2-Asn-tetrahydroisoquinoline-3S-formyl-Ala, and preparation and application thereof

Abstract

The invention discloses four 3S-2-Asn-tetrahydroisoquinoline-3-formyl-AA (AA in the formula is L-Ala residue) with the following structure, a preparation method of the four 3S-2-Asn-tetrahydroisoquinoline-3-formyl-AA, and application of the four 3S-2-Asn-tetrahydroisoquinoline-3-formyl-AA in preparation of a TNF-alpha inhibitor and an anti-inflammatory agent. Experiments prove that the TNF-alpha inhibitor disclosed by the invention has a good anti-inflammatory effect. Therefore, the invention provides an effective technical means for anti-inflammation. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to four 3S-2-Asn-tetrahydroisoquinoline-3-carbonyl-AA compounds, their preparation methods, and the use of these compounds in the preparation of TNF-α inhibitors and anti-inflammatory agents. The present invention belongs to the field of biomedicine. Background Art

[0002] Inflammation can trigger organ fibrosis. Inflammation in the central nervous system can trigger brain fibrosis. Cancer metastasis is another fatal disease triggered by inflammation. Coronary inflammation or vascular inflammation is associated with atherosclerotic cardiovascular disease. Another fatal problem of inflammation is its association with the development of various tumors. The development of anti-inflammatory agents without toxic side effects has received increasing attention.

[0003] Inflammatory factors related to the onset of inflammation include IL-2, IL-8, IL-10, and TNF-α. In order to find TNF-α inhibitors, the inventors docked 3S-2-Asn-tetrahydroisoquinoline-3-carbonyl-AA (where AA is an L-Gln residue, an L-Asn residue, an L-Leu residue, and an L-Ala residue) with the active sites of IL-2, IL-8, IL-10, and TNF-α. Molecular docking showed that the docking free energy of 2-Asn-tetrahydroisoquinoline-3S-carbonyl-AA with TNF-α was the lowest. Activity evaluation showed that 3S-2-Asn-tetrahydroisoquinoline-3-carbonyl-AA downregulated the blood TNF-α concentration. Activity evaluation also showed that 3S-2-Asn-tetrahydroisoquinoline-3-carbonyl-AA had good anti-inflammatory effects. Based on these findings, the applicant proposed the present invention.

[0004] Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide 3S-2-Asn-tetrahydroisoquinoline-3-carbonyl-AA (where AA is an L-Gln residue, an L-Asn residue, an L-Leu residue, and an L-Ala residue), and to confirm the use of these four compounds in the preparation of TNF-α inhibitors. The TNF-α inhibitors of the present invention have excellent anti-inflammatory effects. To achieve the above object, the present invention adopts the following four technical means.

[0006] First, the present invention provides four 3S-2-Asn-tetrahydroisoquinoline-3-carbonyl-AA compounds having the following structure,

[0007]

[0008] where AA is an L-Gln residue, an L-Asn residue, an L-Leu residue, and an L-Ala residue.

[0009] Secondly, dock 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-AA with the active sites of IL-2, IL-8, IL-10, and TNF-α to screen for TNF-α inhibitors.

[0010] The third technical means is to propose a preparation method for 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-AA, and its preparation method is characterized by including the steps:

[0011] 1) Prepare 3S-tetrahydroisoquinoline-3-carboxylic acid;

[0012] 2) Prepare methyl 3S-tetrahydroisoquinoline-3-carboxylate;

[0013] 3) Prepare methyl 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylate;

[0014] 4) Prepare 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid;

[0015] 5) Prepare 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxamide-AA-OBzl;

[0016] 6) Prepare 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3S-carboxamide-AA;

[0017] 7) Prepare 3S-2-Asn-tetrahydroisoquinoline-3S-carboxamide-AA.

[0018] The fourth technical means is to confirm that 3S-2-Asn-tetrahydroisoquinoline-3S-carboxamide-AA downregulates the blood TNF-α concentration.

[0019] The fifth technical means is to confirm the application of 3S-2-Asn-tetrahydroisoquinoline-3S-carboxamide-AA in the preparation of anti-inflammatory drugs.

