Patent Application
20250034089
The present invention relates to the field of pharmaceutical technology, specifically to a tetrahydrocarbazole compound, and pharmaceutical composition and use thereof.
Cancer stem cells (CSC) are a small subgroup of tumor cells, accounting for only a small fraction (usually 1% or less) of tumor tissue, and they possess several key characteristics similar to normal stem cells. The first characteristic is self-renewal, CSCs maintain the ability to undergo cell division continuously and differentiate into various types of tumor cells. The second characteristic is the ability to undergo asymmetric division, i.e., dividing to produce a daughter CSC with the same characteristics as the mother CSC, while a differentiated daughter cell is also produced, those daughter cells rapidly proliferates to occupy a large portion of the tumor volume, and eventually, the CSCs and their differentiated daughter cells together constitute tumor tissue with the abilities of self-renewal and rapid proliferation (Clevers H. The cancer stem cell: Premises, promises and challenges. Nat Med 011, 17:313-319). This means that the contribution of CSCs to the long-term survival of tumors is much greater than that of their daughter differentiated tumor cells.
In recent years, the transformation between adult stem cells (ASC) and CSCs has also received widespread attention. Mutations in normal ASC cause dysregulation of self-renewal and induce cell to transform to CSC. These mutated CSCs exhibit typical malignant tumor characteristics such as stronger ability of proliferation, anti-apoptosis and immune escape, which are important reasons for tumor resistance and recurrence.
CSCs have been reported to have extensive metastatic potential, and regenerate tumors and generate disseminated metastatic tumors (Wang Y J, Herlyn M. The emerging roles of OCT4 in tumor-initiating cells. Am J Physical Cell Physical 2015, 309: C709-718). Currently, almost all drugs and radiation treatments can only kill or inhibit rapidly proliferating differentiated tumor cells, but cannot effectively remove or inhibit CSCs. Since the CSCs and differentiated tumor cells can transform into each other under specific conditions, only combination therapies that simultaneously target the inhibition or killing of CSCs and differentiated tumor cells, and completely blocks the bidirectional transformation between them, may make it possible to eradicate the tumor.
So far, there have been no drugs specifically targeting CSCs on the market, and a series of candidate drugs (such as Notch, Wnt inhibitors) that can promote CSCs differentiation or inhibit their proliferation are at various stages of clinical trials (Maurort 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 exhibit resistance to conventional radiotherapy, chemotherapy, and molecular targeted therapy drugs, making specifically targeting CSCs become an important strategy for tumor treatment and anti-tumor resistance research.
The core stem transcription factor OCT4 is a member of the POU transcription factor family, and its coding gene POU5F1 has multiple transcription start sites, which can transcribe different mRNA isoforms and then translate them into various OCT4 subtype proteins. OCT4A (commonly referred to as OCT4), as a classical transcription factor, has a POU domain that can specifically recognize and bind to the octamer motifs (ATGC (A/T) AAT) of numerous target gene promoter regions. The OCT4 protein regulates the transcription of hundreds of downstream target genes through collaboration with interacting proteins such as SOX2, and these target genes can usually be divided into two categories: one is the stem factors that positively activate its transcription by OCT4/SOX2, such as NANOG genes, and the other is the specific genes which are differentiated from various germ layers and negatively inhibit its transcription by OCT4/SOX2. Therefore, OCT4/SOX2 maintains self-renewal and pluripotency of pluripotent Stem Cells (PSC) by simultaneously promoting stem gene expression and shutting down germ gene expression, 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).
The expression of OCT4 is closed in normal differentiated tissues, but it is slightly expressed 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 cancer, etc. And its expression is significantly up-regulated under the stimulation of some growth factors and hypoxia conditions, which further suggests the plasticity of OCT4 expression in these cells. The expression of OCT4 in tumor tissues and cells exhibits heterogeneity: relative to most differentiated tumor cells, OCT4 is mainly expressed in a small fraction of CSCs, and thus has gradually been recognized as a biomarker of CSCs in recent years (Wang Y J, Herlyn M. The emerging roles of OCT4 in tumor initiating cells. Am J Physical Cell Physiol 2015, 309: C709-718).
The research conducted by the applicant's laboratory has shown that knocking out the OCT4 gene with CRISPR/Cas9 in tumor cells such as hepatocellular and cervical carcinomas cell significantly inhibits the proliferation and metastasis of CSCs, promotes its differentiation and apoptosis, thus confirming that OCT4 is a potential drug target for anti-tumor cells, especially for 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 crucial role in CSCs self-renewal and survival, tumor metastasis, and tumor resistance, small molecule compounds that can specifically target and inhibit OCT4 have hardly been reported.
It is known that compounds such as retinoic acid can promote PSC differentiation through inhibiting transcription of OCT4 gene, but these compounds have multiple targets and mechanisms of action, and they are not specifically targeted to inhibit OCT4. KRIBB53, the only small molecule compound reported so far that can specifically target the OCT4 protein, downregulates the level of OCT4 protein in cells by promoting ubiquitination degradation of OCT4 protein, but the specific site and mechanism of action are still unclear. Moreover, KRIB53 has a relatively weak efficacy with an IC50 of 10-20 μM in inhibiting the proliferation of two types of PSCs, NCCIT and TERA1. (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).
In addition, due to the low average expression level of OCT4 in tumor cells, limited drug efficacy, and negative feedback compensatory mechanisms in tumor signaling pathways, it is difficult to completely inhibit the role of OCT4 in tumor cells by targeting the expression and transcription factor function of OCT4 alone. Among the regulatory pathway of OCT4, the JAK/STAT pathway not only directly regulates the transcription of OCT4 and NANOG to maintain the multifunctionality of CSC, but also becomes an important therapeutic target for differentiated tumor cells because of its high activation in various tumors and its involvement in malignant transformation of tumor cells. Currently, the molecular targeted drugs against the JAK/STAT pathway that commonly used in clinic are mainly used to treat autoimmune diseases and bone marrow fibrosis, and their efficacy in targeted treatment for tumors remains to be determined. Therefore, the development of JAK/STAT inhibitors with tumor suppressive effects has good application prospects.
In view of all or part of the shortcomings of the prior art mentioned above, the present invention provides a class of tetrahydrocarbazole compound, and pharmaceutical composition and use thereof. On the one hand, the provided tetrahydrocarbazole compounds can jointly inhibit OCT4 at the transcriptional and functional levels by means of targeting OCT4 and JAK to promote CSCs differentiation, and on the other hand, inhibit differentiated tumor cells by means of inhibiting the JAK/STAT pathway. The compounds provided in the present invention act on two drug targets of OCT4 and JAK/STAT in tumor cells, and synergistically inhibit tumor proliferation and metastasis by means of a dual-targeting effect, and can be used for preparing drug for treating and/or preventing of cancers.
