Thiophene acetyl tetrahydro-beta-carboline compound and application thereof

Abstract

The invention relates to the field of antitumor drugs, in particular to a thiophene acetyl tetrahydro-beta-carboline compound and application thereof. The thiophene acetyl tetrahydro-beta-carboline compound or the pharmaceutically acceptable salt thereof, and the pharmaceutical composition containing the compound can be used for preparing medicines for preventing and / or treating various cancers. The cancer is at least one of breast cancer, lung cancer, osteosarcoma, melanoma, gastric cancer, pancreatic cancer, kidney cancer, glioma and ovarian cancer. Activity screening is carried out on various compounds, it is found for the first time that the 12 compounds provided by the invention have remarkable broad-spectrum inhibitory activity on various human cancer cell lines, and key candidate compounds are provided for developing novel anti-cancer drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antitumor drugs, in particular to a thienylacetyl tetrahydro-beta-carboline compound and application thereof. BACKGROUND

[0002] Malignant tumor is one of the major diseases leading to death worldwide. The commonly used chemotherapeutic drugs in clinic generally have poor selectivity, large toxic side effects and easy drug resistance. Therefore, developing antitumor drugs with novel structure, high activity and low toxicity has always been the core challenge of drug research and development.

[0003] Beta-carboline alkaloids have been confirmed to have significant anti-proliferation, pro-apoptosis and anti-angiogenesis activities due to their unique indole and pyridine structure, and have become important lead compounds for the design of antitumor drugs. Among them, tetrahydro-beta-carboline skeleton has attracted much attention due to its good structure modifiability and potential biocompatibility. Existing studies have shown that some benzeneacetyl substituted tetrahydro-beta-carboline derivatives exhibit certain in vitro antitumor activity, but their inhibitory potency is limited and the antitumor spectrum is narrow, which is difficult to meet the clinical demand for high-efficiency broad-spectrum antitumor drugs.

[0004] In recent years, heterocyclic substituents have been proved to significantly enhance the targeting and cell permeability of drug molecules due to their unique electronic effect and spatial configuration. However, the research on introducing thienylacetyl into tetrahydro-beta-carboline skeleton to optimize the antitumor activity has not been systematically reported, and the structure-activity relationship and mechanism of action still need to be further explored. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a thienylacetyl tetrahydro-beta-carboline compound and its application in the preparation of drugs for preventing and / or treating cancer. The present application develops a new type of compound with strong and broad-spectrum antitumor activity and good drug-likeness by innovative structural modification of the tetrahydro-beta-carboline skeleton, which provides a better anticancer candidate drug for clinical use.

[0006] The technical scheme of the present application is as follows: a thienylacetyl tetrahydro-beta-carboline compound, the structure of the compound is shown in formula (I) or formula (II): (I) (II) R1 and R2 do not form a ring, i.e. when R1, R2 are independently selected from any one of the following groups: hydrogen, C2-C8 alkoxy, -(CH2) n -OH, -(CH2) n -SH, -(CH2) n -NH2, -(CH2)n -NHCH3, -(CH2) n -N(CH3)2, C 3-8 cycloalkyl, C 3-8 cycloalkyl, piperidinyl, oxazinanyl, morpholinyl, piperazinyl and tetrahydrofuranyl; or R1and R2form a ring, i.e. is selected from any one of the following groups: aziridine, azetidine, tetrahydropyrrole, hydroxytetrahydropyrrole, oxazolidine, isoxazolidine, piperidine, 2-hydroxypiperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, morpholine, 1,3-oxazinane, [1,2]oxazinane, piperazine, hexahydropyrimidine, hydroxyhexahydropyrimidine, hexahydropyridazine, hexamethyleneimine, 1,4-oxaazepane and 1,4-diazepane; n is an integer between 0 and 8 (0, 1, 2, 3, 4, 5, 6, 7 or 8).

[0007] As a preference, the compound is selected from one of the following compounds: N -(2-hydroxyethyl)-2-(3-(thiophen-2-yl)propionyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b ]indole-8-carboxamide; 1-(8-((3-hydroxyhexahydropyridin-1-yl)carbonyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b ]indol-2-yl)-3-(thiophen-2-yl)propan-1-one; N -(2-hydroxycyclopentyl)-2-(3-(thiophen-2-yl)propionyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b ]indole-8-carboxamide; N -(3-hydroxycyclopentyl)-2-(3-(thiophen-2-yl)propionyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b ]indole-8-carboxamide; N -(2-hydroxycyclopentyl)-2-(thiophen-2-ylcarbonyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b ]indole-8-carboxamide; N -(3-hydroxycyclopentyl)-2-(thiophen-2-ylcarbonyl)-2,3,4,9-tetrahydro-1H pyrido[3,4- b ]indole-8-carboxamide; N (2-hydroxyethyl)-2-(2-(thiophen-3-yl)acetyl)-2,3,4,9-tetrahydro-1 H pyrido[3,4- b ]indole-8-carboxamide; 1-(8-((3-hydroxyhexahydropyridin-1-yl)carbonyl)-2,3,4,9-tetrahydro-1 H pyrido[3,4- b ]indol-2-yl)-2-(thiophen-3-yl)ethan-1-one; N (2-hydroxycyclopentyl)-2-(2-(thiophen-3-yl)acetyl)-2,3,4,9-tetrahydro-1 H pyrido[3,4- b ]indole-8-carboxamide; N (3-hydroxycyclopentyl)-2-(2-(thiophen-3-yl)acetyl)-2,3,4,9-tetrahydro-1 H pyrido[3,4- b ]indole-8-carboxamide; N (2-hydroxyethyl)-2-((thiophen-2-ylcyclopropyl)carbonyl)-2,3,4,9-tetrahydro-1 H pyrido[3,4- b ]indole-8-carboxamide; N (3-hydroxycyclopentyl)-2-((thiophen-2-ylcyclopropyl)carbonyl)-2,3,4,9-tetrahydro-1 H pyrido[3,4- b ]indole-8-carboxamide.