[0020] The beneficial effects of the present invention are:

[0021] Four 3S-2-Asn-tetrahydroisoquinoline-3S-carboxamide-AA compounds with the above structure are prepared by the present invention. Through experiments, it is proved that these four compounds have the lowest docking free energy with TNF-α, can effectively downregulate the blood TNF-α concentration, and have excellent anti-inflammatory effects. Therefore, the present invention provides new therapeutic drugs for the treatment of inflammation and related diseases triggered by inflammation, and enriches the drug options for the treatment of inflammation. Description of the Drawings

[0022] Figure 1 Synthetic route diagram of 3S-2-Asn-tetrahydroisoquinoline-3S-carboxamide-AA: i) CH 2 O, H2 SO 4 ; ii) SOCl 2 , methanol; iii) DCC, HOBt, N-methylmorpholine, Boc-Ala; iv) 0 °C, aqueous NaOH solution (2N); v) palladium on carbon, continuous hydrogen gas introduction; vi) ethyl acetate solution of hydrogen chloride (4N). Detailed implementation manners

[0023] To further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative and are only used to specifically describe the present invention and should not be construed as limiting the present invention.

[0024] Example 1 Preparation of 3S-tetrahydroisoquinoline-3-carboxylic acid (1)

[0025] Slowly add 0.2 mL of concentrated sulfuric acid to 400 mL of water. Add 5.0 g (24.5 mmol) of L-Trp to the obtained dilute sulfuric acid aqueous solution, and ultrasonically oscillate until L-Trp is completely dissolved. Add 10 mL of 35% aqueous formaldehyde solution to the obtained solution. The reaction mixture is stirred at room temperature for 6 hours. When thin layer chromatography (ethyl acetate / ether, 20 / 1) monitors the disappearance of L-Trp, the reaction is terminated. Slowly add concentrated ammonia water to the reaction solution to adjust the pH to 6, and let it stand for 30 minutes. Filter out the generated precipitate and wash it with water. The filtered colorless solid is spread flat in a petri dish and dried in air to obtain 5.05 g (95%) of the title compound, which is a colorless solid. ESI-MS (m / e): 217 [M+H] + .

[0026] Example 2 Preparation of methyl 3S-tetrahydroisoquinoline-3-carboxylate (2)

[0027] Cool 50 mL of methanol to 0 °C, and slowly add 10 mL of SOCl 2 dropwise to the cooled methanol, and stir for 30 minutes to obtain a cooled methanol-SOCl 2 solution system. Add 3S-tetrahydroisoquinoline-3-carboxylic acid (4.2 g, 23.7 mmol) to the cooled methanol-SOCl 2 solution system. The reaction mixture is stirred at 0 °C for 24 hours. When thin layer chromatography (CHCl 3 / CH 3 OH, 5 / 1) shows the disappearance of 3S-tetrahydroisoquinoline-3-carboxylic acid, the reaction mixture is concentrated to dryness under reduced pressure. The obtained residue is dissolved in 30 mL of methanol and then concentrated to dryness under reduced pressure. This operation is repeated 3 times to completely remove the remaining SOCl 2 and hydrogen chloride to obtain 4.3 g (95%) of the title compound, which is a colorless solid. ESI-MS (m / e): 192 [M+H] + ;1 HNMR (300 MHz, DMSO-d 6 ): δ / ppm = 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 4.36 (m, J = 4.5 Hz, 2H), 3.88 (t, J = 5.6 Hz, 1H), 3.66 (s, 3H), 3.02 (dd, J 1 = 8.2 Hz, J 2 = 7.2 Hz, 2H), 1.92 (s, 1H); 13 CNMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.82, 135.91, 130.63, 127.51, 126.93, 126.34, 56.63, 51.92, 46.44, 35.52, 30.23。

[0028] Example 3 Preparation of Methyl 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylate (3)