The first aspect of the present invention provided is a compound as shown in formula (I), an optical isomer or a pharmaceutically acceptable salt thereof:
Preferably, the compound does not include compounds selected from the group consisting of
In some embodiments, the compound has a structure as shown in formula (II) or formula (III):
In another preferred embodiment, the compound has a structure selected from the group consisting of:
In another preferred embodiment, the compound has a structure selected from the group consisting of:
In some embodiments, R2 is a unsubstituted or one or more Rc substituted phenyl, or a unsubstituted or one or more Rc substituted 5-10 membered heteroaryl;
In another preferred embodiment, R2 is selected from the group consisting of phenyl,
In some embodiments, R2 is selected from the group consisting of hydrogen, halogen, hydroxyl, carboxyl, cyano, sulfonyl, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl, C1-4 haloalkoxy, C2-4 ester group and C1-4 carbonyl; and the alkyl, alkoxy, haloalkyl, haloalkoxy, ester group and carbonyl are independently and optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, carboxyl, nitro, cyano, sulfonyl, C1-3 alkyl and C1-3 amino.
In some embodiments, Ra is selected from the group consisting of hydrogen, halogen, hydroxyl, carboxyl, cyano, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy, C2-4 ester group, C1-4 carbonyl, 3-8-membered saturated or partially unsaturated heterocyclyl, C6-10 aryl, and 5-12 membered heteroaryl; and the alkyl, alkenyl, alkynyl, alkoxy, ester group, carbonyl, heterocyclyl, aryl and heteroaryl are independently and optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, carboxyl, amino, nitro, cyano, sulfonyl, C1-3 alkyl and C1-3 amino.
The second aspect of the present invention provided is a compound shown in formula (IA), an optical isomer, or a pharmaceutically acceptable salt thereof:
In some embodiments, in the compound shown in formula I, R1 is an oxygen atom and is connected to the carbon atom at position 1 via a carbon-oxygen double bond; the carbon atom at position 2 is connected to the carbon atom at position 3 via a carbon-carbon double bond, and comprises the compound shown in formula (IIA):
In some embodiments, in the compound shown in formula IIA, R2 is a phenyl, and at least one hydrogen atom on the phenyl is substituted by hydroxyl.
In some embodiments, the compound is selected from the group consisting of:
The second aspect of the present invention provided is a pharmaceutical composition, comprising (1) a compound according to the first aspect of the present invention, an optical isomer or a pharmaceutically acceptable salt thereof; and optional (2) pharmaceutically acceptable carriers, excipients or other active drugs.
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.
The third aspect of the present invention provided is a use of a compound according to the first aspect of the present invention, an optical isomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the second aspect of the present invention in the preparation of a drug for treating and/or preventing cancers.
In some embodiments, the cancers are selected from the group consisting of liver cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, cervical cancer, ovarian cancer, head and neck cancer.
In some embodiments, the treating and/or preventing cancers comprises: administrating the compound as shown in formula I according to any one of claims 1-9, an optical isomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, according to claim 7 in combination with a DNA methyltransferase inhibitor.
In some embodiments, the DNA methyltransferase inhibitor is SGI-1027.
It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the specific technical features described below (as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated herein.
As used herein, the term “alkyl” includes straight or branched alkyl groups. For example, C1-6 alkyl refers to a straight or branched alkyl having 1-6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert-butyl, cyclobutyl, n-amyl, isopentyl, neopentyl, and the like.
As used herein, the term “alkenyl” includes straight or branched alkenyl groups. For example, C2-6 alkenyl refers to a straight or branched alkenyl having 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, and the like.
As used herein, the term “alkynyl” includes straight or branched alkynyl groups. For example, C2-6 alkyne refers to a straight or branched alkynyl having 2-6 carbon atoms, such as ethynyl, propenyl, butenyl, and the like.
As used herein, the term “cycloalkyl” refers to a cyclic saturated aliphatic hydrocarbyl having a specific number of carbon atoms. For example, C3-C10 alkenyl refers to a cyclic saturated aliphatic hydrocarbyl having 3-10 carbon atoms. It can be a monocycle, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. “Cycloalkyl” can also be in the form of a bicyclic ring, such as a bridged ring or a spiro ring.
As used herein, the term “alkylamino” refers to the an amine group substituted by alkyl groups. For example, “C1-6 alkylamino” refers to an amino substituted by a C1-6 alkyl, which can be mono- or di-substituted; for example, methylamino, ethylamino, propylamino, iso-propylamino, butylamino, iso-butylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, di-iso-butylamino, di-tert-butylamino, and the like.
As used herein, the term “alkoxy” refers to a group having an alkyl-oxy structure. For example, “C1-8 alkoxy” refers to a straight or branched alkoxy having 1-8 carbon atoms, including methoxy, ethoxy, propoxy, iso-propoxy, butoxy, iso-butyloxy, tert-butoxy, sec-butoxy, pentoxy, neopentoxy, and the like.
As used herein, the term “haloalkyl” refers to an alkyl in which one or more hydrogen atoms are substituted by halogens, wherein the definition of alkyl is as described above.
As used herein, the term “haloalkoxy” refers to an alkoxy in which one or more hydrogen atoms are substituted by halogens, wherein the definition of alkoxy is as described above.
As used herein, the term “carbonyl” refers to a group having a specific number of carbon atoms, and an alkyl-C(═O)— or alkyl-C(═O)-alkyl- structure.
As used herein, the term “heterocyclyl “heterocyclylalkyl” refers to a saturated or partially saturated cyclic group having a specific number of ring atoms (such as 3-10 ring atoms), wherein 1-3 atoms are heteroatoms selected from N, S, and O. It can be a monocyclic, a bicyclic or a polycyclic form, such as bridged ring or spiro ring form. Specific examples can include oxetanyl, azetidinyl, tetrahydro-2H-pyranoyl, piperidyl, tetrahydrofuryl, morpholinyl, and pyrrolidinyl.
As used herein, the terms “aryl”, “aromatic ring” or “aromatic-ring” refer to a aromatic cyclic group. For example, the term “C6-C10 aryl” refers to a aromatic group with 6-10 carbon atoms, such as phenyl or naphthyl. “Aryl” can also be a fused aryl.