[0008] The present application also provides a pharmaceutical composition comprising the thienyl acetyl tetrahydro-beta-carboline compound or a hydrate thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0009] The pharmaceutically acceptable salts include acid and base salts, groups which are suitable for contact with the human or animal body without undue toxicity or carcinogenicity and are particularly ones which are nontoxic, allergenically or otherwise. Alkaline or organic salts of acidic residues (such as carboxylic acids) and inorganic and organic salts of basic residues (such as amines).

[0010] Anions useful for salt formation include, but are not limited to, chloride, bromide, sulfate, phosphate, acetate, tartrate, salicylate, citrate, methanesulfonate, p-toluenesulfonate, lactate, pyruvate, maleate, succinate, ascorbate, benzoate, bicarbonate, ethylenediaminetetraacetate, formate, glutamate, glycolate, hydroxymaleate, malate, mandelate, sulfamate, sulfonate, chloride, and bromide, and the like.

[0011] Similarly, cations useful for salt formation include, but are not limited to, ammonium, ethylenediamine, choline, diethanolamine, procaine, and metal sodium, potassium, magnesium, aluminum, zinc, and lithium, and the like.

[0012] As preferred, the pharmaceutical composition further comprises a second agent for preventing and / or treating cancer.

[0013] As preferred, the pharmaceutical composition further comprises an excipient, diluent, adjuvant, vehicle, or a combination thereof.

[0014] As preferred, the pharmaceutical composition is formulated as an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup, or a transdermal patch.

[0015] The present application also provides use of the compound or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating cancer. The thienylacetyl tetrahydro-β-carboline compound and pharmaceutically acceptable salts, metabolites, or prodrugs, or pharmaceutical compositions thereof can be used to inhibit the proliferation, growth, migration, invasion, clonogenicity, and metastasis of cancer cells, promote the apoptosis of cancer cells, promote the autophagy of tumor cells, and / or prolong the survival of tumor patients.

[0016] As preferred, the cancer is at least one of breast cancer, lung cancer, osteosarcoma, melanoma, gastric cancer, pancreatic cancer, renal cancer, glioma, and ovarian cancer.

[0017] That is, the compound or the pharmaceutical composition can inhibit the activity of at least one of human breast cancer cells, human lung cancer cells, human osteosarcoma cells, human malignant melanoma cells, human gastric adenocarcinoma cells, human pancreatic cancer cells, human renal cancer cells, human glioma cell lines, and human ovarian adenocarcinoma.

[0018] The present application also provides use of the compound or the pharmaceutical composition in the preparation of a tumor stemness regulator.

[0019] The present application is to develop a new type of antitumor compound based on tetrahydro-beta-carboline skeleton, and the inventors find that a class of phenylacetyl tetrahydro-beta-carboline compounds have certain anticancer activity through a series of previous careful research work, and the inhibitory activity of the compound is obviously improved after replacing phenylacetyl with thienylacetyl, and the results of multiple experiments verify that the compound shows excellent antitumor effect.

[0020] Compared with the prior art, the present application has the following beneficial effects: the present application performs activity screening on a plurality of compounds, and for the first time discovers that the 12 compounds provided by the present application have significant broad-spectrum inhibitory activity on a plurality of human cancer cell lines, and provides key candidate compounds for developing new anticancer drugs. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Inhibition results of thienylacetyl tetrahydro-beta-carboline compounds I-XII on human breast cancer cell lines HCC1806 (A) and SUM149PT cell lines (B); significant differences are indicated by *. p <0.05, p <0.01, p <0.001, ns represents no significant difference, p >0.05. DETAILED DESCRIPTION

[0022] The present application will be further described in conjunction with the following specific examples and drawings, and the protection scope of the present application is not limited to the following examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

[0023] The instruments used in Example 1 include EYELA (N-1100) rotary evaporator, etc.; the purity results of the obtained compounds are from Agilent 1200 series LC system high performance liquid chromatograph (chromatographic conditions: Zorbax XDB-C18 (4.6x150 nm, 5 μm), column 40℃, mobile phase is MeOH / H2O or MeCN / H2O, running flow rate is 1.5 mL / min, ultraviolet detection wavelength is 254 nm, injection amount is 10 μL); nuclear magnetic resonance spectrometer is Bruker 300 or Bruker 500 type (internal standard: TMS, using solvent is DMSO-d6, chemical shift is ppm). d 6) and the purification of the reaction intermediates and final products were carried out by chromatography on silica gel (200-300 mesh, purchased from Qingdao Haoyang Chemical Factory). All solvents were redistilled before use, and the anhydrous solvents were dried according to standard methods. Unless otherwise stated, all reactions were carried out under nitrogen and the progress of the reactions was followed by TLC, and the work-up was carried out by washing with saturated brine and drying over anhydrous sodium sulfate.

[0024] The human breast cancer cells used in Examples 2-3 were human breast cancer cells HCC1806 and SUM149PT, the human lung cancer cells were human lung cancer cells H460, the human osteosarcoma cells were human osteosarcoma cells MG63, the melanoma was human malignant melanoma cells A375, the human gastric cancer cells were human gastric adenocarcinoma cells SGC-7901, the human pancreatic cancer cells were human pancreatic cancer cells SW1990, the human renal cancer cells were human renal cancer cells G401, and the human glioma cells were human glioma cells U251. The above-mentioned conventional cancer cells were all obtained from Kunming Medical University. The cell strains were cultured in DMEM or RPMI1640 medium containing 5% fetal bovine serum at 37°C, 5% CO2 and 90% humidity.