[0029] To a solution of methyl 3S-tetrahydroisoquinoline-3-carboxylate (4.30 g, 22.5 mmol), Boc-Asn (4.89 g, 22.5 mmol) and 150 mL of anhydrous tetrahydrofuran at 0 °C was added N-hydroxybenzotriazole (HOBt, 3.04 g, 22.5 mmol) and dicyclohexylcarbodiimide (DCC, 4.71 g, 22.5 mmol). The reaction mixture was stirred at 0 °C for 30 minutes. Then the pH was adjusted to 8 with N-methylmorpholine (NMM). The reaction mixture was stirred at 0 °C for 6 hours. Thin layer chromatography (ethyl acetate / methanol, 20 / 1) showed the disappearance of methyl 3S-tetrahydroisoquinoline-3-carboxylate, and the reaction was terminated. The reaction mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in 200 mL of ethyl acetate. The resulting solution was washed successively with 5% aqueous sodium bicarbonate solution (30 mL × 3), saturated aqueous sodium chloride solution (30 mL × 3), 5% aqueous hydrochloric acid solution (30 mL × 3), and saturated aqueous sodium chloride solution (30 mL × 3). The ethyl acetate layer was separated, dried over anhydrous sodium sulfate for 12 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 8.48 g (93%) of the title compound as a colorless powder. ESI-MS (m / e): 406 [M+H] + ; 1 HNMR (300 MHz, DMSO-d 6): δ / ppm = 8.29 (s, 1H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.52 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 3.66 (d, 3H), 2.56 (d, J = 4.5 Hz, 2H), 2.52 (d, J = 4.8 Hz, 2H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 172.65, 172.13, 171.68, 169.12, 134.28, 129.63, 127.81, 127.45, 127.32, 126.85, 80.02, 68.13, 65.40, 55.27, 44.44, 37.19, 28.57, 28.56, 28.55, 27.49.

[0030] Example 4 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid (4)

[0031] A solution of methyl 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylate (8.48 g, 20.92 mmol) and 50 mL of methanol was mixed with 5 mL of an aqueous NaOH solution (3 N) at 0 °C. The reaction mixture was stirred at 0 °C for 6 hours, and the reaction was terminated when thin-layer chromatography (ethyl acetate / methanol, 20 / 1) monitored the disappearance of methyl 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylate. The reaction mixture was adjusted to pH 2 with dilute hydrochloric acid, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in 200 mL of ethyl acetate. The resulting solution was washed successively with 5% aqueous sodium bicarbonate solution (30 mL × 3), saturated sodium chloride aqueous solution (30 mL × 3), 5% hydrochloric acid aqueous solution (30 mL × 3), and saturated sodium chloride aqueous solution (30 mL × 3). The ethyl acetate layer was separated, dried over anhydrous sodium sulfate for 12 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 7.61 g (93%) of the title compound as a colorless powder. ESI-MS (m / e): 390 [M-H] - ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 12.89 (s, 1H), 8.29 (s, 1H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.52 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.52 (d, J = 4.8 Hz, 2H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 172.65, 172.13, 171.68, 169.12, 134.28, 129.63, 127.81, 127.45, 127.32, 126.85, 80.02, 68.13, 65.40, 44.44, 37.19, 28.57, 28.56, 28.55, 27.49.

[0032] Example 5 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxaldehyde-Gln-OBzl (5a)

[0033] Using the method of Example 3, 5.05 g (83%) of the title compound was obtained as a colorless powder from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid (3.91 g, 10 mmol) and Gln-OBzl (2.36 g, 10 mmol). ESI-MS (m / z): 610 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d 6 ): 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 8.29 (s, 1H), 8.25 (s, 1H), 8.20 (s, 2H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 6.18 (s, 2H), 4.84 (t, J = 4.5 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 4.52 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.52 (d, J = 4.8 Hz, 2H), 2.18 (m, J = 5.7 Hz, 2H), 2.01 (t, J = 5.7 Hz, 2H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 159.17, 159.15, 134.97, 133.55, 133.17, 128.68, 128.41, 127.59, 127.33, 127.20, 127.19, 126.38, 125.99, 125.96, 80.02, 67.56, 55.58, 51.69, 46.34, 40.61, 31.46, 28.36, 28.35, 28.34, 28.33, 24.61, 24.33.

