A beta-carboline oral small molecule anti-inflammatory drug, its preparation method and application

Abstract

The present invention discloses a beta-carboline oral small molecule anti-inflammatory drug, its preparation method and application. The structure of the carboline oral small molecule anti-inflammatory drug is shown in general formula I. (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid represented by general formula I, wherein R is NHOH, and AA1-AA2 is Val-Phe, Thr-Phe, Ser-Phe or Leu-Phe, namely FV-KLNHOH, FT-KLNHOH, FS-KLNHOH, FL-KLNHOH. The present invention also discloses a preparation method of the above compound and the application of the compound in the preparation of TNF-α down-regulators and anti-inflammatory drugs, especially oral small molecule anti-inflammatory drugs. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a class of beta-carboline oral small molecule anti-inflammatory drugs and their preparation methods, and also relates to their applications in the preparation of anti-inflammatory drugs. The present invention belongs to the field of biomedical technology. Background Art

[0002] Inflammation is a reaction of the body to tissue damage, physical damage, ischemic damage, bacterial or viral infections, etc. The body's inflammatory response causes cellular changes and immune responses, leading to the repair of damaged tissues and cell proliferation at the damaged tissue site. If inflammation persists, it will lead to the occurrence of chronic inflammation. In a chronic inflammatory environment, cells will mutate and proliferate, usually creating an environment conducive to the development of cancer. Therefore, regulating inflammation is crucial.

[0003] TNF-α is mainly produced by activated macrophages and T lymphocytes. High levels of TNF-α are associated with various inflammatory diseases such as rheumatoid arthritis (RA), Crohn's disease, ulcerative colitis, diabetes, multiple sclerosis, and atherosclerosis. Currently, mainly by injecting biological agents such as adalimumab, infliximab, etanercept and other drugs to bind to TNF-α in the body, reducing the level of TNF-α in the human body, but such biological agents have problems such as high cost, inability to be taken orally, and poor patient compliance.

[0004] Carboline alkaloids have the activity of inhibiting the production of pro-inflammatory cytokines. Therefore, the inventor conjectures that a novel carboline derivative may have good anti-inflammatory activity in vivo.

[0005] Therefore, the inventor modified the structure of carboline and introduced a dipeptide chain. Experiments found that this class of compounds has good anti-inflammatory activity. Therefore, the inventor proposed the present invention. Summary of the Invention

[0006] The purpose of the present invention is to provide a class of novel oral small molecule anti-inflammatory drugs and their preparation methods and applications.

[0007] To achieve the above purpose, the present invention adopts the following technical means:

[0008] The first object of the present invention is to provide a (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid having the structure of general formula I, wherein R is NHOH in general formula I, and AA1-AA2 is Val-Phe, Thr-Phe, Ser-Phe or Leu-Phe, namely FV-KLNHOH, FT-KLNHOH, FS-KLNHOH, FL-KLNHOH;

[0009]

[0010] The second object of the present invention is to provide a method for preparing the said (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, and the method comprises the following steps:

[0011] (1) Under the catalysis of 1M H2SO4, L-Trp and formaldehyde undergo Pictet-Spengler condensation to obtain 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid;

[0012] (2) Using methanol as a solvent, under the catalysis of thionyl chloride, the carboxyl group at the 3-position of 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid is introduced with a methyl ester protection to obtain methyl 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylate, namely KLSS-OMe;

[0013] (3) Using anhydrous tetrahydrofuran as a solvent, under the conditions of DCC and HOBt, KLSS-OMe and Boc-Phe-Val-OH are subjected to amide condensation to form methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-valine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate, namely Boc-FV-KLSS-OMe;

[0014] (4) In methanol, under the catalysis of sodium hydroxide, the methyl ester of Boc-FV-KLSS-OMe is hydrolyzed to obtain (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-valine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, namely Boc-FV-KLSS;

[0015] (5) Using dichloromethane as a solvent, under the conditions of EDC and HOBt, Boc-FV-KLSS reacts with hydroxylamine hydrochloride to form (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-valine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid oxime, namely Boc-FV-KLNHOH;

[0016] (6) Boc-FV-KLNHOH is deprotected by Boc under the condition of 4N EA / HCl to obtain the target compound (3S)-2-(L-phenylalanine-L-valine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid oxime, namely FV-KLNHOH;

[0017] (7) Using anhydrous tetrahydrofuran as a solvent, under the conditions of DCC and HOBt, KLSS-OMe and Boc-Phe-Leu-OH are subjected to amide condensation to form methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-leucine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate, namely Boc-FL-KLSS-OMe;

[0018] (8) In methanol, catalyzed by sodium hydroxide, the methyl ester of Boc-FL-KLSS-OMe is hydrolyzed to obtain (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-leucine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, which is Boc-FL-KLSS;

[0019] (9) Using dichloromethane as the solvent, under the conditions of EDC and HOBt, Boc-FL-KLSS reacts with hydroxylamine hydrochloride to form (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-leucine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid oxime, which is Boc-FL-KLNHOH;

[0020] (10) Boc-FL-KLNHOH is deprotected by Boc under the condition of 4N EA / HCl to obtain the target compound (3S)-2-(L-phenylalanine-L-valine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid oxime, which is FL-KLNHOH;

[0021] (11) Using anhydrous tetrahydrofuran as the solvent, under the conditions of DCC and HOBt, KLSS-OMe and Boc-Phe-Ser-OH are amide condensed to form methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate, which is Boc-FS-KLSS-OMe;

[0022] (12) In methanol, catalyzed by sodium hydroxide, the methyl ester of Boc-FS-KLSS-OMe is hydrolyzed to obtain (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, which is Boc-FS-KLSS;

[0023] (13) Using dichloromethane as the solvent, under the conditions of EDC and HOBt, Boc-FS-KLSS reacts with hydroxylamine hydrochloride to form (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid oxime, which is Boc-FS-KLNHOH;

[0024] (14) Boc-FS-KLNHOH is deprotected by Boc under the condition of 4N EA / HCl to obtain the target compound (3S)-2-(L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid oxime, which is FS-KLNHOH;

[0025] (15) Using anhydrous tetrahydrofuran as the solvent, under the conditions of DCC and HOBt, KLSS-OMe and Boc-Phe-Thr-OH are subjected to amide condensation to form methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-threonyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate, which is Boc-FT-KLSS-OMe;

[0026] (16) In methanol, catalyzed by sodium hydroxide, the methyl ester of Boc-FT-KLSS-OMe is hydrolyzed to obtain (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-threonyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, which is Boc-FT-KLSS;

[0027] (17) Using dichloromethane as the solvent, under the conditions of EDC and HOBt, Boc-FT-KLSS reacts with hydroxylamine hydrochloride to form (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-threonyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid oxime, which is Boc-FT-KLNHOH;

[0028] (18) Boc-FT-KLNHOH is deprotected by Boc under the condition of 4N EA / HCl to obtain the target compound (3S)-2-(L-phenylalanyl-L-threonyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid oxime, which is FT-KLNHOH.