As used herein, the term “heteroaryl” or “heteroaromatic ring” refers to an aromatic cyclic 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 in the form of a mono- or a fused-ring. Specific examples can be furanyl, thienyl, pyrrolyl, thiazolyl, thiadiazolyl, azolyl, diazolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, 1,4-dioxacyclohexadienyl, 2H-1,2-azinyl, 4H-1,3-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, oxacycloheptatrienyl, thiacycloheptatrienyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, benzofuranyl, benzoisofuranyl, benzothienyl, indolyl, benzozolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, isoquinolinyl, pyridinopyrazolyl, pyridinopyrrolyl, pyrimidinopyrazolyl, pyrimidinopyrrolyl, pyridazinopyrazolyl, pyridazinopyrrolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenazinyl, pteridinyl, purinyl, naphthyridinyl, and the like.
As used herein, “halogen” or “halogen atom” refers to F, Cl, Br and I. Preferably, the halogen or halogen atoms are selected from F, C1, and Br. “Halogenated” refers to substituted by atoms selected from F, Cl, Br, and I.
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): such as R and S configurations with asymmetric centers, (Z), (E) isomers with double bonds, etc. Therefore, mixtures of individual stereochemical isomers or their enantiomers, diastereomers, or geometric isomers (or conformational isomers) of the compounds of the present invention all fall within the scope of the present invention.
As used herein, the term “tautomer” refers to structural isomers having different energies that can exceed lower energy barriers and thus transform into each other. For example, proton tautomer (i.e. proton transfer) includes interconversion through proton transfer, such as 1H-indole and 2H-indole. The covalent tautomer include interconversion through the recombination of some bonding electrons.
As used herein, the term “solvate” refers to a complex formed by coordinating a compound of the invention with a solvent molecule in specific proportion.
As used herein, the term “hydrate” refers to a complex formed by coordinating a compound of the invention with water.
After extensive and intensive research, the inventor unexpectedly discovered a class of tetrahydrocarbazole compound and conducted a series of biological activity tests on it. It was found that it can jointly inhibit OCT4 at the transcriptional and functional levels by targeting OCT4 and JAK to promote CSC differentiation, and on the other hand, it can inhibit differentiated tumor cells by inhibiting the JAK/STAT pathway. Based on this, the present invention has been completed.
The main advantages of the present invention are:
The present invention will be further illustrated below in conjunction with examples. Those skilled in the art will understand that the following examples are only intended to illustrate the present invention and should not be regarded as limiting the scope of the present invention. The specific technology or conditions with no specific conditions described in the examples are performed under the technology or conditions described in the literature in this field, or according to the product manual. The reagents or instruments used with no specific manufacturer are conventional products that can be commercially available.
KZT (200 mg, 1.0 mmol), 1-1 (1.5 mmol), concentrated hydrochloric acid (10 mL) and methanol (5 mL) were added to a 25 mL single neck bottle in sequence, and they were refluxed for 10 hours until KZT disappears. The reaction solution was cooled down to room temperature and poured into crushed ice, the 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 yellow green solid compound 1, yield 53.6%, m.p.=191-193° C. 1H 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); 13C 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 C22H2ONO3− (M−H)− 346.1449, found 346.1455.
KZT (200 mg, 1.0 mmol), 2-1 (1.5 mmol), concentrated hydrochloric acid (10 mL) and methanol (5 mL) were added to a 25 mL single neck bottle in sequence, and they were refluxed for 10 hours until KZT disappears. The reaction solution was cooled down to room temperature and poured into crushed ice. The 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 gel 2, yield 43.7%. 1H 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)13C 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 C22H20NO4− (M−H)− 362.1398, found 362.1405.
KZT (200 mg, 1.0 mmol), 3-1 (1.5 mmol), concentrated hydrochloric acid (10 mL) and methanol (5 mL) were added to a 25 mL single neck bottle in sequence, and they were refluxed for 10 hours until KZT disappears. The reaction solution was cooled down to room temperature and poured into crushed ice, the 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%. 1H 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) 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 C20H16NO3− (M−H)− 318.1136, found 3118.1140.
KZT (200 mg, 1.0 mmol), 4-1 (1.5 mmol), concentrated hydrochloric acid (10 mL) and methanol (5 mL) were added to a 25 mL single neck bottle in sequence, and they were refluxed for 10 hours until KZT disappears. The reaction solution was cooled down to room temperature and poured into crushed ice, the solid was precipitate, 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 solid 4, yield 52.7%, m.p.=195-197° C. 1HNMR (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). 13C 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 C22H20NO2− (M−H)− 330.1500, found 330.1505.
KZT (200 mg, 1.0 mmol), 5-1 (1.5 mmol), concentrated hydrochloric acid (10 mL) and methanol (5 mL) were added to a 25 mL single neck bottle in sequence, and they were refluxed for 10 hours until KZT disappears. The reaction solution was cooled down to room temperature and poured into crushed ice, the 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 yellow solid 5, yield 45.7%, m.p.=169-171° C. 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). 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 C21H18NO3− (M−H)− 332.1292, found 332.1296.
Palladium hydroxide 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) at room temperature under a hydrogen atmosphere. The reaction was stirred overnight at room temperature, and then filtered, the filter cake was washed with ethyl acetate (2×10 mL). After the filtrate was concentrated, it was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a white solid compound 8 (54.8 mg, 17.85%). ESI/MS (m/z): 366.1 [M+H]+. 1HNMR (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), 2.89 5-2.71 (m, 2H), 2.59-2.52 (m, 1H), 2.37 (s, 3H), 2.15-2.01 (m, 1H), 1.78 (m, 1H).
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) at room temperature. The reaction solution was stirred at 60° C. overnight, and then adjusted to pH 8 with sodium hydroxide. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a yellow solid compound 10 (49.3 mg, 22.79%). ESI/MS (m/z): 288.2 [M+H]+. 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).
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) at room temperature. The reaction solution was stirred overnight at 60° C., and then adjusted to pH 8 with sodium hydroxide. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a 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]+. 1HNMR (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).
KZT (200 mg, 1.0 mmol), 12-1 (1.5 mmol), concentrated hydrochloric acid (10 mL) and methanol (5 mL) were added to a 25 mL single neck bottle in sequence, and they were refluxed for 10 hours until KZT disappears. The reaction solution was cooled down to room temperature and poured into crushed ice, the 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 yellow green solid compound 12, yield 46.7%, m.p.=200-202° C. 1HNMR (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) 13C 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 C21H18NO2− (M−H)− 316.1343, found 316.1346.