[0025] Unless otherwise stated, the reagents used in the following examples can be purchased commercially. Fetal bovine serum was purchased from ROyacel; RPMI1640 medium, DMEM medium, phosphate buffer (PBS) and 0.25% trypsin (containing EDTA) were purchased from Gibco Company.

[0026] The English meanings used in the following examples are as follows: THF tetrahydrofuran, MeOH methanol, EA ethyl acetate, EtOH ethanol, HATU 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, DIEA N,N-diisopropylethylamine, DMF N,N-dimethylformamide, EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), HOBT 1-hydroxybenzotriazole.

[0027] The structural formulas and preparation methods of compounds I-X used in Examples 2-3 are shown in Example 1.

[0028] Example 1: Preparation of each compound

[0029] Example 1-1 N -(2-hydroxyethyl)-2-(3-(thiophene-2-yl)propionyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b ]indole-8-carboxamide (I) Structural formula and synthesis route of compound I

[0030]

[0031] Sodium borohydride (2.1 g, 54.45 mmol) was dissolved in a mixed solvent of THF (96 mL) and MeOH (24 mL) at 0 °C, and (Z)-3-(2-nitrovinyl)-1H-indole-7-carboxylic acid methyl ester (4.5 g, 19.09 mmol) was added to the reaction solution at 0 °C. After reaction at 0 °C for 2 h and then at room temperature for 2 h, the reaction was monitored by TLC. After the reaction was completed, the organic phase was extracted with EA, and the solvent was evaporated under reduced pressure to obtain a crude product. The product, 3-(2-nitroethyl)-1H-indole-7-carboxylic acid methyl ester (2813 mg, yield 60%), was obtained by column chromatography separation and purification.

[0032] Methyl 3-(2-nitroethyl)-1H-indole-7-carboxylate (2813 mg, 11.33 mmol) was dissolved in a mixed solvent of EtOH (10 mL) and H2O (60 mL), and iron powder (1898 mg, 33.99 mmol) and NH4Cl (6060 mg, 113.3 mmol) were added. The reaction was carried out at 60 °C for 2 h under nitrogen protection. After the reaction was completed, the iron powder was removed by filtration, and the EtOH was removed by evaporation under reduced pressure. The organic phase was extracted with EA, and the solvent was evaporated under reduced pressure to obtain a crude product. The product, 7-methoxycarbonyltryptamine (878 mg, yield: 36%), was obtained by column chromatography separation and purification.

[0033] 7-Methoxycarbonyltryptamine (878 mg, 4.023 mmol) was dissolved in acetic acid (20 mL) under ice bath conditions, and formaldehyde (0.33 mL, 4.425 mmol) was slowly added. The reaction was carried out at room temperature overnight. After the reaction was completed, most of the solvent was evaporated under reduced pressure, and NaOH solution was added to adjust the pH to > 12. The organic phase was extracted with EA, and the solvent was evaporated under reduced pressure to obtain a crude product, 8-methoxycarbonyl-1,3,4,9-tetrahydro-β-carboline, which was directly used in the next step.

[0034] The crude product 8-methoxycarbonyl-1,3,4,9-tetrahydro-β-carboline (300 mg, 1.302 mmol), 3-(thiophen-2-yl)propanoic acid (244 mg, 1.563 mmol), HATU (741.4 mg, 1.953 mmol) and DIEA (841.9 mg, 6.510 mmol) were added into DMF (4 mL) and stirred at room temperature for 6 h. After the reaction was completed as monitored by TLC, the reaction mixture was extracted with EA, the organic phases were combined and the solvent was evaporated under reduced pressure to obtain the crude product. The product was purified by column chromatography to obtain 2-(3-(thiophen-2-yl)propyl-2,3,4,9-tetrahydro-1 H - pyrido [3, 4- b ] indole-8-carboxylic acid methyl ester (192 mg, yield: 40%).

[0035] 2-(3-(thiophen-2-yl)propyl-2,3,4,9-tetrahydro-1 H - pyrido [3, 4- b ] indole-8-carboxylic acid methyl ester (192 mg, 0.5211 mmol) was dissolved in MeOH (5 mL) and water (1 mL), and lithium hydroxide monohydrate (100 mg) was added to the reaction mixture, which was then reacted at 60 °C overnight. After the reaction was completed as monitored by TLC, the solvent was evaporated under reduced pressure, 1 N dilute hydrochloric acid was added, and the mixture was extracted with EA, the organic phases were combined, and the solvent was evaporated under reduced pressure to obtain the crude product 2-(3-(thiophen-2-yl)propyl-2,3,4,9-tetrahydro-1 H - pyrido [3, 4- b ] indole-8-carboxylic acid, which was used directly in the next step without further purification.