[0029] The third object of the present invention is to provide the use of the described (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in the preparation of anti-inflammatory drugs.

[0030] The use of the described (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in down-regulating the pro-inflammatory cytokine TNF-α in vitro. And the use of the described (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid in the preparation of a drug for down-regulating the pro-inflammatory cytokine TNF-α in vivo.

[0031] Among them, preferably, the anti-inflammatory drug is an oral small molecule anti-inflammatory drug.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] Based on the structure of carboline, the present invention modifies it and introduces a dipeptide chain to prepare a class of (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids with the structure of general formula I. Experiments have found that this class of compounds exhibits good TNF-α inhibitory activity both in vivo and in vitro. Among them, the inhibitory effect of FV-KLSS is comparable to that of the known TACE inhibitor TMI-1 at the same concentration, and shows a certain dose-dependent relationship. The present invention provides a new therapeutic drug for the treatment of inflammation and enriches the drug options for treating inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the synthetic route of (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid.

[0035] i: 37% HCHO, H2SO4, H2O; ii: SOCl2, MeOH; iii: DCC, HOBt, NMM, THF; iv: 2N NaOH, MeOH; v: EDC, HOBt, TEA, DCM; vi: 4N EA / HCl. 3a AA1-AA2 = Ser-Phe, 3b AA1-AA2 = Thr-Phe, 3c AA1-AA2 = Val-Phe, 3d AA1-AA2 = Leu-Phe, 4a AA1-AA2 = Ser-Phe, 4b AA1-AA2 = Thr-Phe, 4c AA1-AA2 = Val-Phe, 4d AA1-AA2 = Leu-Phe, 5a AA1-AA2 = Ser-Phe, 5b AA1-AA2 = Thr-Phe, 5c AA1-AA2 = Val-Phe, 5d AA1-AA2 = Leu-Phe, 6a AA1-AA2 = Ser-Phe, 6b AA1-AA2 = Thr-Phe, 6c AA1-AA2 = Val-Phe, 6d AA1-AA2 = Leu-Phe, 7a AA1-AA2 = Ser-Phe, 7b AA1-AA2 = Thr-Phe, 7c AA1-AA2 = Val-Phe, 7d AA1-AA2 = Leu-Phe. DETAILED DESCRIPTION OF THE INVENTION

[0036] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention and should not be construed as a limitation of the present invention.

[0037] Example 1 Preparation of 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (KLSS)

[0038] Under ice bath conditions, 0.2 mL of concentrated sulfuric acid was added to 400 mL of water, and then 5.00 g (24.5 mmol) of L-tryptophan was added. After dissolution, 10 mL of formaldehyde solution was added, and the mixture was stirred at room temperature for 4 - 5 hours. After the reaction was completed, the pH was adjusted to 7, filtered, the filter cake was collected and washed three times with a small amount of methanol to obtain 5.15 g (99%) of the filter cake, which was the target compound 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid. ESI / MS: 217 [M+H] + ; 1 H NMR (300 MHz, DMSO-d6) δ 10.95 (s, 1H), 7.47 (q, J = 8.5 Hz, 2H), 7.07 (m, 2H), 5.42 (s, 1H), 4.42 (d, J = 15.8 Hz, 1H), 4.25 (m, 1H), 3.63 (m, 1H), 3.14 (m, 1H), 2.85 (m, 1H).

[0039] Example 2 Preparation of methyl 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (KLSS-OMe)

[0040] Under ice bath conditions, 3 mL of SOCl2 was added to 40 mL of methanol and activated under ice bath. After the activation was completed, 3.00 g (15 mmol) of 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid was weighed and added to the reaction solution. The raw materials attached to the wall were rinsed with a small amount of methanol, and a drying tube was added. The ice bath was removed, and the reaction was stirred at room temperature for 48 hours. After the reaction was completed, the methanol was pumped dry with a water pump, and an appropriate amount of methanol was added successively, and then dried with petroleum ether. It was redissolved with ethyl acetate, an appropriate amount of saturated sodium bicarbonate solution was added to remove salts, and then it was placed in a separatory funnel and allowed to stand for layering. It was washed successively with saturated NaHCO3 and saturated NaCl. The ester layer was dried with anhydrous Na2SO4 and then filtered and concentrated by rotary evaporation. Silica gel column chromatography purification was carried out to obtain 1.7 g (54%) of a white solid compound, which was methyl 3S-1,2,3,4-tetrahydro-β-carboline-3-carboxylate, KLSS-OMe. ESI / MS: 231.1 [M+H] + ; 1 H NMR (300 MHz, DMSO-d6) δ 10.73 (s, 1H), 7.37 (m, 1H), 7.28 (m, 1H), 6.99 (m, 2H), 3.97 (m, 2H), 3.74 (dd, J = 8.8, 4.8 Hz, 1H), 3.68 (s, 3H), 2.95 (m, 1H), 2.75 (m, 2H).