Potassium hydroxide (211 mg, 3.765 mmol) was added to a solution of compound KZT (150 mg, 0.753 mmol) and 13-1 (132 mg, 0.904 mmol) in methanol (3 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with 2M hydrochloric acid. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L+0.1% ammonia water), mobile phase B: acetonitrile) to obtain a yellow solid compound 13 (5.0 mg, 2.03%). ESI/MS (m/z): 328.1 [M+H]+. 1HNMR (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), 7.45-7.40 (m, 1H) 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).
Concentrated sulfuric acid (0.4 mL, 7.504 mmol) was added to a solution of compound KZT (409 mg, 2.053 mmol) and 14-1 (300 mg, 2.053 mmol) in methanol (4 mL) at room temperature. After stirred overnight at 65° C., the reaction solution was adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L), mobile phase B: acetonitrile) to obtain a brown solid compound 14 (6.3 mg, 0.94%). ESI/MS (m/z): 328.0 [M+H]+.
Potassium hydroxide (437 mg, 7.805 mmol) was added to a solution of compound KZT (311 mg, 1.561 mmol) and 15-1 (150 mg, 1.561 mmol) in methanol (4 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with 2M hydrochloric acid. After concentration, the reaction solution was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate+0.1% ammonia water), mobile phase B: acetonitrile) to obtain a yellow solid compound 15 (32.3 mg, 7.41%). ESI/MS (m/z): 278.1 [M−H]−. HNMR (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).
Potassium hydroxide (42.5 mg, 0.759 mmol) was added to a mixture of KZT (37.0 mg, 0.253 mmol) and 16-1 (60.5 mg, 0.303 mmol) in 1,4-dioxane (2 mL) at room temperature. The reaction was heated to 100° C. for 3 hours. It was then quenched with water, and the obtained mixture was extracted with ethyl acetate (3×20 mL). The combined organic layer was washed with saturated salt water (5 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (methanol:dichloromethane=1:20) to obtain a yellow solid compound 16 (2.0 mg, 2.41%). ESI/MS (m/z) 327.9 [M+H]+.
An aqueous solution (20 mL) of sodium nitrite (1.25 g, 18.162 mmol) was added slowly 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) under zero temperature conditions. The reaction solution was stirred at 0° C. for 30 minutes, then a solution (80 mL, 1:1) of compound 17-2 (1.15 g, 9.081 mmol) and sodium acetate (1.64 g, 19.978 mmol) in methanol and water was added slowly to the reaction solution. The reaction solution was stirred at 0° C. for additional 0.5 hours, and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was subjected to silica gel column chromatography (dichloromethane:methanol=5:1) to obtain a yellow solid compound 17-3 (400 mg, 20.09%). ESI/MS (m/z): 220.1 [M+H]+.
Acetic acid (12 mL, 209.359 mmol) and concentrated sulfuric acid (1.20 mL, 22.508 mmol) were added to a solution of compound 17-3 in methanol (3 mL) at room temperature. The reaction solution was refluxed and stirred at 65° C. for 2 hours, and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by silica gel column chromatography (dichloromethane:methanol=5:1) to obtain a yellow solid compound 17-4 (300 mg, 43.42%). ESI/MS (m/z): 203.1 [M+H]+.
Pyridine (587 mg, 7.420 mmol) and trifluoromethanesulfonic anhydride (935 mg, 4.452 mmol) were added to a mixture of compound 17-4 (300 mg, 1.484 mmol) in dichloromethane (10 mL) at room temperature, and stirred for 2 hours. After concentration, the crude product was subjected to silica gel column chromatography (petroleum ether:ethyl acetate=1:1) to obtain a yellow solid compound 17-5 (210 mg, 42.35%). ESI/MS (m/z): 334.8 [M+H]+.
Under a nitrogen atmosphere conditions, trimethylaluminum (226 mg, 3.143 mmol) was added slowly to a mixture of compounds 17-5 (150 mg, 0.449 mmol) and Pd(PPh3)4 (52 mg, 0.045 mmol) in tetrahydrofuran (5 mL) at 0° C. The reaction solution was refluxed at 80° C. for 1 hour. It was then concentrated, and subjected to silica gel column chromatography (petroleum ether:ethyl acetate=1:1) to obtain a yellow solid compound 17-6 (70 mg, 77.90%). ESI/MS (m/z): 200.9 [M+H]+.
Concentrated sulfuric acid (0.1 mL) was added to a mixture of compounds 17-6 (60 mg, 0.300 mmol) and 17-7 (50 mg, 0.330 mmol) in methanol (1 mL) at room temperature. The reaction solution was stirred and refluxed overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column to obtain a yellow solid compound 17 (43.8 mg, 42.54%). ESI/MS (m/z): 335.0 [M+H]+. 1HNMR (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).
Concentrated hydrochloric acid (1 mL) was added to a solution of compounds 18-1 (1 g, 7.237 mmol) and 18-2 (0.97 g, 8.684 mmol) in 10 mL acetic acid at room temperature. After stirred at 65° C. for 2 hours, the reaction solution was directly concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=8:1) to obtain a light brown solid compound 18-3 (110 mg, 7.06%). ESI/MS (m/z): 216.2 [M+H]+. 1H 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).
Concentrated sulfuric acid (0.3 mL, 5.628 mmol) was added to a solution of compounds 18-3 (110 mg, 0.511 mmol) and 18-4 (93 mg, 0.613 mmol) in methanol (3 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution, and extracted with ethyl acetate (3×20 mL). The organic phase was washed with saturated saline water (30 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a yellow solid compound 18 (5.9 mg, 3.30%). ESI/MS (m/z): 350.0 [M+H]+. 1H 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) H).
AcOH acetate (15 mL) and concentrated hydrochloric acid (3 mL) were added to a solution of compounds 19-1 (5 g, 22.371 mmol) and 19-2 (3.01 g, 26.845 mmol) in methanol (60 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, it was separated by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain a light yellow compound 19-3 (700 mg, 11.85%). ESI/MS (m/z): 263.8 [M+H]+.