[0034] Example 6 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carbonyl-Gln (6a)

[0035] Dissolve 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carbonyl-Gln-OBzl (4.64 g, 8 mmol) in 50 mL of methanol, add 240 mg of Pd / C, introduce hydrogen gas, and perform hydrogenolysis for 48 hours. Filter off the Pd / C, concentrate under reduced pressure to remove methanol, and wash the residue with petroleum ether (30 mL × 3) to obtain 3.95 g (95%) of the title compound as a colorless powder. ESI-MS (m / z): 520 [M + H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 11.92 (s, 1H), 8.29 (s, 1H), 8.25 (s, 1H), 8.20 (s, 2H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 4.52 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.52 (d, J = 4.8 Hz, 2H), 2.18 (m, J = 5.7 Hz, 2H), 2.01 (t, J = 5.7 Hz, 2H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H); 13 C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 172.45, 171.63, 159.34, 159.17, 159.15, 134.97, 128.68, 127.59, 127.33, 126.38, 125.96, 80.02, 67.56, 51.69, 46.34, 40.61, 31.46, 29.71, 28.36, 28.35, 28.34, 28.33, 24.61, 24.33.

[0036] Example 7 Preparation of 3S-2-Asn-tetrahydroisoquinoline-3-carboxyl-Gln (7a)

[0037] Dissolve 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxyl-Gln (3.63 g, 7 mmol) in 100 mL of an anhydrous ethyl acetate solution of hydrogen chloride with a concentration of 4N, and stir at 0 °C for 50 minutes. Add anhydrous ether to the reaction mixture and concentrate under reduced pressure until all hydrogen chloride gas is completely removed. 2.79 g (95%) of the title compound is obtained as a colorless powder. FT-ICR-MS (m / e): 420.1883 [M + H] + ; 1 H NMR (300 MHz, DMSO-d 6): δ / ppm = 11.92 (s, 1H), 8.29 (s, 1H), 8.20 (s, 2H), 8.19 (s, 2H), 8.12 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 4.52 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.52 (d, J = 4.8 Hz, 2H), 2.18 (m, J = 5.7 Hz, 2H), 2.01 (t, J = 5.7 Hz, 2H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 172.45, 171.63, 159.34, 159.17, 159.15, 134.97, 128.68, 127.59, 127.33, 126.38, 125.96, 67.56, 51.69, 46.34, 43.21, 40.61, 31.46, 29.71, 28.33.

[0038] Example 8 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carbonyl-Asn-OBzl (5b)

[0039] Using the method of Example 3, 5.06 (85%) of the title compound was obtained as a colorless powder from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid (3.91 g, 10 mmol) and Asn-OBzl (2.22 g, 10 mmol). ESI-MS (m / z): 596 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 8.29 (s, 1H), 8.25 (s, 1H), 8.20 (s, 2H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 6.18 (s, 2H), 4.84 (t, J = 4.5 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 4.52 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.52 (d, J = 4.8 Hz, 2H), 2.18 (d, J = 5.7 Hz, 2H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 159.17, 159.15, 134.97, 133.55, 133.17, 128.68, 128.41, 127.59, 127.33, 127.20, 127.19, 126.38, 125.99, 125.96, 80.02, 67.56, 55.58, 51.69, 46.34, 40.61, 31.46, 28.36, 28.35, 28.34, 28.33, 24.33.

[0040] Example 9 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxamide-Asn (6b)

[0041] Using the method of Example 6, 3.41 g (95%) of the title compound was obtained from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxamide-Asn-OBzl (4.31 g, 8 mmol) as a colorless powder. ESI-MS (m / z): 449 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 11.28 (s, 1H), 8.29 (s, 1H), 8.25 (s, 1H), 8.20 (s, 2H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 4.52 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.52 (d, J = 4.8 Hz, 2H), 2.18 (d, J = 5.7 Hz, 2H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 159.17, 159.15, 134.97, 133.17, 128.68, 127.33, 127.19, 125.99, 80.02, 67.56, 51.69, 46.34, 40.61, 31.46, 28.36, 28.35, 28.34, 28.33, 24.33.

[0042] Example 10 Preparation of 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-Asn (7b)

[0043] Using the method of Example 7, 2.69 g (95%) of the title compound was obtained from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxamide-Asn (3.14 g, 7 mmol) as a colorless powder. FT-ICR-MS (m / e): 406.1727 [M + H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 11.28 (s, 1H), 8.29 (s, 2H), 8.25 (s, 1H), 8.20 (s, 2H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 4.52 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.52 (d, J = 4.8 Hz, 2H), 2.18 (d, J = 5.7 Hz, 2H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 159.17, 134.97, 133.17, 128.68, 127.33, 127.19, 125.99, 67.56, 51.69, 46.34, 40.61, 31.46, 28.36, 24.33.