[0041] Example 3 Preparation of methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-valyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (Boc-FV-KLSS-OMe)

[0042] Under ice bath conditions, 1.9 g (5.22 mmol) of Boc-FV-OH, 1.08 g (5.22 mmol) of DCC, and 0.7 g (5.22 mmol) of HOBt were added to 40 mL of anhydrous tetrahydrofuran solution and activated for 40 min. Then 1 g (4.35 mmol) of KLSS-OMe was added, and the reaction was carried out overnight at room temperature. The reaction solution was filtered and concentrated by rotary evaporation, redissolved in an appropriate amount of ethyl acetate, and washed three times successively with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The aqueous layer was discarded, and the ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography gave 1.2 g (47%) of the title compound as a white solid. ESI / MS: 577.2 [M+H] + ; 1 1H NMR (300 MHz, DMSO) δ 10.87 (d, J = 7.0 Hz, 1H), 8.15 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.30 (q, J = 7.9, 5.9 Hz, 3H), 7.23–7.10 (m, 2H), 7.13–7.03 (m, 1H), 7.00 (q, J = 9.5, 7.7 Hz, 2H), 5.71–5.58 (m, 1H), 5.23 (dd, J = 24.0, 16.4 Hz, 1H), 4.73 (s, 1H), 4.33–4.14 (m, 2H), 4.03 (q, J = 7.1 Hz, 1H), 3.61–3.50 (m, 3H), 3.41 (d, J = 14.8 Hz, 2H), 2.88 (d, J = 13.9 Hz, 1H), 2.76–2.62 (m, 1H), 2.17 (s, 1H), 1.29 (s, 9H), 1.03–0.77 (m, 6H).

[0043] Example 4 Preparation of (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-valine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (Boc-FV-KLSS) Synthesis

[0044] Under ice bath conditions, 500 mg (0.87 mmol) of Boc-FV-KLSS-OMe was dissolved in methanol solution, and an appropriate amount of NaOH solution was added to the system. After the reaction was completed, the acid was added for neutralization, and a solid precipitated. The methanol in the reaction system was removed by rotary evaporation and then transferred to a separatory funnel. Ethyl acetate was added for extraction, and the ester layer was collected. The ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation to obtain 318 mg (65%) of the title compound as a yellow solid. ESI / MS: 563.3 [M+H] + ; 11H NMR (300 MHz, DMSO) δ 10.94 (dd, J = 14.0, 5.1 Hz, 1H), 8.26–8.07 (m, 1H), 7.38–7.19 (m, 4H), 7.20 (s, 2H), 7.22–6.92 (m, 4H), 5.71–5.57 (m, 1H), 5.22 (dd, J = 24.0, 16.4 Hz, 1H), 4.80–4.64 (m, 1H), 4.33–4.13 (m, 2H), 3.68–3.55 (m, 1H), 3.31 (dd, J = 24.5, 14.8 Hz, 2H), 3.17 (s, 1H), 2.88 (d, J = 14.1 Hz, 1H), 2.71 (q, J = 12.8 Hz, 1H), 1.35 (s, 9H), 1.03–0.76 (m, 6H).

[0045] Example 5 Preparation of (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-valyl)-1,2,3,4-tetrahydro-β-carboline-3-hydroxamic acid (Boc-FV-KLNHOH)

[0046] Under ice bath conditions, 200 mg (0.35 mmol) of Boc-FV-KLSS was dissolved in dichloromethane, and then 80 mg (0.42 mmol) of EDC and 57 mg (0.42 mmol) of HOBt were added. After activation for 2 h, 37 mg (0.525 mmol) of solid hydroxylamine hydrochloride was added, and the pH was adjusted to 9 with triethylamine. The ice bath was removed, and the reaction was carried out at room temperature for 48 h. The reaction was monitored by TLC (dichloromethane:methanol 15:1). After 72 h, the reaction still could not proceed completely, and the reaction was terminated. The reaction solution was filtered and concentrated by rotary evaporation, redissolved in an appropriate amount of ethyl acetate, and washed successively with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl three times. The aqueous layer was discarded. The ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography (dichloromethane-methanol system) gave 65 mg (32%) of the title compound as a white solid. ESI / MS: 578.4 [M + H] + ; 11H NMR (300 MHz, DMSO) δ 10.86 (d, J = 11.4 Hz, 1H), 8.19 (dt, J = 32.4, 9.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.38–7.18 (m, 5H), 7.15 (s, 1H), 7.10–6.98 (m, 1H), 7.01–6.84 (m, 1H), 5.78 (d, J = 15.6 Hz, 1H), 4.86–4.64 (m, 1H), 4.19 (d, J = 17.4 Hz, 1H), 3.61–3.48 (m, 3H), 2.99 (d, J = 17.5 Hz, 2H), 2.90 (s, 1H), 2.73 (s, 1H), 1.32–1.15 (s, 9H), 0.84 (dd, J = 10.6, 5.9 Hz, 6H).

[0047] Example 6 Preparation of (3S)-2-(L-phenylalanine-L-valine)-1,2,3,4-tetrahydro-β-carboline-3-isohydroxamic acid (FV-KLNHOH) Synthesis

[0048] Under ice bath conditions, 100 mg (0.17 mmol) of Boc-FV-KLNHOH was dissolved in 5 mL of 4N EA / HCl, keeping it dry throughout. The disappearance of the starting material spot was monitored by TLC. Then the reaction solution was dried with a circulating water pump, and then redissolved and dried three times successively with dried ethyl acetate and petroleum ether. Purification was carried out using a reverse-phase C 18 column (methanol, water system). After purification, the methanol was evaporated to dryness, and the remaining water was freeze-dried to obtain 34.9 mg (43%) of the title compound as a white solid. m.p.: 168.8 - 170.3 °C; ESI / MS: 478.3 [M+H] + ; 1 1H NMR (300 MHz, DMSO) δ 10.97–10.83 (m, 1H), 8.84 (ddd, J = 43.4, 17.8, 8.3 Hz, 1H), 8.27 (s, 2H), 7.43 (d, J = 7.9 Hz, 1H), 7.39–7.20 (m, 6H), 7.13–6.92 (m, 3H), 5.80 (dd, J = 14.2, 5.7 Hz, 1H), 5.29 (dd, J = 31.0, 16.2 Hz, 1H), 4.76–4.59 (m, 1H), 4.15 (s, 1H), 3.57 (d, J = 4.4 Hz, 3H), 3.19–3.03 (m, 1H), 3.07–2.94 (m, 2H), 2.94–2.83 (m, 1H), 0.96–0.62 (m, 6H).