[0036] 2-(3-(thiophen-2-yl)propyl-2,3,4,9-tetrahydro-1 H - pyrido [3, 4- b ] indole-8-carboxylic acid (crude product 172 mg, 0.4852 mmol), 2-hydroxyethylamine (36 mg, 0.5823 mmol), HATU (332 mg, 0.8733 mmol) and DIEA (627 mg, 4.850 mmol) were dissolved in DMF (3 mL) and stirred at room temperature for 6 h. After the reaction was completed as monitored by TLC, the reaction mixture was extracted with EA, the organic phases were combined, and the solvent was evaporated under reduced pressure to obtain the crude product. The product was purified by column chromatography to obtain N -(2-hydroxyethyl)-2-(3-(thiophen-2-yl)propyl)-2,3,4,9-tetrahydro-1 H - pyrido [3, 4- bIndole-8-carboxamide (N-(2-Hydroxyethyl)-2-[3-(thiophen-2-yl)propanoyl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-8-carboxamide) (64 mg, yield: 33.5%). 1 H NMR (500 MHz, DMSO) δ 11.08 - 10.96 (m, 1H), 8.48 - 8.42 (m, 1H), 7.63 - 7.55 (m, 2H), 7.31 - 7.26 (m, 1H), 7.07 - 7.01 (m, 1H), 6.94 - 6.88 (m, 2H), 4.78 - 4.71 (m, 3H), 3.86 - 3.75 (m, 2H), 3.59 - 3.54 (m, 2H), 3.40 (td, J = 6.4, 6.2 Hz, 2H), 3.08 (td, J = 7.4, 7.3 Hz, 2H), 2.83 (t, J = 7.4 Hz, 1H), 2.78 - 2.72 (m, 2H), 2.72 - 2.67 (m, 1H).

[0037] Example 1-2 1-(8-((3-Hydroxyhexahydropyridin-1-yl)carbonyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indol-2-yl)-3-(thiophen-2-yl)propan-1-one (II)

[0038] Using a similar method to that for preparing compound I, 2-hydroxyethylamine was replaced by 3-hydroxypiperidine, and compound II was obtained after final purification by column chromatography, yield: 65%. 1 H NMR (500 MHz, DMSO- d 6) δ 10.83 - 10.73 (m, 1H), 7.47 (dd, J = 7.2, 7.0 Hz, 1H), 7.29 (d, J= 1.6 Hz, 1H), 7.07 – 6.99 (m,2H), 6.94 – 6.88 (m, 2H), 4.90 (br, 1H), 4.73 – 4.63 (m, 2H), 3.88 – 3.74 (m,2H), 3.49 (br, 2H), 3.14 – 3.02 (m, 3H), 2.90 – 2.67 (m, 5H), 1.93 – 1.61 (m,2H), 1.49 – 1.20 (m, 3H)。

[0039] Example 1-3 Synthesis of N-(2-hydroxycyclopentyl)-2-(3-(thiophen-2-yl)propionyl)- 2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-8-carboxamide (III)

[0040] To a solution of 2-(3-(thiophen-2-yl)propyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4- b]indole-8-carboxylic acid (crude product 172 mg, 0.4852 mmol), 2-aminocyclopentan-l- ol (59 mg, 0.5823 mmol), HATU (332 mg, 0.8733 mmol) and DIEA (627 mg, 4,850 mmol) in DMF (5 mL) was stirred at room temperature for 6 h. After completion of the reaction as monitored by TLC, the reaction mixture was extracted with EA and the organic layers were combined and evaporated under reduced pressure to obtain the crude product. The compound III (82 mg, yield: 48%) was purified by column chromatography.1H NMR (500 MHz, DMSO-d6) δ 11.06 - 10.94 (m, 1H), 8.26 (dd, J = 8.8, 6.5 Hz, 1H), 7.67 - 7.60 (m, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.31 - 7.27 (m, 1H), 7.07 - 7.00 (m, 1H), 6.94 - 6.88 (m, 2H), 4.79 (d, J = 3.9 Hz, 1H), 4.73 (s, 2H), 4.11 - 4.03 (m, 2H), 3.87 - 3.74 (m, 2H), 3.11 - 3.05 (m, 2H), 2.86 - 2.68 (m, 4H), 2.09 - 2.02 (m, 1H), 1.93 - 1.86 (m, 1H), 1.72 - 1.65 (m, 2H), 1.59 - 1.47 (m, 2H).

[0041] Example 1-4 Synthesis of N-(3-hydroxycyclopentyl)-2-(3-(thiophen-2-yl)propionyl)- 2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-8-carboxamide (IV)

[0042] Using similar method as for the preparation of compound III, 2-aminocyclopentan-1-ol was replaced by 3-aminocyclopentan-1-ol, compound IV was obtained after purification by column chromatography in 43% yield. 1 H NMR (500 MHz, DMSO- d 6) δ 11.07 - 10.95 (m, 1H), 8.31 (t, J = 7.7 Hz, 1H), 7.64 - 7.52 (m, 2H), 7.31 - 7.26 (m, 1H), 7.07 - 6.99 (m, 1H), 6.95 - 6.88 (m, 2H), 4.73 (s, 2H), 4.57 - 4.50 (m, 1H), 4.28 - 4.21 (m, 1H), 3.87 - 3.73 (m, 2H), 3.12 - 3.03 (m, 2H), 2.83 (t, J = 7.4 Hz, 1H), 2.78 - 2.67 (m, 3H), 2.14 - 2.06 (m, 1H), 1.98 - 1.85 (m, 2H), 1.80 - 1.71 (m, 1H), 1.60 - 1.45 (m, 2H).

[0043] Example 1-5 Synthesis of N-(2-hydroxycyclopentyl)-2-(thiophen-2-ylcarbonyl)- 2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-8-carboxamide (V)

[0044] Crude product 2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-8-carboxylic acid methyl ester (300 mg, 1.30 mmol), thiophene-2-carboxylic acid (200 mg, 1.56 mmol), EDCI (422 mg, 2.21 mmol), HOBt (299 mg, 2.21 mmol) and DIEA (842 mg, 6.510 mmol) were taken in DMF (20 mL) and stirred at room temperature for 6 h. After completion of the reaction as monitored by TLC, the reaction mixture was extracted with EA and the organic layers were combined and evaporated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain 2-(thiophen-2-ylcarbonyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-8-carboxylic acid methyl ester (295 mg, yield: 67%).