Concentrated sulfuric acid (1.5 mL, 28.143 mmol) was added to a mixture of compounds 19-3 (300 mg, 1.136 mmol) and 19-4 (259 mg, 1.704 mmol) in MeOH (15 mL) at room temperature, and the reaction solution was stirred overnight at 80° C. The reaction solution was then cooled down to room temperature, and adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate and concentrated to obtain a crude product, it was then separated by reverse phase preparation column to obtain a yellow solid compound 19 (66.8 mg, 14.80%). ESI/MS (m/z): 398.0 [M+H]+. 1HNMR (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)).
Concentrated hydrochloric acid (2.4 mL, 78.991 mmol) was added to solution of 20-1 (2.5 g, 15.020 mmol) and 20-2 (2.02 g, 18.024 mmol) in an acetic acid (11.8 mL) at room temperature. The reaction solution was refluxed at 120° C. for 1 hour. The reaction solution was then concentrated, and separated by silica gel column (petroleum ether:ethyl acetate=1:1) to obtain a yellow solid compound 20-3 (400 mg, 12.67%). ESI/MS (m/z): 211.1 [M+H]+.
Concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a solution of 20-3 (200 mg, 0.951 mmol) and 20-4 (173.69 mg, 1.141 mmol) in methanol (5 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate (3×20 mL), and the organic phase was washed with saturated salt water, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was prepared and separated by reverse phase column (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]+. 1H 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).
Concentrated sulfuric acid (1 mL, 18.762 mmol) was added to a solution of compounds 21-1 (920 mg, 4.540 mmol) and 21-2 (610 mg, 5.448 mmol) in acetic acid (10 mL) at room temperature. The reaction solution was stirred at 65° C. for 2 hours, and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate (3×50 mL), 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]+.
Concentrated sulfuric acid (0.4 mL, 7.505 mmol) was added to a solution of compounds 21-3 (150 mg, 0.617 mmol) and 21-4 (112 mg, 0.740 mmol) in methanol (4 mL) at room temperature. The reaction solution was stirred overnight at 80° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (0.05% formic acid), mobile phase B: acetonitrile) to obtain a yellow solid compound 21 (20.04 mg, 8.35%). ESI/MS (m/z): 378.2 [M+H]+. 1HNMR (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).
Under a nitrogen atmosphere, sodium hydride NaH (29.9 mg, 1.250 mmol) was added to a solution of compounds 22-1 (300 mg, 1.136 mmol) and 22-2 (162 mg, 1.136 mmol) in DMF (5 mL) at 0° C. The reaction solution was stirred at room temperature for 4 hours. The reaction was then quenched with water, extracted with ethyl acetate (3×20 mL), washed with saturated salt water, dried over anhydrous sodium sulfate, concentrated, and then the crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain a white solid compound 22-3 (300 mg, 68.73%). ESI/MS (m/z): 384.0 [M+H]+.
Under a nitrogen atmosphere, catalyst Pd PEPPSI-IHeptCl3-chloropyridine (76 mg, 0.078 mmol) was added to a solution of compounds 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) at room temperature. The reaction solution was stirred overnight at 100° C. under a nitrogen atmosphere, then cooled and concentrated, the crude product was then separated by silica gel column chromatography (dichloromethane:methanol=1:1) to obtain a light yellow solid compound 22-5 (150 mg, 47.50%). ESI/MS (m/z): 405.2 [M+H]+.
Concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a solution of compounds 22-5 (150 mg, 0.371 mmol) and 22-6 (68 mg, 0.445 mmol) in methanol (5 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate (3×20 mL), washed with saturated salt water (30 mL), dried over anhydrous sodium sulfate, concentrated, and then the crude product was separated by reverse phase preparation column (mobile phase A: water (0.05% formic acid), mobile phase B: acetonitrile) to obtain a yellow solid compound 22 (25.3 mg, 15.99%). ESI/MS (m/z): 419.1 [M+H]+. 1HNMR (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), 6.86 (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).
Under a nitrogen atmosphere, 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 a solution of compound 23-2 (116 mg, 0.624 mmol) in 1,4-dioxane (5 mL) were added at room temperature. Under a nitrogen atmosphere, the reaction solution was stirred at 100° C. for 4 hours and then quenched with water, extracted with ethyl acetate (3×10 mL), washed with saturated saline water (10 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 a white solid compound 23-3 (182 mg, 71.42%). ESI/MS (m/z): 490.3 [M+H]+.
Concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a mixture of compounds 23-3 (150 mg, 0.286 mmol) and 23-4 (56 mg, 0.367 mmol) in methanol (5 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 7 with a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate (3×20 mL), washed with saturated salt water (20 mL), dried over anhydrous sodium sulfate, concentrated, and the crude product was separated by a reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a light yellow solid compound 23 (10.1 mg, 8.17%). ESI/MS (m/z): 404.0 [M+H]+. 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).
Under a nitrogen atmosphere, potassium acetate (371 mg, 3.786 mmol) was added to a solution of compounds 24-1 (500 mg, 1.893 mmol), 24-2 (734 mg, 3.786 mmol), catalyst Pd(dppf)Cl2CH2Cl2 (231 mg, 0.284 mmol) in 1,4-dioxane (10 mL) and water (2 mL) at room temperature. Under a nitrogen atmosphere, the reaction solution was stirred overnight at 100° C., then quenched with water, extracted with ethyl acetate (3×20 mL), washed with saturated salt water (20 mL), dried over anhydrous sodium sulfate and concentrated, and the crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate=1:1) to obtain a brown solid compound 24-3 (100 mg, 21.02%). ESI/MS (m/z): 252.2 [M+H]+.
Concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a solution of compounds 24-3 (100 mg, 0.398 mmol) and 24-4 (72 mg, 0.478 mmol) in methanol (5 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a brown compound 24 (12.2 mg, 7.80%). ESI/MS (m/z): 386.1 [M+H]+. 1HNMR (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=5.76 Hz, 2H), 3.07 (t, J=6.2 Hz, 2H).
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)C12CH2Cl2 (123 mg, 0.151 mmol) and cuprous iodide (14 mg, 0.075 mmol) in DMF (5 mL) at room temperature under a nitrogen atmosphere. The reaction solution was stirred at 80° C. for 2 hours, then quenched with water (1 mL), extracted with ethyl acetate (3×10 mL), washed with saturated salt water (15 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column (petroleum ether:ethyl acetate=1:1) to obtain a yellow solid compound 25-3 (150 mg, 47.92%). ESI/MS (m/z): 416.0 [M+H]+.