[0049] Example 7 Preparation of Methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-leucyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (Boc-FL-KLSS-OMe)

[0050] Under ice bath conditions, 1.97 g (5.22 mmol) of Boc-FL-OH, 1.08 g (5.22 mmol) of DCC, and 0.7 g (5.22 mmol) of HOBt were added to 40 mL of anhydrous tetrahydrofuran solution and activated for 40 min. Then 1 g (4.35 mmol) of KLSS-OMe was added, and the reaction was carried out overnight at room temperature. The reaction solution was filtered and concentrated by rotary evaporation, redissolved in an appropriate amount of ethyl acetate, and washed three times successively with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The aqueous layer was discarded, and the ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography gave 1.7 g (67%) of the title compound as a white solid. ESI / MS: 591.5 [M+H] + ; 1 1H NMR (300 MHz, DMSO) δ 10.95 (s, 1H), 8.26 (dd, J = 15.3, 8.5 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.37–7.24 (m, 2H), 7.27–7.12 (m, 3H), 7.10 (s, 1H), 7.15–6.93 (m, 2H), 6.83 (d, J = 8.7 Hz, 1H), 5.20 (d, J = 15.4 Hz, 1H), 5.11–4.92 (m, 1H), 4.68 (d, J = 15.4 Hz, 2H), 4.35–4.12 (m, 1H), 3.55 (d, J = 10.0 Hz, 3H), 3.36 (d, J = 15.7 Hz, 2H), 2.91 (qd, J = 10.3, 9.8, 6.0 Hz, 2H), 1.74 (dt, J = 11.0, 5.2 Hz, 1H), 1.64–1.52 (m, 1H), 1.29 (d, J = 3.9 Hz, 7H), 1.26–1.12 (m, 3H), 0.99 (dt, J = 6.6, 4.0 Hz, 4H), 0.88 (d, J = 6.2 Hz, 2H).

[0051] Example 8 Preparation of (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-leucyl)-1,2,3,4-tetrahydro-β-carboxylic acid (Boc-FL-KLSS)

[0052] Under ice bath conditions, 1 g (1.7 mmol) of Boc-FL-KLSS-OMe was dissolved in a methanol solution, and then 2N NaOH solution was added to the system to adjust the pH to 13. After the reaction was completed, the system was neutralized with 1N aqueous HCl solution. The methanol in the reaction system was evaporated and the residue was transferred to a separatory funnel. Ethyl acetate was added for extraction, and the ester layer was collected. The ester layer was dried over anhydrous Na2SO4, filtered, and evaporated to obtain 705 mg (72%) of the title compound as a white solid. ESI / MS: 577.4 [M+H] + ; 1 H NMR (300 MHz, DMSO) δ 12.87 (s, 1H), 10.93 (s, 1H), 8.17 (dd, J = 15.5, 8.3 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.37–7.11 (m, 6H), 7.15–6.98 (m, 1H), 7.01–6.79 (m, 1H), 5.62 (ddd, J = 17.5, 6.3, 1.6 Hz, 1H), 5.21–5.01 (m, 1H), 4.97 (dd, J = 11.5, 5.3 Hz, 1H), 4.83 (d, J = 10.1 Hz, 1H), 4.37–4.13 (m, 2H), 4.03 (q, J = 7.1 Hz, 1H), 3.33 (dt, J = 26.7, 16.3 Hz, 1H), 3.04–2.85 (m, 1H), 2.90–2.61 (m, 2H), 1.51 (s, 3H), 1.34–1.19 (s, 9H), 1.02–0.86 (m, 3H), 0.90–0.76 (m, 3H).

[0053] Example 9 Preparation of (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-leucyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid hydroxamic acid (Boc-FL-KLNHOH)

[0054] Under ice bath conditions, 202 mg (0.35 mmol) of Boc-FL-KLSS was dissolved in dichloromethane, then 80 mg (0.42 mmol) of EDC and 57 mg (0.42 mmol) of HOBt were added, and the mixture was activated for 2 h. Subsequently, 37 mg (0.525 mmol) of solid hydroxylamine hydrochloride was added, and the pH was adjusted to weakly basic with triethylamine. The ice bath was removed, and the reaction was carried out at room temperature for 48 h to terminate the reaction. The reaction solution was filtered and concentrated by rotary evaporation, redissolved in an appropriate amount of ethyl acetate, and washed three times successively with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The aqueous layer was discarded, and the ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography (dichloromethane-methanol system) gave 54 mg (26%) of the title compound as a white solid. ESI / MS: 592.5 [M+H] + ; 1 H NMR (300 MHz, DMSO) δ 11.03–10.42 (m, 1H), 8.77 (d, J = 19.3 Hz, 0H), 8.52–8.06 (m, 1H), 7.52–6.71 (m, 8H), 5.28–4.58 (m, 3H), 4.56–3.81 (m, 2H), 2.91 (d, J = 14.1 Hz, 2H), 2.73 (s, 2H), 1.55 (d, J = 20.1 Hz, 2H), 1.40–1.12 (m, 10H), 1.01–0.78 (m, 6H).

[0055] Example 10 Preparation of (3S)-2-(L-phenylalanine-L-leucine)-1,2,3,4-tetrahydro-β-carboline-3-hydroxamic acid (FL-KLNHOH)

[0056] Under ice bath conditions, 100 mg (0.17 mmol) of Boc-FV-KLNHOH was dissolved in 5 mL of 4N EA / HCl, and the whole process was kept dry. The disappearance of the starting material spot was monitored by TLC. The reaction solution was then dried by a circulating water pump, and then redissolved three times successively with dried ethyl acetate and petroleum ether and dried by suction respectively. Purification was carried out using a reverse-phase C 18 column. After purification, methanol was concentrated by rotary evaporation, and the remaining water was freeze-dried to obtain 51 mg (60%)(43%) of the title compound as a white solid. m.p.: 202.8 - 203.9 °C; ESI / MS: 492.3 [M+H] + ; 11H NMR (300 MHz, DMSO) δ 11.01 (d, J = 13.5 Hz, 1H), 8.99 (t, J = 8.9 Hz, 1H), 8.89 (s, 1H), 8.29 (s, 2H), 7.40–7.23 (m, 5H), 7.18–6.93 (m, 4H), 5.22–5.03 (m, 1H), 4.85 (s, 1H), 4.14–3.92 (m, 2H), 3.12–3.02 (m, 2H), 3.02–2.84 (m, 1H), 1.72 (s, 1H), 1.58 (d, J = 7.6 Hz, 1H), 1.40–1.24 (m, 1H), 0.98 (s, 1H), 0.88 (dq, J = 13.7, 7.7, 7.0 Hz, 4H), 0.79–0.71 (m, 1H).