[0045] 2-(thiophen-2-ylcarbonyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-8-carboxylic acid methyl ester (295 mg, 867 pmol) was taken in MeOH (10 mL) and H20 (2 mL) and lithium hydroxide monohydrate (100 mg) was added to the reaction mixture and stirred at 60 °C overnight. After completion of the reaction as monitored by TLC, the reaction mixture was evaporated under reduced pressure to obtain the crude product. The pH was adjusted to 5 by adding 1 N dilute hydrochloric acid and the reaction mixture was extracted with EA and the organic layers were combined and evaporated under reduced pressure to obtain the crude product 2-(thiophen-2-ylcarbonyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-8-carboxylic acid which was used as such in the next step without further purification.

[0046] To a solution of 2-(thiophene-2-carbonyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4- b]indole-8-carboxylic acid (crude product 256 mg, 784 µmol), 2-aminocyclopentan-l-ol (95 mg, 941 µmol), HATU (537 mg, 1.41 mmol) and DIEA (1 g, 7.84 mmol) in DMF (10 mL) was stirred at room temperature for 6 h. After the reaction was completed by TLC monitoring, the organic phase was extracted with EA, combined and evaporated to dryness under reduced pressure to obtain the crude product. Compound V (148 mg, yield: 46%) was obtained by column chromatography separation and purification.1H NMR (500 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.27 (d, J = 6.5 Hz, 1H), 7.80 (d, J = 5.0 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.53 (d, J = 3.7 Hz, 1H), 7.19 (t, J = 5.0 Hz, 1H), 7.05 (dd, J = 7.6, 7.6 Hz, 1H), 4.92 (br, 2H), 4.79 (d, J = 3.9 Hz, 1H), 4.10 - 4.04 (m, 2H), 3.96 (t, J = 5.7 Hz, 2H), 2.85 (s, 2H), 2.09 - 2.01 (m, 1H), 1.93 - 1.86 (m, 1H), 1.72 - 1.65 (m, 2H), 1.57 - 1.47 (m, 2H).

[0047] Example 1-6 Synthesis of N-(3-hydroxycyclopentyl)-2-(thiophene-2-carbonyl)-2,3,4,9- tetrahydro-lH-pyrido[3,4-b]indole-8-carboxamide (VI)

[0048] Using a similar method to that for preparing compound V, 2-aminocyclopentan-l-ol was replaced by 3-aminocyclopentan-l-ol, and compound VI was obtained after column chromatography purification, yield: 18%. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 (s, 1H),8.31 (d, J = 7.5 Hz, 1H), 7.80 (d, J= 5.0 Hz, 1H), 7.65 – 7.55 (m, 2H), 7.52(d, J = 3.6 Hz, 1H), 7.19 (dd, J = 5.0, 3.6 Hz, 1H), 7.04 (dd, J = 7.6, 7.6Hz, 1H), 4.92 (br, 2H), 4.60 – 4.46 (m, 2H), 4.29 – 4.20 (m, 1H), 3.95 (t, J = 5.7 Hz, 2H), 2.85 (s, 2H), 2.15 – 2.05 (m, 1H), 2.00 – 1.85 (m, 2H), 1.80 –1.72 (m, 1H), 1.59 – 1.46 (m, 2H)。

[0049] Example 1 -7 Synthesis of N-(2-hydroxyethyl)-2-(2-(thiophen-3-yl)acetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-8-carboxamide (VII)

[0050] Using a similar method to that used for the preparation of compound I, 3-(thiophen-2-yl)propanoic acid was replaced by 2-(thiophen-3-yl)acetic acid, and compound VII was obtained after purification by column chromatography in a yield of 33%. 1 H NMR (500 MHz, DMSO- d 6) δ 11.08 –10.98 (m, 1H), 8.45 (dt, J = 16.4, 5.7 Hz, 1H), 7.64 – 7.52 (m, 2H), 7.50 –7.45 (m, 1H), 7.34 – 7.24 (m, 1H), 7.07 – 6.98 (m, 2H), 4.82 – 4.70 (m, 2H),3.89 – 3.75 (m, 4H), 3.56 (t, J = 6.3 Hz, 2H), 3.42 – 3.38 (m, 3H), 2.74 –2.60 (m, 2H)。

[0051] Example 1-8 Synthesis of 1-(8-((3-hydroxyhexahydropyridin-l-yl)carbonyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indol-2-yl)-2-(thiophen-3-yl)ethan-l-one (VIII)

[0052] Using a similar method to that for preparing compound II, 3-(thiophen-2-yl)propanoic acid was replaced by 2-(thiophen-3-yl)acetic acid, and compound VIII was obtained after final purification by column chromatography in a yield of 82%.1H NMR (500 MHz, DMSO-d6) δ 10.83 - 10.73 (m, 1H), 7.49 - 7.42 (m, 2H), 7.34 - 7.21 (m, 1H), 7.06 - 6.98 (m, 3H), 4.85 (br, 1H), 4.74 - 4.68 (m, 2H), 3.87 - 3.81 (m, 4H), 3.44 - 3.34 (m, 3H), 3.12 - 2.81 (m, 2H), 2.73 - 2.60 (m, 2H), 1.93 - 1.64 (m, 2H), 1.45 - 1.31 (m, 2H).