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. The reaction solution was stirred at 60° C. for 2 hours, then quenched with 1 mL of water, extracted with ethyl acetate (3×10 mL), and the organic phase was washed with saturated salt water (15 mL), dried over anhydrous sodium sulfate and concentrated, and the crude product was separated by reverse phase preparation column to obtain a yellow solid compound 25 (20.3 mg, 16.38%). ESI/MS (m/z): 344.0 [M+H]+. 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).
Lithium hydroxide (3 mg, 0.130 mmol) was added to a solution of compound 26-1 (10 mg, 0.026 mmol) in methanol (1 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with 2M hydrochloric acid, concentrated, and the crude product was separated by reverse phase preparation column (mobile phase A: water (0.05% formic acid), mobile phase B: acetonitrile) to obtain a brown solid compound 26 (2 mg, 20.77%). ESI/MS (m/z): 364.0 [M+H]+.
Concentrated hydrochloric acid (0.3 mL, 9.874 mmol) was added to a 12 mL aqueous solution of compounds 27-1 (300 mg, 1.845 mmol) and 27-2 (248 mg, 2.214 mmol) at room temperature. The reaction solution was stirred overnight at 120° C. The obtained solid was collected and washed with water (30 mL) to obtain a brown solid compound 27-3 (180 mg, 48.01%). ESI/MS (m/z): 204.0 [M+H]+. 1HNMR (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).
Concentrated sulfuric acid (0.3 mL, 5.628 mmol) was added to a mixture of compounds 27-3 (180 mg, 0.886 mmol) and 27-4 (594 mg, 5.302 mmol) in methanol (3 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain a yellow solid compound 27 (20.4 mg, 6.83%). ESI/MS (m/z): 338.0 [M+H]+. 1HNMR (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), 3.02 1 (t, J=6.3 Hz, 2H).
Triethylamine (0.19 mL, 1.347 mmol) was added to compound 28-1 (150 mg, 0.449 mmol) and catalyst Pd(dppf)Cl2·CH2Cl2 (73 mg, 0.090 mmol) in methanol (20 mL) at room temperature. Under a carbon monoxide gas atmosphere, the reaction solution was stirred overnight at 80° C., then concentrated, and the crude product was separated by silica gel column chromatography (dichloro:methanol=20:1) to obtain a light yellow solid compound 28-2 (70 mg, 63.87%). ESI/MS (m/z): 245.0 [M+H]+. 1HNMR (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).
Concentrated sulfuric acid (0.3 mL) was added to a mixture of compounds 28-2 (50 mg, 0.205 mmol) and 28-3 (37 mg, 0.246 mmol) in methanol (3 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by a reverse phase preparation column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain a yellow solid compound 28 (12.5 mg, 16.14%). ESI/MS (m/z): 379.0 [M+H]+. 1HNMR (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).
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) at room temperature. The reaction solution was stirred at room temperature for 2 hours, then quenched with saturated ammonium chloride aqueous 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]−. 1HNMR (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).
Concentrated sulfuric acid (0.3 mL, 5.628 mmol) was added to a solution of compounds 29-2 (150 mg, 0.657 mmol) and 29-3 (120 mg, 0.788 mmol) in methanol (3 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation 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]−. 1HNMR (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=5.78 Hz, 2H, 3.08 (t, J=6.3 Hz, 2H).
DIEA (1.14 mL, 6.545 mmol) and HATU (597 mg, 1.571 mmol) were added to a solution of compounds 30-1 (300 mg, 1.309 mmol) and 30-2 (106 mg, 1.571 mmol) in DMF (3 mL) at room temperature. The reaction solution was stirred at room temperature for 2 hours, then quenched with water, extracted with ethyl acetate (3×10 mL), concentrated, and the crude product was separated by silica gel column chromatography (dichloromethane:methanol=10:1) to obtain a brown compound 30-3 (180 mg, 56.77%). ESI/MS (m/z): 243.0 [M+H]+. 1HNMR (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).
Concentrated sulfuric acid (0.3 mL, 5.629 mmol) was added to a solution of compounds 30-3 (180 mg, 0.743 mmol) and 30-4 (135 mg, 0.892 mmol) in methanol (3 mL) at room temperature. The reaction solution was stirred overnight at 80° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain a yellow solid compound 30 (11.3 mg, 4.04%). ESI/MS (m/z): 377.1 [M+H]+. 1HNMR (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).
Concentrated hydrochloric acid (5 mL) was added to an aqueous (100 mL) solution of compounds 31-1 (1.2 g, 5.945 mmol) and 31-2 (1 g, 8.918 mmol) at room temperature. The reaction solution was refluxed overnight at 120° C. It was then cooled down to room temperature, filtered. The obtained solid was collected, washed with ice water, and subjected to silica gel column chromatography (petroleum ether:ethyl acetate=1:1) to obtain a yellow solid compound 31-3 (120 mg, 8.23%). ESI-MS (m/z): 245.95 [M+H]+.
Concentrated sulfuric acid (0.5 mL, 9.381 mmol) was added to a solution of compounds 31-3 (120 mg, 0.489 mmol) and 31-4 (89 mg, 0.587 mmol) in methanol (5 mL) at room temperature. The reaction solution was stirred at 65° C. overnight, and then adjusted to pH 8 with a saturated sodium carbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column to obtain a yellow solid compound 31 (74.0 mg, 38.31%). ESI/MS (m/z): 380.0 [M+H]+. 1H 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).
Concentrated hydrochloric acid (0.5 mL, 16.456 mmol) was added to an aqueous (20 mL) solution of compounds 32-1 (300 mg, 1.695 mmol) and 32-2 (228 mg, 2.034 mmol) at room temperature. The reaction solution was stirred overnight at 120° C. The obtained solid was collected, washed with water (30 mL) to obtain a brown solid compound 32-3 (170 mg, 39.48%). ESI/MS (m/z): 255.1 [M+H]+. 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).
Concentrated sulfuric acid (0.3 mL, 5.629 mmol) was added to a solution of compounds 32-3 (170 mg, 0.669 mmol) and 32-4 (122 mg, 0.803 mmol) in methanol (3 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted pH to 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain a yellow solid compound 32 (27 mg, 10.40%). ESI/MS (m/z): 388.1 [M+H]+. 1HNMR (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).
Concentrated hydrochloric acid (2.40 mL, 78.989 mmol) was added to a aqueous (100 mL) solution of compounds 33-1 (1.2 g, 6.833 mmol) and 33-2 (0.92 g, 8.200 mmol) at room temperature. The reaction solution was stirred overnight at 120° C., concentrated, and the crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate=1:1) to obtain a yellow solid compound 33-3 (400 mg, 27.07%). ESI/MS (m/z): 217.1 [M+H]+. 1HNMR (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).