[0057] Example 11 Preparation of Methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanyl-L-threonyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (Boc-FT-KLSS-OMe)

[0058] Under ice bath conditions, 1.91 g (5.22 mmol) of Boc-FT-OH, 1.08 g (5.22 mmol) of DCC, and 0.7 g (5.22 mmol) of HOBt were added to an anhydrous tetrahydrofuran solution and activated for 40 min. Then 1 g (4.35 mmol) of KLSS-OMe was added. The ice bath was removed, and the reaction was carried out overnight at room temperature. After the reaction was complete, the reaction was terminated. The reaction solution was filtered and concentrated by rotary evaporation, redissolved in an appropriate amount of ethyl acetate, and washed three times successively with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The aqueous layer was discarded. The ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography gave 1.6 g (64%) of the title compound as a white solid. ESI / MS: 579.1 [M+H] + ; 11H NMR (300 MHz, DMSO) δ 10.90 (d, J = 14.9 Hz, 1H), 7.94 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.30 (q, J = 6.6, 5.0 Hz, 4H), 7.25–7.03 (m, 3H), 7.00 (q, J = 9.8, 7.5 Hz, 1H), 5.76 (s, 1H), 5.03 (t, J = 13.0 Hz, 1H), 4.75 (dd, J = 14.2, 6.1 Hz, 1H), 4.24 (t, J = 14.1 Hz, 2H), 3.97 (q, J = 5.7, 5.1 Hz, 1H), 3.55 (q, J = 5.5, 4.1 Hz, 3H), 3.38 (d, J = 15.7 Hz, 1H), 2.90 (s, 1H), 2.71 (q, J = 14.2 Hz, 1H), 1.31 (s, 9H), 1.04 (d, J = 6.6 Hz, 3H).

[0059] Example 12 Preparation of (3S)-2-((tert-butyl ester)-L-phenylalanine-L-threonine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (Boc-FT-KLSS)

[0060] Under ice bath conditions, 1 g (1.73 mmol) of Boc-FT-KLSS-OMe was dissolved in a methanol solution, and then 2N NaOH solution was added to the system. The reaction was terminated after completion. The pH was adjusted with 1N aqueous HCl. The methanol in the reaction system was evaporated and transferred to a separatory funnel. Ethyl acetate was added for extraction, and the ester layer was collected. The ester layer was dried over anhydrous Na2SO4, filtered, and evaporated to obtain 625 mg (64%) of the title compound as a white solid. ESI / MS: 565.1 [M+H] + ; 1 1H NMR (300 MHz, DMSO) δ 12.72 (s, 1H), 10.99 (d, J = 8.1 Hz, 1H), 7.93 (dd, J = 24.1, 8.5 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.37–7.23 (m, 5H), 7.26–7.11 (m, 2H), 7.01 (dt, J = 21.0, 1H), 5.45 (m, 1H), 5.02 (d, J = 17.4 Hz, 1H), 4.82–4.67 (m, 1H), 4.39–4.17 (m, 1H), 3.98 (s, 1H), 3.16 (m, 2H), 2.99 (m, 2H), 2.83–2.68 (m, 1H), 1.30 (s, 9H), 1.16–0.97 (m, 3H).

[0061] Example 13 Preparation of (3S)-2-((tert-Butoxycarbonyl)-L-phenylalanyl-L-threonine)-1,2,3,4-tetrahydro-β-carboline-3-hydroxamic acid (Boc-FT-KLNHOH)

[0062] Under ice bath conditions, 197 mg (0.35 mmol) of Boc-FT-KLSS was dissolved in dichloromethane, and then 80 mg (0.42 mmol) of EDC and 57 mg (0.42 mmol) of HOBt were added. After activation for 2 h, 37 mg (0.525 mmol) of solid hydroxylamine hydrochloride was added, and the mixture was adjusted to alkaline with triethylamine. The ice bath was removed, and the reaction was carried out at room temperature for 48 h to terminate the reaction. The reaction solution was filtered and concentrated by rotary evaporation, redissolved in an appropriate amount of ethyl acetate, and washed three times successively with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The aqueous layer was discarded, and the ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography gave 55 mg (27%) of the title compound as a white solid. ESI / MS: 580.2 [M+H] + ; 1 1H NMR (300 MHz, DMSO) δ 10.95–10.80 (m, 1H), 9.78–9.62 (m, 1H), 7.42 (dd, J = 13.8, 7.7 Hz, 1H), 7.28 (tt, J = 8.5, 3.9 Hz, 5H), 7.20 (s, 2H), 7.09–6.98 (m, 1H), 7.03–6.89 (m, 2H), 5.47 (s, 1H), 5.18 (d, J = 17.3 Hz, 1H), 5.10–4.92 (m, 1H), 5.00 (m, 1H), 4.55–4.80 (m, 1H), 4.38 (m, 1H), 4.23 (s, 1H), 4.09–3.93 (m, 1H), 3.55 (d, J = 2.6 Hz, 1H), 3.00–2.87 (m, 1H), 2.81–2.66 (m, 1H), 1.36 (s, 9H), 1.17 (d, J = 7.1 Hz, 3H).