[0053] Example 1-9 N Synthesis of 1-(8-((3-hydroxyhexahydropyridin-l-yl)carbonyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indol-2-yl)-2-(2-(thiophen-3-yl)acetyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indol-8-carboxamide (IX) H b ]indole-8-carboxamide (IX)

[0054] Using a similar method to that for preparing compound III, 3-(thiophen-2-yl)propanoic acid was replaced by 2-(thiophen-3-yl)acetic acid, and compound IX was obtained after final purification by column chromatography in a yield of 81%. 1 H NMR (500 MHz, DMSO- d 6) δ 11.07 - 10.97 (m, 1H), 8.26 (dd, J = 14.1, 6.6 Hz, 1H), 7.63 (dd, J = 16.0, 7.5 Hz,1H), 7.56 (dd, J ​= 18.1, 7.7 Hz, 1H), 7.49 – 7.45 (m, 1H), 7.33 – 7.25 (m, 1H), 7.06 – 7.00 (m, 2H), 4.82 – 4.70 (m, 2H), 4.12 – 4.02 (m, 2H), 3.88 – 3.76 (m, 4H), 2.73 – 2.60 (m, 2H), 2.10 – 2.01 (m, 1H), 1.94 – 1.85 (m, 1H), 1.73 – 1.63 (m, 2H), 1.58 – 1.46 (m, 2H).

[0055] Example 1-10 N -(3-hydroxycyclopentyl)-2-(2-(thiophen-3-yl)acetyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b ]indole-8-carboxamide (X)

[0056]

[0057] Using a similar method to that for preparing compound IX, 3-(thiophen-2-yl)propionic acid was replaced by 2-(thiophen-3-yl)acetic acid, and compound X was obtained after purification by column chromatography in a yield of 59%. 1 H NMR (500 MHz, DMSO- d 6) δ 11.07 –10.98 (m, 1H), 8.31 (dd, J = 12.9, 7.6 Hz, 1H), 7.63 – 7.51 (m, 2H), 7.49 –7.45 (m, 1H), 7.33 – 7.25 (m, 1H), 7.06 – 6.98 (m, 2H), 4.80 – 4.71 (m, 2H),4.57 – 4.50 (m, 2H), 4.28 – 4.21 (m, 1H), 3.89 – 3.76 (m, 4H), 2.66 (dt, J =36.7, 5.7 Hz, 2H), 2.14 – 2.05 (m, 1H), 1.98 – 1.85 (m, 2H), 1.79 – 1.72 (m,1H), 1.59 – 1.47 (m, 2H).

[0058] Example 1-11 N- (2-hydroxyethyl)-2-((thiophen-2-ylcyclopropyl)carbonyl)-2, 3, 4, 9-tetrahydro-lH- carbazoie-8-carboxylic acid (VIII) H - pyrido [3, 4- b ] indole-8-carboxylic acid (IX)

[0059] Using a similar method to that for preparing compound I, 3-(thiophen-2-yl)propanoic acid was replaced by 1-(thiophen-2-yl)cyclopropane-l-carboxylic acid, and compound IX was obtained after purification by column chromatography, yield: 55%. 1 H NMR (500 MHz, DMSO- d 6) δ 11.06 (s, 1H), 8.43 (t, J = 5.6 Hz, 1H), 7.59 (d, J = 7.4 Hz, 1H), 7.51 (s,1H), 7.35 (s, 1H), 7.05 – 6.93 (m, 3H), 4.91 – 4.66 (m, 4H), 3.87 (s, 2H),3.56 (dt, J = 6.1, 6.1 Hz, 2H), 3.39 (dt, J = 6.2, 6.1 Hz, 2H), 1.52 – 1.32(m, 3H), 1.24 (s, 2H)。

[0060] Examples 1-12 N - (2-hydroxyethyl)-2-((thiophen-2-ylcyclopropyl)carbonyl)-2, 3, 4, 9-tetrahydro-lH- carbazoie-8-carboxylic acid (VIII) H - pyrido [3, 4- b ] indole-8-carboxylic acid (IX)

[0061]

[0062] Using a similar method to that for preparing compound IV, 3-(thiophen-2-yl)propanoic acid was replaced by 1-(thiophen-2-yl)cyclopropane-l-carboxylic acid, and compound XII was obtained after purification by column chromatography, yield: 44%. 1 H NMR (500 MHz, DMSO- d 6) δ 11.06 (s, 1H), 8.29 (d, J = 7.5 Hz, 1H), 7.58 (d, J= 7.5 Hz, 1H), 7.50 (s,1H), 7.35 (s, 1H), 7.04 - 6.87 (m, 3H), 4.90 - 4.67 (m, 2H), 4.57 - 4.48 (m, 2H), 4.27 - 4.21 (m, 1H), 3.86 (s, 2H), 2.77 - 2.62 (m, 1H), 2.40 (s, 2H), 2.13 - 2.05 (m, 1H), 1.98 - 1.85 (m, 2H), 1.79 - 1.72 (m, 1H), 1.59 - 1.29 (m, 5H).

[0063] Example 2: Anti-tumor activity test of anti-tumor active compounds The CCK-8 method was used to detect cell growth inhibition. The CCK-8 reagent contains WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt). In the presence of an electron coupling agent 1-methoxy-5-methyl phenazine dimethyl sulfate (1-methoxy PMS), WST-8 can be reduced to a highly water-soluble orange yellow formazan product by dehydrogenase in mitochondria. The amount of Formazan generated is directly proportional to the number of living cells, so this property can be used for direct cell proliferation and toxicity analysis. The product produces an absorption peak at 450 nm wavelength: the more and faster the cell proliferation, the darker the color; the greater the cell toxicity, the lighter the color. The CCK-8 detection reagent was purchased from Dalian Melin Biotechnology Co., Ltd., item number MA0218-5.

[0064] Twelve thienylacetyl tetrahydro-β-carboline compounds (I-XII) were dissolved in DMSO (dimethyl sulfoxide) to prepare DMSO solutions with concentrations of 1 mM, 10 μM and 100 μM for each compound.