Concentrated sulfuric acid (0.40 mL, 7.511 mmol) was added to a solution of compounds 33-3 (400 mg, 1.850 mmol) and 33-4 (309 mg, 2.035 mmol) in methanol (5 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a yellow solid compound 33 (110 mg, 16.43%). ESI/MS (m/z): 351.0 [M+H]+. 1H 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).
Sodium ethanol (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. The reaction solution was stirred at room temperature for 1 hour, and then adjusted to pH 2 with 2 M hydrochloric acid, extracted with ethyl acetate, washed with saturated salt water, 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]−.
An aqueous (10 mL) solution of sodium nitrite (643 mg, 9.332 mmol) was added to an aqueous (20 mL) solution of compound p-methylaniline (500 mg, 4.666 mmol) and concentrated hydrochloric acid (3.7 mL, 123.416 mmol) 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 added to the reaction solution at 0° C. The reaction solution was stirred at room temperature for additional 2 hours. The obtained 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]+.
Concentrated sulfuric acid, MeOH (0.1 mL) was added to a solution of compound 34-3 (250 mg, 1.023 mmol) and acetic acid (0.1 mL) in methanol (1 mL) at room temperature, the reaction solution was stirred at 65° C. for 24 hours, then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate (2×10 mL), and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to obtain a yellow solid compound 34-4 (88 mg, 37.84%). ESI/MS (m/z): 228.3 [M+H]+.
Concentrated sulfuric acid (0.2 mL, 3.752 mmol) was added to a mixed solution of compound 34-4 (80 mg, 0.352 mmol) and 34-5 (64 mg, 0.422 mmol) in methanol (2 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a yellow solid compound 34 (28 mg, 22.01%). ESI/MS (m/z): 362.1 [M+H]+. 1H 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).
Sodium hydride (1.71 g, 71.328 mmol) was added to a solution of compound 35-1 (2 g, 23.776 mmol) and ethyl formate (6.28 g, 71.328 mmol) in toluene (23 mL) at 0° C. The reaction solution was stirred at room temperature for 2 hours, and then adjusted to pH 2 with 2 M hydrochloric acid, extracted with ethyl acetate (3×50 mL) and concentrated to obtain a colorless oily liquid 35-2 (1.5 g, 56.26%). ESI/MS (m/z): 113.1 [M+H]+.
An aqueous (10 mL) solution of sodium nitrite (615 mg, 8.918 mmol) was added to a aqueous (20 mL) solution of compound p-methylaniline (477 mg, 4.459 mmol) and concentrated hydrochloric acid (1 mL) 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 obtained solid was collected and washed with water (3×50 mL) to obtain a yellow solid compound 35-3 (800 mg, 88.70%). ESI/MS (m/z): 203.0 [M+H]+.
Concentrated sulfuric acid (0.4 mL, 7.504 mmol) was added to an 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 adjusted to pH 7 with a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate (3×30 mL), and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain a yellow solid compound 35-4 (150 mg, 40.95%). ESI/MS (m/z): 186.2 [M+H]+.
Concentrated sulfuric acid (0.4 mL, 7.504 mmol) was added to a mixed solution of compounds 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 overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate), mobile phase B: acetonitrile) to obtain a yellow solid compound 35 (22 mg, 8.51%). ESI/MS (m/z): 320.0 [M+H]+. 1H 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).
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) at 0° C. The reaction solution was stirred at room temperature for 2 hours, and then adjusted to pH 7 with 2 M hydrochloric acid, extracted with ethyl acetate (3×30 mL), washed with saturated salt water (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 a light brown solid compound 36-2 (490 mg, 39.48%). ESI/MS (m/z): 145.0 [M+H]+.
An aqueous (10 mL) solution of sodium nitrite (431 mg, 6.242 mmol) was added to an aqueous (10 mL) solution of compound p-methylaniline (334 mg, 3.121 mmol) and concentrated hydrochloric acid (1.5 mL) at 0° C., and a solution of compound 36-2 (450 mg, 3.121 mmol) and sodium acetate (563 mg, 6.242 mmol) in aqueous and methanol (20 mL, 1:1) was added. The reaction solution was stirred at room temperature for 2 hours. The obtained solid was collected and used with water (3×100 mL) to obtain a yellow solid compound 36-3 (180 mg, 24.61%). ESI/MS (m/z): 235.0 [M+H]+.
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) at room temperature. The reaction solution was stirred overnight at 65° C. It was then concentrated, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=5:1) to obtain a yellow solid compound 36-4 (100 mg, 67.40%). ESI-MS (m/z): 218.2 [M+H]+.
Concentrated sulfuric acid (0.1 mL, 1.876 mmol) was added to a mixture of compounds 36-4 (80 mg, 0.390 mmol) and 36-5 (71 mg, 0.468 mmol) in methanol (1 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile) to obtain a yellow solid compound 36 (8.1 mg, 5.91%). ESI/MS (m/z): 352.0 [M+H]+. 1HNMR (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).
Sodium ethanol (1.21 g, 17.830 mmol) was added to a mixture of compound 37-1 (1 g, 8.915 mmol) and ethyl formate (1.32 g, 17.830 mmol) in dioxane (10 mL) at 0° C. The reaction solution was stirred at 0° C. for additional 1 hour, and then adjusted to pH 7 with 2 M hydrochloric acid, extracted with ethyl acetate, washed with saturated salt water, 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 a light brown solid oily liquid 37-2 (700 mg, 56.01%). ESI/MS (m/z): 141.1 [M+H]+.
An aqueous (5 mL) solution of sodium nitrite (590 mg, 8.560 mmol) was added to an aqueous (20 mL) solution of compound 4-methylaniline (458 mg, 4.280 mmol) and concentrated hydrochloric acid (1.50 mL, 48.382 mmol) at 0° C. Maintain 0° C. and 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 the mixture. The reaction solution was stirred at room temperature for additional 2 hours. The obtained solid was collected, washed with water (3×50 mL), and separated by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to obtain a brown solid compound 37-3 (300 mg, 30.43%). ESI/MS (m/z): 231.0 [M+H]+.