[0044] Example 11 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carbonyl-Leu-OBzl (5c)

[0045] Using the method of Example 3, 5.11 g (86%) of the title compound was obtained as a colorless powder from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid (3.91 g, 10 mmol) and Leu-OBzl (2.22 g, 10 mmol). ESI-MS (m / z): 595 [M + H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 8.29 (s, 1H), 8.25 (s, 1H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 6.18 (s, 2H), 4.87 (t, J = 4.7 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.58 (t, J = 5.7 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.18 (d, J = 5.7 Hz, 2H), 1.99 (m, J = 5.6 Hz, 2H), 1.98 (m, J = 4.7 Hz, 1H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H), 1.04 (d, J = 4.7 Hz, 3H), 1.02 (d, J = 4.7 Hz, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 159.17, 134.97, 133.55, 133.17, 128.68, 128.41, 127.59, 127.33, 127.20, 127.19, 126.38, 125.99, 125.96, 80.02, 67.56, 66.42, 55.58, 53.37, 46.34, 40.61, 31.46, 28.36, 28.35, 28.34, 28.17, 24.33, 22.63, 22.42.

[0046] Example 12 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carbonyl-Leu(6c)

[0047] Using the method of Example 6, 3.83 g (95%) of the title compound was obtained from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carbonyl-Leu-OBzl (4.75 g, 8 mmol) as a colorless powder. ESI-MS (m / z): 505 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 11.13 (s, 1H), 8.29 (s, 1H), 8.25 (s, 1H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 4.87 (t, J = 4.5 Hz, 1H), 4.84 (t, J = 5.7 Hz, 1H), 4.58 (t, J = 5.6 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.18 (d, J = 5.7 Hz, 2H), 1.99 (m, J = 5.6 Hz, 2H), 1.98 (m, J = 5.6 Hz, 1H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H), 1.04 (d, J = 4.7 Hz, 3H), 1.02 (d, J = 4.7 Hz, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 159.17, 134.97, 128.68, 127.59, 127.19, 126.38, 125.96, 80.02, 67.56, 55.58, 53.37, 46.34, 40.61, 31.46, 28.36, 28.35, 28.34, 28.17, 24.33, 22.63, 22.42.

[0048] Example 13 Preparation of 3S-2-Asn-tetrahydroisoquinoline-3-carboxy-Leu (7c)

[0049] Using the method of Example 7, 2.69 g (95%) of the title compound was obtained from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxy-Leu (3.53 g, 7 mmol) as a colorless powder. FT-ICR-MS (m / e): 405.2138 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 11.13 (s, 1H), 8.29 (s, 1H), 8.25 (s, 2H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 4.87 (t, J = 4.5 Hz, 1H), 4.84 (t, J = 5.7 Hz, 1H), 4.58 (t, J = 5.6 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.18 (d, J = 5.7 Hz, 2H), 1.99 (m, J = 5.6 Hz, 2H), 1.98 (m, J = 5.6 Hz, 1H), 1.04 (d, J = 4.7 Hz, 3H), 1.02 (d, J = 4.7 Hz, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 134.97, 128.68, 127.59, 127.19, 126.38, 125.96, 67.56, 55.58, 53.37, 46.34, 40.61, 31.46, 28.17, 24.33, 22.63, 22.42.

[0050] Example 14 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carbonyl-Ala-OBzl (5d)

[0051] Using the method of Example 3, 4.81 g (87%) of the title compound was obtained as a colorless powder from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid (3.91 g, 10 mmol) and Ala-OBzl (1.79 g, 10 mmol). ESI-MS (m / z): 553 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 8.29 (s, 1H), 8.25 (s, 1H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 6.18 (s, 2H), 4.87 (t, J = 4.7 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.73 (m, J = 5.6 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.18 (d, J = 4.7 Hz, 2H), 1.99 (d, J = 5.6 Hz, 3H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 159.17, 134.97, 133.55, 133.17, 128.68, 128.41, 127.59, 127.33, 127.20, 127.19, 126.38, 125.99, 125.96, 80.02, 67.56, 66.42, 55.58, 53.37, 46.34, 40.61, 31.46, 28.36, 28.35, 28.34, 28.17.

