonies for 3 technical replicates, which is a statistical graph of the number of cell colonies after treatment with DMSO, KZT-A5 single drug, SGI-1027 single drug, and KZT-A5+SGI-1027 combination drug. According to Figure 8 the statistical results, the number of colonies formed after the combination of KZT-A5 and SGI-1027 is much lower than that formed by the KZT-A5 single drug and the SGI-1027 single drug. This is because KZT-A5 and SGI-1027 can act jointly to inhibit the JAK / STAT / OCT4 and DNMT1 pathways simultaneously, so they have a strong effect on inhibiting tumor cell proliferation, inducing cell cycle arrest and apoptosis. Compounds such as KZT-A5 and SGI-1027 have the effect of highly synergistically inhibiting tumor cell proliferation. It can be seen that the combination scheme of KZT-A5 and the DNMT inhibitor provides a new idea for the combination therapy of simultaneously blocking multiple interacting signaling pathways / survival pathways, which can achieve the therapeutic effect with reduced dosage, thereby minimizing the toxicity and other side effects of high-dose single drugs, while reducing the possibility of drug resistance and overlapping toxicity, and has good development prospects.

[0320] The present invention explores the mechanism of action of tetrahydrocarbazole compounds at the molecular level, verifies their dual-target inhibitory effect on cancer stem cells (CSCs), and thus can simultaneously target promoting the differentiation of CSCs and killing differentiated tumor cells, providing the possibility for completely blocking the bidirectional transformation between CSCs and differentiated tumor cells, and has good potential application prospects in eradicating tumors. The tetrahydrocarbazole compounds provided by the present invention have significant inhibitory effects on tumors such as cervical cancer and liver cancer, among which the inhibitory effect on liver cancer is more significant. Compared with other control substances, the inhibitory effect on liver cancer is very prominent and unexpected, and has very good research and application prospects in the field of liver cancer treatment.

[0321] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A compound represented by formula (I), its optical isomers or its pharmaceutically acceptable salts: Among them, R2 is an unsubstituted or one or more R- c substituted phenyl; and when R2 is a benzene ring, at least one hydrogen atom on the benzene ring is substituted by a hydroxyl group; The said R c is selected from the group consisting of: hydrogen, halogen, hydroxy, carboxy, amino, nitro, cyano, sulfonyl, C 1-5 alkyl, C 2-5 alkenyl, C 2-5 alkynyl, C 1-5 alkoxy, C 1-5 haloalkyl, C 2-5 haloalkenyl, C 2-5 haloalkynyl, C 1-5 haloalkoxy, C 1-5 amino, C 2-5 ester group; And the compound does not include the compounds selected from the following group:

2. The compound according to claim 1, its optical isomer or its pharmaceutically acceptable salt, characterized in that, The Rc described above is selected from the following group: halogen, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C 1～5 alkyl, C 1～5 alkoxy, C 1～5 haloalkyl, C 1～5 haloalkoxy.

3. The compound, its optical isomer or its pharmaceutically acceptable salt according to claim 1, characterized in that, The compound is selected from the following group:

4. A compound, its optical isomer or its pharmaceutically acceptable salt, characterized in that, The compound is selected from the following group:

5. A pharmaceutical composition comprising (1) a compound, its optical isomers or its pharmaceutically acceptable salts according to any one of claims 1-4; and optionally (2) a pharmaceutically acceptable carrier, excipient or other active drug.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition further comprises a second therapeutic component, and the second therapeutic component is a DNA methyltransferase inhibitor.

7. The pharmaceutical composition according to claim 6, characterized in that, The DNA methyltransferase inhibitor is SGI-1027.

8. Use of a compound, its optical isomers or its pharmaceutically acceptable salts according to any one of claims 1-4, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for the treatment and / or prevention of cancer.

9. The use according to claim 8, characterized in that, The cancer is selected from the following group: liver cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, cervical cancer, ovarian cancer, head and neck tumors.

10. The use according to claim 8, characterized in that, The treatment and / or prevention of cancer includes: using the compound represented by formula I, its optical isomers or its pharmaceutically acceptable salts according to any one of claims 1-4, or the pharmaceutical composition according to claim 5 in combination with a DNA methyltransferase inhibitor.

11. The use according to claim 10, wherein, The DNA methyltransferase inhibitor is SGI-1027.

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