Concentrated sulfuric acid (0.5 mL, 10.944 mmol) was added to a solution of compound 37-3 (280 mg, 1.216 mmol) and acetic acid (1 mL, 24.320 mmol) in methanol (5 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution, extracted with ethyl acetate, washed with saturated salt water, and concentrated. The crude product was separated by silica gel column chromatography (petroleum ether:ethyl acetate=1:1) to obtain a brown solid compound 37-4 (135 mg, 52.06%). ESI-MS (m/z): 214.2 [M+H]+. 1HNMR (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).
Concentrated sulfuric acid (0.3 mL, 5.628 mmol) was added to a mixture of compounds 37-4 (100 mg, 0.469 mmol) and 37-5 (85 mg, 0.563 mmol) in methanol (3 mL) at room temperature. The reaction solution was stirred overnight at 65° C., and then adjusted to pH 8 with a saturated sodium bicarbonate aqueous solution. After concentration, the crude product was separated by reverse phase preparation column (mobile phase A: water (10 mmol/L ammonium bicarbonate+0.1% ammonia water), mobile phase B: acetonitrile) to obtain a yellow solid compound 37 (32.6 mg, 19.81%). ESI/MS (m/z): 348.1 [M+H]+. 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).
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) at room temperature. The reaction was heated up to 100° C. and stirred for 2 hours. The reaction mixture was cooled down to room temperature, and the reaction was quenched with water. The obtained mixture was extracted with ethyl acetate (3×30 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (ethyl acetate:petroleum ether=1:5) to obtain a yellow solid 38 (21.8 mg, 14.44%). ESI/MS (m/z) 377.9 [M+H]+. 1H 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).
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) at room temperature. The reaction was heated up to 100° C. for 6 hours. It was then quenched with water, and the obtained mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with saturated salt water (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (methanol:dichloromethane=1:20) to obtain an orange solid compound 39 (20.87 mg, 6.29%). ESI/MS (m/z) 330.9 [M+H]+. 1H NMR (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).
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) at room temperature. The reaction was heated up to 100° C. for 6 hours. It was then quenched with water, and the obtained mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with saturated salt water (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (methanol:dichloromethane=1:20) to obtain an orange solid compound 40 (30.79 mg, 8.60%). ESI/MS (m/z) 357.0 [M+H]+. 1H NMR (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).
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) at room temperature. The reaction was heated up to 100° C. for 6 hours. The reaction was quenched with water, and the obtained mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with saturated salt water (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by a silica gel column chromatography (methanol:dichloromethane=1:20) to obtain a deep yellow solid compound 41 (120 mg, 31.0%). ESI/MS (m/z) 385.9 [M+H]+. 1H 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).
The inhibitory effects of the above compounds on the OCT4 protein and JAK1/2-STAT3 signaling pathway in HeLa cells indicate that the series compounds of KZT can simultaneously target OCT4 protein and JAK1/2. On the one hand, they can inhibit the transcription of OCT4 and NANOG stem factors by blocking the formation of OCT4/SOX2/DNA complexes, and on the other hand, they can inhibit the downstream STAT3 transcriptional activity by inhibiting JAK1/2 kinase activity, which are a class of one-drug dual-target candidate drugs that simultaneously exert inhibitory effects on both OCT4 self-transcription and OCT4 transcriptional activity. This dual target inhibition mechanism can simultaneously target the inhibition and clearance of CSC and differentiated tumor cells, providing the possibility of completely blocking the bidirectional transformation between CSC and differentiated tumor cells, and having good potential application prospects in tumor eradication.
By comparing the effects of AH057 and KZT-A5 on tumor microspheres cultured in Huh7 cells, it can be seen that 1 μM KZT-A5 shows more significant inhibitory effect compared with the same concentration of AH057, indicating that KZT-A5 has the potential to have better pharmacological effects in inhibiting the proliferation and differentiation of liver cancer cells than AH057.
By comparing the effects of AH057 and KZT-A5 on tumor microspheres cultured in HeLa and Caski cells, it can be seen that when the concentration of KZT-A5 reaches to 10 μM, its inhibitory effect on tumor microspheres cultured in cervical cancer cells is more significant than that of the control compound AH057.
(2) The Inhibitory Effect of Compounds on Human Tumor Cell Lines from Different Tissue Sources (1)
It can be seen from Table 2 that the IC50 values of KZT-A5 on Huh7 cell lines in human liver cancer cells and HeLa cell lines in human cervical cancer cells are relatively low, both lower than 1 μM, indicating it has superior ability to inhibit the proliferation of liver cancer cells and cervical cancer cells. In addition, IC50 values of compounds 8, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 31, 32, 33, 34, 35, 36, 37, and 38 on HeLa cell lines in human cervical cancer cells are much less than 50 μM.
(3) The Inhibitory Effect of Compounds on Human Tumor Cell Lines from Different Tissue Sources (2)
According to Table 3, AH057 was used as a control to compared in vitro efficacy of KZT-A5 and AH057 on human liver cancer cells and cervical cancer cells. It can be seen that the inhibitory effect of KZT-A5 on Hep3B and HeLa cell lines is comparable to AH057. Compared with AH057, KZT-A5 has lower IC50 values and high significance on Huh7 and HepG2 cell lines of liver cancer cells. This result confirmed that KZT-A5 has a significantly better inhibitory effect on liver cancer cell proliferation than AH057, and has a better in vitro efficacy.
SGI-1027 is an inhibitor of DNA methyltransferase II, with CAS number 1020149-73-8. Bliss independent model was used to screen and determine SGI-1027 as the compound for combination with KZT-A5, and the screening process was carried out according to the methods in the following literature: Goldoni M, Johansson C. A mathematical approach to study combined effects of toxins in vitro: evaluation of the Bliss independence criterion and the Loewe additivity model Toxic In Vitro 2007, 21:759-69.
The present invention explores the action mechanism of tetrahydrocarbazole compounds at the molecular level and verifies their dual target inhibitory effect on tumor stem cells (CSCs), which can simultaneously target on promoting CSC differentiation and killing differentiated tumor cells, providing the possibility of completely blocking the bidirectional transformation between CSC and differentiated tumor cells, and has good potential application prospects in eradicating tumors. The tetrahydrocarbazole compounds provided in the present invention have significant inhibitory effects on tumors such as cervical cancer and liver cancer, wherein the inhibitory effect against liver cancer is more significant, and compared with other control substances, the inhibitory effect of the compounds provided in the present invention on liver cancer is very prominent and unexpected, and have very good research and application prospects in the field of liver cancer treatment.
All references mentioned in the present invention are cited as references in this application, just as each reference is cited separately. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the appended claims of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111410807.9 | Nov 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/132643 | 11/17/2022 | WO |