[0063] Example 14 Preparation of (3S)-2-(L-Phenylalanyl-L-threonine)-1,2,3,4-tetrahydro-β-carboline-3-hydroxamic acid (FT-KLNHOH)

[0064] Under ice bath conditions, 100 mg (0.173 mmol) of Boc-FT-KLNHOH was dissolved in 5 mL of 4N EA / HCl, and the whole process was kept dry. The disappearance of the starting material spot was monitored by TLC. Then the reaction solution was dried by a circulating water pump, and then redissolved and dried three times successively with dried ethyl acetate and petroleum ether. Reverse phase C 18The column was purified, and after lyophilization, 42 mg (51%) of the title compound was obtained as a white solid. m.p.: 246.9 - 248.5 °C; ESI / MS: 480.2 [M+H] + ; 1 H NMR (300 MHz, DMSO) δ 10.91 (d, J = 7.1 Hz, 1H), 10.83 (s, 1H), 7.50–7.38 (m, 1H), 7.29 (q, J = 6.2, 4.5 Hz, 3H), 7.22 (d, J = 15.5 Hz, 3H), 7.22–7.05 (m, 1H), 7.11–6.93 (m, 2H), 5.61 (d, J = 6.3 Hz, 1H), 5.30 (s, 1H), 5.12 (d, J = 16.0 Hz, 1H), 4.97 (d, J = 16.6 Hz, 1H), 4.47 (q, J = 7.5 Hz, 1H), 4.38–4.17 (m, 1H), 4.09–3.92 (m, 1H), 3.54 (s, 1H), 2.90 (dd, J = 12.7, 7.2 Hz, 2H), 2.62 (dd, J = 13.4, 8.0 Hz, 1H), 1.08 (tq, J = 7.0, 6.2 Hz, 3H).

[0065] Example 15 Preparation of Methyl (3S)-2-((tert-Butoxycarbonyl)-L-phenylalanyl-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate (Boc-FS-KLSS-OMe)

[0066] Under ice bath conditions, 1.84 g (5.22 mmol) of Boc-FS-OH, 1.08 g (5.22 mmol) of DCC, and 0.7 g (5.22 mmol) of HOBt were added to an anhydrous tetrahydrofuran solution and activated for 40 min. Then 1 g (4.35 mmol) of KLSS-OMe was added. The ice bath was removed, and the reaction was carried out overnight at room temperature. After the reaction was complete, the reaction was terminated. The reaction solution was filtered and concentrated by rotary evaporation, redissolved in an appropriate amount of ethyl acetate, and washed three times successively with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The aqueous layer was discarded, and the ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography gave 1.3 g (54%) of the title compound as a white solid. ESI / MS: 565.3 [M+H] + ; 11H NMR (300 MHz, DMSO) δ 10.92 (d, J = 22.1 Hz, 1H), 7.45 (dd, J = 8.1, 4.3 Hz, 1H), 7.29 (dq, J = 11.5, 6.5 Hz, 3H), 7.19–6.93 (m, 2H), 5.52 (d, J = 6.1 Hz, 1H), 5.23–4.94 (m, 2H), 4.76 (d, 1H), 4.28 (d, 1H), 4.20 (s, 1H), 3.61–3.49 (m, 3H), 2.90 (d, J = 17.1 Hz, 2H), 2.76–2.62 (m, 2H), 1.31 (s, 9H).

[0067] Example 16 Preparation of (3S)-2-((tert-Butoxycarbonyl)-L-phenylalanyl-L-seryl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (Boc-FS-KLSS)

[0068] Under ice bath conditions, 1 g (1.77 mmol) of Boc-FS-KLSS-OMe was dissolved in a methanol solution, and then 2N NaOH solution was added to the system. The reaction was terminated after completion. The reaction was neutralized with 1N aqueous HCl. After the methanol in the reaction system was evaporated, it was transferred to a separatory funnel, and ethyl acetate was added for extraction. The ester layer was collected, dried over anhydrous Na2SO4, filtered, and evaporated to obtain 584 mg (60%) of the title compound as a white solid. ESI / MS: 551.2 [M+H] + ; 1 1H NMR (300 MHz, DMSO) δ 12.83 (s, 1H), 10.90 (d, J = 16.4 Hz, 1H), 8.14 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.49–6.90 (m, 9H), 5.46 (d, J = 5.6 Hz, 1H), 5.40–5.13 (m, 2H), 5.00 (tt, J = 16.4, 12.8 Hz, 2H), 4.78 (dt, J = 23.0, 9.2 Hz, 1H), 4.38–4.10 (m, 2H), 4.08–3.87 (m, 1H), 2.98 (dddd, J = 27.9, 19.7, 12.9, 6.0 Hz, 2H), 2.71 (ddd, J = 18.5, 13.9, 10.7 Hz, 2H), 1.30 (s, 9H).

[0069] Example 17 Preparation of (3S)-2-((tert-Butoxycarbonyl)-L-phenylalanyl-L-seryl)-1,2,3,4-tetrahydro-β-carboline-3-hydroxamic acid (Boc-FS-KLNHOH)

[0070] Under ice bath conditions, 192 mg (0.35 mmol) of Boc-FS-KLSS was dissolved in dichloromethane, then 80 mg (0.42 mmol) of EDC and 57 mg (0.42 mmol) of HOBt were added, and the mixture was activated for 2 h. Subsequently, 37 mg (0.525 mmol) of solid hydroxylamine hydrochloride was added, and the pH was adjusted to weakly basic with triethylamine. The ice bath was removed, and the reaction was carried out at room temperature for 48 h. Then the reaction solution was concentrated by rotary evaporation, redissolved with an appropriate amount of ethyl acetate, and washed three times successively with saturated aqueous NaHCO3, saturated aqueous NaCl, 5% aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The aqueous layer was discarded, and the ester layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation. Purification by silica gel column chromatography gave 63 mg (32%) of the title compound as a white solid. ESI / MS: 566.2 [M+H] + ; 1 1H NMR (300 MHz, DMSO) δ 10.81 (d, J = 40.5 Hz, 1H), 9.04 (s, 1H), 7.59–6.54 (m, 9H), 5.46–4.99 (m, 2H), 4.59–3.89 (m, 2H), 3.71–3.46 (m, 3H), 3.18–2.81 (m, 3H), 2.73 (m, 1H), 1.34 (s, 9H).