[0065] Breast cancer cells HCC1806 and SUM149PT (4 x 10 3Cells were seeded in 96-well plates at 37°C and 5% CO2 for 24 hours. Then, 1 μL of each of 12 different concentrations of thiophene acetyl tetrahydro-β-carboline compounds (I-XII) prepared with DMSO were added to each well, resulting in final concentrations of 10 μM, 1 μM, and 0.1 μM for each compound in the cell culture solution. The control group received only the same volume of DMSO and was incubated for 72 hours. 10 μL of LCK-8 solution was added to each well and gently mixed until no bubbles were generated. Incubation continued for 0.5-2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula. The percentage of absorbance in the experimental group relative to the absorbance in the control group represents the cell viability or proliferation level; the control group is assumed to be 100%.

[0066] Calculation formula: Cell viability = [(As-Ab) / (Ac-Ab)] × 100%; Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%; As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drugs); Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0067] The results are as follows Figure 1 As shown, among the 12 thiophene acetyl tetrahydro-β-carboline compounds, compounds I-XII exhibited good inhibitory activity at a concentration of 10 μM in human breast cancer cells HCC1806, with survival rates all below 50%; in human breast cancer cells SUM149PT, compounds I-IX, XI, and XII all showed breast cancer inhibitory activity at a concentration of 10 μM.

[0068] Example 3 Thiophene acetyltetrahydro-β-carboline compounds (I-XII) were dissolved in DMSO, and then solutions of each compound with concentrations of 1000 μM, 600 μM, 300 μM, 100 μM, 60 μM, 30 μM, 10 μM, and 6 μM were prepared using DMSO. These solutions were used as test solutions.

[0069] Human breast cancer cells HCC1806 and SUM149PT (4×10⁶) were used. 3HCC1806 and SUM149PT cell lines were inoculated in 96-well plates, respectively, and incubated in a 37℃, 5% CO2 constant temperature incubator for 24 hours. Then 1 μL of the test solution was added to each well of the cell culture, so that the final concentration of each compound in the cell culture solution was 10 μM, 6 μM, 3 μM, 1 μM, 0.6 μM, 0.3 μM, 0.1 μM and 0.06 μM. The control group only added 1 μL of DMSO to the cell culture, and then incubated in the incubator for 72 hours. The proliferation effect was detected by CCK8. The results are shown in Table 1.

[0070] Table 1 Half maximal inhibitory concentration IC of thienylacetyl tetrahydro-β-carboline compounds (I-X) on human breast cancer cell HCC1806 and SUM149PT cell lines 50 (μM)

[0071] According to the results in Table 1, thienylacetyl tetrahydro-β-carboline compounds (I-X) have strong cancer cell toxicity activity on human breast cancer cell HCC1806 and SUM149PT cell lines.

[0072] Example 4 The human breast cancer cell HCC1806 was replaced by human lung cancer cell H460, and the others were the same as in Example 3. The results are shown in Table 2.

[0073] Table 2 Half maximal inhibitory concentration IC of thienylacetyl tetrahydro-β-carboline compounds (I-X) on human lung cancer cell H460 cell line 50 (μM)

[0074] According to the results in Table 2, thienylacetyl tetrahydro-β-carboline compounds (I-X) have strong cancer cell toxicity activity on human lung cancer cell H460 cell line.

[0075] Example 5 The human breast cancer cell HCC1806 was replaced by human osteosarcoma cell MG63. The others were the same as in Example 3. The results of IC 50 values are shown in Table 3.

[0076] Table 3 Half maximal inhibitory concentration IC of thienylacetyl tetrahydro-β-carboline compounds (I-X) on human osteosarcoma cell MG63 cell line 50 (μM)

[0077] According to the results of Table 3, the thienylacetyl tetrahydro-β-carboline compounds (I-X) have strong cancer cell toxicity activity on human osteosarcoma cell MG63 cell strain.

[0078] Example 6 The human breast cancer cell HCC1806 is replaced by human malignant melanoma cell A375. The others are the same as Example 3. IC 50 The results of the values are shown in Table 4.

[0079] Table 4 Half growth inhibition concentration IC of thienylacetyl tetrahydro-β-carboline compounds (I-X) on human malignant melanoma cell A375 cell strain 50 (μM)

[0080] According to the results of Table 4, the thienylacetyl tetrahydro-β-carboline compounds (I-X) have strong cancer cell toxicity activity on human malignant melanoma cell A375 cell strain, IC 50 are 3.296 μM, 4.116 μM, 0.359 μM, 0.687 μM, 0.310 μM, 8.915 μM, 2.742 μM, 5.521 μM, 1.703 μM, 11.370 μM, 1.313 μM and 0.284 μM respectively.

[0081] Example 7 The human breast cancer cell HCC1806 is replaced by human gastric adenocarcinoma cell SGC-7901. The others are the same as Example 3. IC 50 The results of the values are shown in Table 5.

[0082] Table 5 Half growth inhibition concentration IC of thienylacetyl tetrahydro-β-carboline compounds (I-X) on human gastric adenocarcinoma cell SGC-7901 cell strain 50 (μM)

[0083] According to the results of Table 5, the thienylacetyl tetrahydro-β-carboline compounds (I-X) have strong cancer cell toxicity activity on human malignant melanoma cell A375 cell strain.

[0084] Example 8 The human breast cancer cell HCC1806 is replaced by human renal cancer cell G401. The others are the same as Example 3. IC 50 The results of the values are shown in Table 6.

[0085] Table 6 Half growth inhibition concentration IC of thienylacetyl tetrahydro-β-carboline compounds (I-X) on human renal cancer cell G401 cell strain 50(μM)

[0086] According to the results of Table 6, the thienylacetyl tetrahydro-β-carboline compounds have strong cancer cell toxicity activity on human renal cancer cell G401 cell strain. 50 respectively, 22.860 μM, 5.112 μM, 0.056 μM, 1.040 μM, 2.740 μM, 41.160 μM, 13.500 μM, 12.430 μM, 6.185 μM, 36.373 μM, 9.980 μM and 15.160 μM. Among them, compounds II, III, IV, V, IX and XI have strong toxicity on human renal cancer cell G401 cell strain.