[0052] Example 15 Preparation of 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid-Ala(6d)

[0053] Using the method of Example 6, 3.08 g (95%) of the title compound was obtained as a colorless powder from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid-Ala-OBzl (4.42 g, 8 mmol). ESI-MS (m / z): 463 [M+H] + ; 1 1H NMR (300 MHz, DMSO-d 6): δ / ppm = 12.89 (s, 1H), 8.29 (s, 1H), 8.25 (s, 1H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 4.87 (t, J = 4.7 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.73 (m, J = 5.6 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.18 (d, J = 4.7 Hz, 2H), 1.99 (d, J = 5.6 Hz, 3H), 1.37 (s, 3H), 1.36 (s, 3H), 1.35 (s, 3H); 13 13C NMR (75 MHz, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 159.17, 134.97, 133.17, 127.59, 127.33, 126.38, 125.96, 80.02, 67.56, 55.58, 53.37, 46.34, 40.61, 31.46, 28.36, 28.35, 28.34, 28.17.

[0054] Example 16 Preparation of 3S-2-Asn-tetrahydroisoquinoline-3-carboxyl-Ala (7d)

[0055] Using the method of Example 7, 2.41 g (95%) of the title compound was obtained from 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxyl-Ala (3.24 g, 7 mmol) as a colorless powder. FT-ICR-MS (m / e): 363.1668 [M + H] + ; 1 1H NMR (300 MHz, DMSO-d 6 ): δ / ppm = 12.89 (s, 1H), 8.29 (s, 1H), 8.25 (s, 2H), 8.19 (s, 2H), 7.41 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.5 Hz, 1H), 4.87 (t, J = 4.7 Hz, 1H), 4.84 (t, J = 4.5 Hz, 1H), 4.73 (m, J = 5.6 Hz, 1H), 4.36 (s, 2H), 2.56 (d, J = 4.5 Hz, 2H), 2.18 (d, J = 4.7 Hz, 2H), 1.99 (d, J = 5.6 Hz, 3H); 13CNMR(75MHZ, DMSO-d 6 ): δ / ppm = 173.35, 172.45, 171.63, 159.34, 134.97, 133.17, 127.59, 127.33, 126.38, 125.96, 67.56, 55.58, 53.37, 46.34, 40.61, 31.46, 28.17。

[0056] Example 17 Molecular Docking of 3S-2-Asn-Tetrahydroisoquinoline-3-carboxamide-AA

[0057] To predict the effects of 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-Gln (7a), 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-Asn (7b), 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-Leu (7c), and 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-Ala (7d) on blood inflammatory factors in inflammatory mice, this application completed the molecular docking of the active sites of 7a-d and IL-2, IL-8, IL-10, and TNF-α. IL-2, IL-8, IL-10, and TNF-α are all from the European Protein Bank. When docking, LigandFit of DiscoveryStudio was used to dock 7a-d to the active sites of IL-2, IL-8, IL-10, and TNF-α. Four steps were experienced during docking. The first step was to select the cavity using the flood-filling algorithm to select and determine the active sites of IL-2, IL-8, IL-10, and TNF-α as the docking regions. The second step was to select sites for 7a-d. First, the conformational search of 7a-d was performed by randomly sampling the flexible values of the variable torsion angles, and then the detection of the sites was performed using a three-dimensional regular grid and the energy required to dock to the active sites of IL-2, IL-8, IL-10, and TNF-α was estimated. The third step was to compare the fractional values of the Coulomb force, van der Waals force, binding energy, atomic distance, hydrogen bond energy, spatial interaction, lipophilic interaction, solvation effect, and entropy effect between IL-2, IL-8, IL-10, and TNF-α and 7a-d to obtain a comprehensive evaluation result. The fourth step was to calculate the docking free energy of 7a-d. The data in Table 1 show that the binding free energy of 7a-d to TNF-α is significantly lower than that to IL-2, IL-8, and IL-10 when docking to the active sites of IL-2, IL-8, IL-10, and TNF-α. It can be seen that TNF-α is a potential molecular target of 7a-d.