[0071] Example 18 Preparation of (3S)-2-(L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-hydroxamic acid (FS-KLNHOH)

[0072] Under ice bath conditions, 100 mg (0.177 mmol) of Boc-FS-KLNHOH was dissolved in 5 mL of 4N EA / HCl, and the whole process was kept dry. The disappearance of the starting material spot was monitored by TLC. Then the reaction solution was concentrated by a circulating water pump, and then redissolved and concentrated three times successively with dried ethyl acetate and petroleum ether. Purification by a reverse-phase C 18 column gave 35 mg (42%) of the title compound as a white solid. m.p.: 246.9 - 248.5 °C; ESI / MS: 466.6 [M+H] + ; 11H NMR (300 MHz, DMSO) δ 11.07 (s, 1H), 9.73 (d, J = 26.3 Hz, 1H), 8.43 (s, 1H), 8.28 (s, 1H), 7.82 (d, J = 6.5 Hz, 1H), 7.47 (dd, J = 20.3, 10.8 Hz, 1H), 7.30 (q, J = 6.2 Hz, 6H), 7.02 (s, 3H), 5.42–4.69 (m, 2H), 4.69–4.04 (m, 2H), 3.99–3.41 (m, 3H), 3.39–3.23 (m, 2H), 3.18–2.57 (m, 3H).

[0073] Example 19 Evaluate the effect of the above compound on down-regulating the content of TNF-α in vitro

[0074] Experimental materials

[0075] DMEM medium, PBS (Jiangsu Kaygene Biotech Co., Ltd.), TMI-1 (CAS: 287403-39-8, MedChemExpress), lipopolysaccharide (Sigma-Aldrich), fetal bovine serum, DMSO (Tianjin Fuyu Fine Chemical Co., Ltd.)

[0076] Experimental instruments

[0077] Microplate reader (Molecular Devices SpectraMax), microscope (Zeiss), 0.22 μm filter membrane (Millex), high-pressure steam sterilizer (TOMY SX-700), cell incubator (INC153, memmer), centrifuge (Eppendorf), laminar flow hood (Thermo MSC-ADVANTAGE), incubator (Thermo HERACELL 150i), 24-well cell culture plate, 25 cm 2 Culture flask (Costar).

[0078] Cell line

[0079] RAW264.7 (mouse mononuclear macrophage leukemia cells).

[0080] Grouping and dosing

[0081] The concentrations of the compounds of the present invention are 50.00 μM, 25.00 μM, 12.50 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM in sequence; positive control: TMI-1, formulated into the target concentrations (400 nM, 200 nM, 100 nM, 50 nM, 25 nM) with PBS buffer containing 0.5% DMSO; blank control: PBS buffer containing 0.5% DMSO.

[0082] Experimental operation

[0083] RAW264.7 cells were placed in a T75 culture flask. After the cells were cultured stably, the experimental operation began. RAW264.7 cells were seeded in a 24-well plate, 2×10 5 cells per well, and the cells adhered to the wall after overnight incubation in an incubator. The supernatant was discarded, and the culture medium was replaced with 400 μL of incomplete culture medium. A negative control group, a positive control group, an LPS model group, and an experimental group were set up in the cell plate, with 3 replicate wells in each group. 50 μL of incomplete DMEM culture medium was added to the negative control group and the LPS model group, 50 μL of the target compound was added to the experimental group, and 50 μL of TMI-1 was added to the positive control group. Incubate in an incubator for 1 hour. Subsequently, 50 μL of LPS (1 μg / mL) was added to each group except the negative control group, and the cells were incubated in an incubator for 24 h. After the culture was completed, the supernatant culture medium was collected, centrifuged for 10 min, and the supernatant was collected. The content of soluble TNF-α in the cell supernatant was measured using an Elisa kit.

[0084] Inhibition rate = [(average OD value of the model group - average OD value of the sample group) / average OD value of the model group]×100%. A growth curve was plotted with the inhibition rate against the concentration of the test compound on Origin software, and the IC 50 (half-maximal inhibitory concentration) value of the test compound was calculated.

[0085] Experimental results

[0086] The results are shown in Table 1. It can be seen that different test compounds all showed good inhibitory activity against the secretion of soluble TNF-α. The ability of the target compound to inhibit the release of sTNF-α was measured, and it was found that the IC 50 of FS-KLNHOH, FV-KLNHOH, FT-KLNHOH, and FL-KLNHOH was between 1.67 μM and 5.80 μM.

[0087] Table 1 Inhibition rate of LPS-stimulated RAW264.7 cells to secrete TNF-α, IC 50 (μM)

[0088]

[0089] Note: n = 3.

[0090] Example 20 Evaluation of the application of the above compounds in down-regulating TNF-α in vivo

[0091] Experimental materials

[0092] Normal saline (Sichuan Kelun Pharmaceutical Co., Ltd.), CMC-Na (Tianjin Fuchen Chemical Reagent Factory), LPS (Sigma-Aldrich), TMI-1 (CAS: 287403-39-8, MedChemExpress)

[0093] Experimental animals

[0094] ICR strain male mice, weighing 20 ± 2 g, were purchased from Beijing Huafukang Experimental Animal Technology Co., Ltd.

[0095] Grouping of mice and administration doses

[0096] The doses of the compounds of the present invention were 6.25 μmol / kg, 12.5 μmol / kg, and 25 μmol / kg, the positive control TMI-1 doses were 6.25 μmol / kg, 12.5 μmol / kg, and 25 μmol / kg, and the negative control and LPS model groups were both CMC-Na.

[0097] Experimental operations

[0098] The mice were randomly grouped, with 6 mice in each group. They were placed in the animal house and allowed to rest for 48 hours, and fasted 12 h before the operation. According to the groups, the mice were given intragastric administration. 1 h after the administration, the negative control group was intraperitoneally injected with normal saline, and the other groups were intraperitoneally injected with 0.1 mL LPS. 1 hour after the LPS injection, blood was taken from the orbital cavity of the mice. The content of soluble TNF-α in the peripheral blood serum was detected using an Elisa kit.

[0099] Experimental results

[0100] The results are shown in Table 2. In the LPS-induced mouse inflammation model, the 5 target compounds at a dose of 25 μmol / kg could inhibit the production of TNF-α in the serum of the test mice.