[0087] Example 9 The human breast cancer cell HCC1806 is replaced by human glioma cell U251. The others are the same as Example 3. IC 50 The results of IC values are shown in Table 7.

[0088] Table 7 Half growth inhibition concentration IC of thienylacetyl tetrahydro-β-carboline compounds (I-X) on human glioma cell U251 cell strain 50 (μM)

[0089] According to the results of Table 7, the thienylacetyl tetrahydro-β-carboline compounds (I-X) have strong cancer cell toxicity activity on human glioma cell U251 cell strain.

[0090] Example 10 The human breast cancer cell HCC1806 is replaced by human ovarian cancer cell SKOV3. The others are the same as Example 3. IC 50 The results of IC values are shown in Table 8.

[0091] Table 8 Half growth inhibition concentration IC of thienylacetyl tetrahydro-β-carboline compounds (I-X) on human ovarian cancer cell SKOV3 cell strain 50 (μM)

[0092] According to the results of Table 8, the thienylacetyl tetrahydro-β-carboline compounds (I-X) have strong cancer cell toxicity activity on human ovarian cancer cell SKOV3 cell strain.

[0093] In summary, the thienylacetyl tetrahydro-β-carboline compounds (I-X) have good inhibitory effect on a variety of different types of human tumor cells, which provides an optional solution for broad-spectrum anticancer drugs.

[0094] It should be noted that the foregoing examples have been provided merely for the purposes of illustration and are not intended to limit the application in any way. Descriptions and examples of materials and processes of the application are intended to be illustrative not limiting. Any modifications of the application and other applications of the application will occur to those skilled in the art to which the application pertains and many options for modification of the application will suggest themselves. The application lies in the broadest scope envisaged by the appended claims and equivalents thereof.

Claims

1. A thiophene acetyl tetrahydro-β-carboline compound, characterized in that, The structure of the compound is shown in formula (I) or formula (II): (I) (II) R1 and R2 do not form a cycle, that is In this case, R1 and R2 are each independently selected from any one of the following groups: hydrogen, C2-C8 alkoxy, -(CH2). n -OH, -(CH2) n -SH, -(CH2) n -NH2、-(CH2) n -NHCH3, -(CH2) n -N(CH3)2、C 3-8 Cycloalkyl groups, C-type compounds substituted with one or two hydroxyl groups 3-8 Cycloalkyl, piperidinyl, oxazinyl, morpholinyl, piperazinyl, and tetrahydrofuranyl; or R1 and R2 form a loop, that is When, it is selected from any one of the following groups: aziridine, cyclobutane, tetrahydropyrrole, hydroxytetrahydropyrrole, oxazolidine, isoxazolidine, piperidine, 2-hydroxypiperidine, 3-hydroxypiperidine, 4-hydroxypiperidine, morpholine, 1,3-oxazine, [1,2]oxazine, piperazine, hexahydropyrimidine, hydroxyhexahydropyrimidine, hexahydropyridazine, hexamethyleneimine, 1,4-oxazazecycloheptane and 1,4-diazacycloheptane; n is an integer between 0 and 8.

2. The thiophene acetyl tetrahydro-β-carboline compound according to claim 1, characterized in that, The compound is selected from one of the following compounds: N -(2-hydroxyethyl)-2-(3-(thien-2-yl)propionyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; 1-(8-((3-hydroxyhexahydropyridin-1-yl)carbonyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indol-2-yl)-3-(thien-2-yl)prop-1-one; N -(2-hydroxycyclopentyl)-2-(3-(thien-2-yl)propionyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; N -(3-hydroxycyclopentyl)-2-(3-(thien-2-yl)propionyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; N -(2-hydroxycyclopentyl)-2-(thiophene-2-ylcarbonyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; N -(3-hydroxycyclopentyl)-2-(thiophene-2-ylcarbonyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; N -(2-hydroxyethyl)-2-(2-(thiophene-3-yl)acetyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; 1-(8-((3-hydroxyhexahydropyridin-1-yl)carbonyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indol-2-yl)-2-(thiophen-3-yl)ethyl-1-one; N -(2-hydroxycyclopentyl)-2-(2-(thiophen-3-yl)acetyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; N -(3-hydroxycyclopentyl)-2-(2-(thiophen-3-yl)acetyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; N -(2-hydroxyethyl)-2-((thiophene-2-ylcyclopropyl)carbonyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide; N -(3-hydroxycyclopentyl)-2-((thiophene-2-ylcyclopropyl)carbonyl)-2,3,4,9-tetrahydro-1 H -pyrido[3,4- b Indole-8-carboxamide.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a thiophene acetyl tetrahydro-β-carboline compound as described in any one of claims 1-2, or its hydrate or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition further includes a second agent for the prevention and / or treatment of cancer.

5. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition further includes excipients, diluents, adjuvants, carriers, or combinations thereof.

6. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition is formulated as an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, or transdermal patch.

7. The use of the compound of any one of claims 1-2 or the pharmaceutical composition of any one of claims 3-6 in the preparation of a medicament for the prevention and / or treatment of cancer.

8. The application according to claim 7, characterized in that, The cancer is at least one of breast cancer, lung cancer, osteosarcoma, melanoma, stomach cancer, pancreatic cancer, kidney cancer, glioma, and ovarian cancer.

9. The use of the compound of any one of claims 1-2 or the pharmaceutical composition of any one of claims 3-6 in the preparation of a tumor stemness modulator.

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