[0058] Table 1 Binding Energies of 7a-d to the Active Sites of IL-2, IL-8, IL-10, and TNF-α

[0059]

[0060] Example 18 Evaluation of the anti-inflammatory activity of 3S-2-Asn-tetrahydroisoquinoline-3-carboxyl-AA

[0061] Xylene-induced ear swelling in mice is recognized as an acute inflammation model. In this invention, the anti-inflammatory activity of 3S-2-Asn-tetrahydroisoquinoline-3-carboxyl-AA was determined using the xylene-induced ear swelling model in mice. Aspirin is a positive drug for treating acute inflammation, and aspirin was selected as the positive control in this invention. ICR male mice (body weight 35±2 g) were rested in an environment at 25 °C for 2 days, with free access to water and food. Then, they were randomly grouped. The mice were orally administered with normal saline (dose: 10 mL / kg, 10 mice), or aspirin (dose: 1110 μmol / kg, 10 mice), or 3S-2-ASn-tetrahydroisoquinoline-3-carboxyl-Gln (7a, 10 nmol / kg, 10 mice), or 3S-2-Asn-tetrahydroisoquinoline-3-carboxyl-Asn (7b, 10 nmol / kg, 10 mice), or 3S-2-Asn-tetrahydroisoquinoline-3-carboxyl-Leu (7c, 10 nmol / kg, 10 mice), or 3S-2-Asn-tetrahydroisoquinoline-3-carboxyl-Ala (7d, 10 nmol / kg, 10 mice). 30 minutes after oral administration, 30 μL of xylene was evenly applied to the left auricle of the mice. After 2 hours, the mice were anesthetized with ether and sacrificed by cervical dislocation. Blood was collected, and then the left and right ears were cut off. Circular ear pieces were taken from the same position of both ears using a punch with a diameter of 7 mm and weighed. The difference in weight between the two ears was used as the degree of swelling. That is, the degree of swelling = weight of the left ear circular piece – weight of the right ear circular piece.

[0062] The data in Table 2 show that 7a-d effectively inhibited xylene-induced inflammation at an oral dose of 10 nmol / kg (p<0.01 compared with normal saline). The data in Table 2 also show that the activity of 7a-d in inhibiting xylene-induced inflammation at an oral dose of 10 nmol / kg was significantly stronger than the activity of aspirin in inhibiting xylene-induced inflammation at an oral dose of 1110 μmol / kg (p<0.01 compared with aspirin). It can be seen that 7a-d has outstanding technical effects.

[0063] Table 2 Anti-inflammatory activity of 3S-2-Asn-tetrahydroisoquinoline-3-carboxyl-AA

[0064] Therapeutic agent Dose Ear swelling degree, mean ± SD mg Normal saline 10 mL / kg 12.95±1.61 Aspirin 1110 μmol / kg 8.42±1.31 7a 10 nmol / kg <![CDATA[4.35±1.11 a > 7b 10 nmol / kg <![CDATA[4.53±1.12 a > 7c 10 nmol / kg <![CDATA[4.54±1.10 a > 7d 10 nmol / kg <![CDATA[4.61±1.21 a >

[0065] a) p<0.01 compared with normal saline and aspirin; n = 10.

Claims

1. 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-AA having the following structural formula, wherein AA is an L-Ala residue.

2. The method for preparing 3S-2-Asn-tetrahydroisoquinoline-3-carboxamide-AA according to claim 1, characterized in that the method comprises the following steps: 1) Prepare 3S-tetrahydroisoquinoline-3-carboxylic acid; 2) Prepare methyl 3S-tetrahydroisoquinoline-3-carboxylate; 3) Prepare methyl 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylate; 4) Prepare 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxylic acid; 5) Prepare 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3-carboxamide-AA-OBzl; 6) Prepare 3S-2-(Boc-Asn)-tetrahydroisoquinoline-3S-carboxamide-AA; 7) Prepare 3S-2-Asn-tetrahydroisoquinoline-3S-carboxamide-AA.

3. The use of 3S-2-Asn-tetrahydroisoquinoline-3S-carboxamide-AA according to claim 1 in the preparation of an anti-inflammatory agent.

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