[0101] Table 2. Effects of the test compounds on the content of TNF-α in the serum of LPS-induced mouse inflammation

[0102]

[0103] Note: n = 6; a: Compared with 25 μmol / kg of TMI-1, P > 0.05; b: Compared with 12.5 μmol / kg of TMI-1, P > 0.05; c: Compared with 6.25 μmol / kg of TMI-1, P > 0.05.

Claims

1. (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid having the structure of general formula I, characterized in that, In general formula Ⅰ, R is NHOH, and AA1 - AA2 is Val - Phe, Thr - Phe, Ser - Phe or Leu - Phe, which are named compound FV - KLNHOH, FT - KLNHOH, FS - KLNHOH, FL - KLNHOH respectively.

2. The preparation method of (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid according to claim 1, characterized in that, It includes the following steps: (1) L - Trp and formaldehyde undergo Pictet - Spengler condensation under the catalysis of 1M H2SO4 to obtain 3S - 1,2,3,4 - tetrahydro - β - carboline - 3 - carboxylic acid; (2) Using methanol as the solvent, the carboxyl group at the 3 - position of 3S - 1,2,3,4 - tetrahydro - β - carboline - 3 - carboxylic acid is introduced with a methyl ester protection under the catalysis of thionyl chloride to obtain methyl 3S - 1,2,3,4 - tetrahydro - β - carboline - 3 - carboxylate, which is KLSS - OMe; (3) Using anhydrous tetrahydrofuran as the solvent, under the conditions of DCC and HOBt, KLSS - OMe and Boc - Phe - Val - OH undergo amide condensation to form methyl (3S) - 2 - ((tert - butyl ester) - L - phenylalanine - L - valine) - 1,2,3,4 - tetrahydro - β - carboline - 3 - carboxylate, which is Boc - FV - KLSS - OMe; (4) In methanol, under the catalysis of sodium hydroxide, the methyl ester of Boc - FV - KLSS - OMe is hydrolyzed to obtain (3S) - 2 - ((tert - butyl ester) - L - phenylalanine - L - valine) - 1,2,3,4 - tetrahydro - β - carboline - 3 - carboxylic acid, which is Boc - FV - KLSS; (5) Using dichloromethane as the solvent, under the conditions of EDC and HOBt, Boc - FV - KLSS reacts with hydroxylamine hydrochloride to form (3S) - 2 - ((tert - butyl ester) - L - phenylalanine - L - valine) - 1,2,3,4 - tetrahydro - β - carboline - 3 - isohydroxamic acid, which is Boc - FV - KLNHOH; (6) Boc - FV - KLNHOH is deprotected by Boc under the condition of 4N EA / HCl to obtain the target compound (3S) - 2 - (L - phenylalanine - L - valine) - 1,2,3,4 - tetrahydro - β - carboline - 3 - isohydroxamic acid, which is FV - KLNHOH; (7) Using anhydrous tetrahydrofuran as the solvent, under the conditions of DCC and HOBt, KLSS - OMe and Boc - Phe - Leu - OH undergo amide condensation to form methyl (3S) - 2 - ((tert - butyl ester) - L - phenylalanine - L - leucine) - 1,2,3,4 - tetrahydro - β - carboline - 3 - carboxylate, which is Boc - FL - KLSS - OMe; (8) In methanol, under the catalysis of sodium hydroxide, the methyl ester of Boc - FL - KLSS - OMe is hydrolyzed to obtain (3S) - 2 - ((tert - butyl ester) - L - phenylalanine - L - leucine) - 1,2,3,4 - tetrahydro - β - carboline - 3 - carboxylic acid, which is Boc - FL - KLSS; (9) Using dichloromethane as a solvent, under the conditions of EDC and HOBt, Boc-FL-KLSS reacts with hydroxylamine hydrochloride to form (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-leucine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid hydroxamic acid, which is Boc-FL-KLNHOH; (10) Boc-FL-KLNHOH is deprotected by Boc under the condition of 4N EA / HCl to obtain the target compound (3S)-2-(L-phenylalanine-L-valine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid hydroxamic acid, which is FL-KLNHOH; (11) Using anhydrous tetrahydrofuran as a solvent, under the conditions of DCC and HOBt, KLSS-OMe and Boc-Phe-Ser-OH are amide condensed to form methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate, which is Boc-FS-KLSS-OMe; (12) In methanol, catalyzed by sodium hydroxide, the methyl ester of Boc-FS-KLSS-OMe is hydrolyzed to obtain (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, which is Boc-FS-KLSS; (13) Using dichloromethane as a solvent, under the conditions of EDC and HOBt, Boc-FS-KLSS reacts with hydroxylamine hydrochloride to form (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid hydroxamic acid, which is Boc-FS-KLNHOH; (14) Boc-FS-KLNHOH is deprotected by Boc under the condition of 4N EA / HCl to obtain the target compound (3S)-2-(L-phenylalanine-L-serine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid hydroxamic acid, which is FS-KLNHOH; (15) Using anhydrous tetrahydrofuran as a solvent, under the conditions of DCC and HOBt, KLSS-OMe and Boc-Phe-Thr-OH are amide condensed to form methyl (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-threonine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylate, which is Boc-FT-KLSS-OMe; (16) In methanol, catalyzed by sodium hydroxide, the methyl ester of Boc-FT-KLSS-OMe is hydrolyzed to obtain (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-threonine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, which is Boc-FT-KLSS; (17) Using dichloromethane as a solvent, under the conditions of EDC and HOBt, Boc-FT-KLSS reacts with hydroxylamine hydrochloride to form (3S)-2-((tert-butoxycarbonyl)-L-phenylalanine-L-threonine)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid hydroxamic acid, which is Boc-FT-KLNHOH; (18) Deprotect Boc-FT-KLNHOH under the condition of 4N EA / HCl to obtain the target compound (3S)-2-(L-phenylalanine-L-threonine)-1,2,3,4-tetrahydro-β-carboline-3-isohydroxamic acid, namely FT-KLNHOH.

3. Use of the (3S)-2-(AA1-AA2)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid according to claim 1 in the preparation of an anti-inflammatory drug.

4. The application according to claim 3, characterized in that, The anti-inflammatory drug is an oral small molecule anti-inflammatory drug.

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