Four 2-Thr-tetrahydroisoquinoline-3S-formyl-AA compounds as IL-2 agonists, and their preparation and use

Abstract

The present invention discloses four compounds of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-AA of the following structure (wherein AA is L-Gln residue, L-Asn residue, L-Leu residue and L-Ala residue), discloses their preparation methods and applications in treating inflammation, and further discloses the application of the 3S-2-Thr-tetrahydroisoquinoline-3-formyl-AA in preparing IL-2 agonists. Experiments have shown that the IL-2 agonists prepared by the compounds of the present invention have good anti-inflammatory effects. Therefore, it is proposed that the present invention provides an effective technical means for anti-inflammatory.

Description

Technical Field

[0001] The present invention relates to four 3S-2-Thr-tetrahydroisoquinoline-3-formyl-AA compounds, to their preparation methods, to their use as IL-2 agonists, and to their use in the preparation of anti-inflammatory drugs. The present invention belongs to the field of biomedicine. Background Art

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

[0003] Inflammatory factors associated with the onset of inflammation include IL-2, IL-8, IL-10 and TNF-ɑ. In order to find an IL-10 agonist, the inventor docked the following structure of 2-Gly-tetrahydroisoquinoline-3S-formyl-AA (AA is L-Gln residue, L-Asn residue, L-Leu residue and L-Ala residue) with the active sites of IL-2, IL-8, IL-10 and TNF-ɑ. Molecular docking showed that 2-Thr-tetrahydroisoquinoline-3S-formyl-AA (AA is L-Gln residue, L-Asn residue, L-Leu residue and L-Ala residue) has the lowest docking free energy with IL-2. Activity evaluation showed that 2-Thr-tetrahydroisoquinoline-3S-formyl-AA (AA is L-Gln residue, L-Asn residue, L-Leu residue and L-Ala residue) can effectively upregulate blood IL-2 concentration. Activity evaluation also shows that 2-Thr-tetrahydroisoquinoline-3S-formyl-AA (AA is L-Gln residue, L-Asn residue, L-Leu residue and L-Ala residue) has good anti-inflammatory effect. 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 four 2-Thr-tetrahydroisoquinoline-3S-formyl-AA compounds and confirm the application of these four compounds in the preparation of IL-2 agonists. The IL-2 agonists prepared by the compounds of the present invention have excellent anti-inflammatory effects.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical means:

[0007] The first technical means is to provide four 2-Thr-tetrahydroisoquinoline-3S-formyl-AA compounds having the following structures:

[0008]

[0009] Wherein AA is selected from: L-Gln residue, L-Asn residue, L-Leu residue or L-Ala residue.

[0010] The second technical approach is to dock the four 2-Thr-tetrahydroisoquinoline-3S-formyl-AA compounds with the active sites of IL-2, IL-8, IL-10 and TNF-ɑ respectively to screen IL-2 agonists.

[0011] The third technical means is to propose a preparation method of the above four 2-Thr-tetrahydroisoquinoline-3S-formyl-AA compounds, which comprises the following steps:

[0012] 1) preparing 3S-tetrahydroisoquinoline-3-carboxylic acid;

[0013] 2) preparing 3S-tetrahydroisoquinoline-3-carboxylic acid methyl ester;

[0014] 3) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid methyl ester;

[0015] 4) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid;

[0016] 5) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-AA-OBzl;

[0017] 6) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3S-formyl-AA;

[0018] 7) Preparation of 3S-2-Thr-tetrahydroisoquinoline-3S-formyl-AA.

[0019] The fourth technical approach is to confirm that the four 2-Thr-tetrahydroisoquinoline-3S-formyl-AA with the above structure upregulate blood IL-2 concentration.

[0020] The fifth technical means is to confirm the application of the four 2-Thr-tetrahydroisoquinoline-3S-formyl-AA with the above structure in the preparation of anti-inflammatory agents.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention prepares four 2-Thr-tetrahydroisoquinoline-3S-formyl-AA compounds having the above structure. Experiments show that the compounds have the lowest docking free energy with IL-2 and have the effect of effectively increasing the blood IL-2 concentration. Therefore, the present invention provides a new therapeutic drug for anti-inflammatory treatment and enriches people's drug selection for treating inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The synthetic route of 3S-2-Thr-tetrahydroisoquinoline-3S-formyl-AA is as follows: i) CH 2 O,H 2 SO 4 ii) SOCl 2 , methanol; iii) DCC, HOBt, N-methylmorpholine, Boc-Ala; iv) 0℃, NaOH aqueous solution (2N); v) palladium carbon, continuously passing hydrogen; vi) hydrogen chloride in ethyl acetate solution (4N). DETAILED DESCRIPTION

[0024] In order to further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention and should not be construed as limiting the present invention.

[0025] Example 1 Preparation of 3S-tetrahydroisoquinoline-3-carboxylic acid (1)

[0026] L-Phe (4.0 g, 24.2 mmol) was stirred with 21.6 mL of formaldehyde and 36 mL of concentrated hydrochloric acid (35%) in sequence to make them uniformly mixed. The obtained suspension was stirred at 80°C. After 2 hours, L-Phe was completely dissolved. After 2.5 hours, a colorless precipitate began to form. After 7 hours, thin layer chromatography (CHCl 3 / CH 3 OH, 5 / 1) showed that L-Phe disappeared. 4.2 g (98%) of the title compound was filtered out as a colorless solid and used directly in the next step. ESI-MS (m / z) 176 [MH] - ; 1 HNMR (300 MHZ, DMSO-d 6 ): δ / ppm=12.89(s,1H),7.41(d,J=7.5Hz,1H),7.31(t,J=7.5Hz,1H),7.22(d,J=7.5H z,1H),7.21(t,J=7.5Hz,1H),4.36(m,J=4.5Hz,2H),3.88(t,J=5.6Hz,1H),3.02(dd,J 1 =8.2Hz,J 2=7.2Hz,2H),1.92(s,1H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=173.2,135.9,130.6,127.5,126.9,126.3,56.6,46.4,35.5,30.2.

[0027] Example 2 Preparation of 3S-tetrahydroisoquinoline-3-carboxylic acid methyl ester (2)

[0028] Cool 50 mL of methanol to 0 °C and slowly add 10 mL of SOCl 2 , stir for 30 minutes to obtain cooled methanol-SOCl 2 Solution system. To the cooled methanol-SOCl 2 3S-Tetrahydroisoquinoline-3-carboxylic acid (4.2 g, 23.7 mmol) was added to the solution system, and the reaction mixture was stirred at 0°C for 24 hours. 3 / CH 3 OH, 5 / 1) showed that 3S-tetrahydroisoquinoline-3-carboxylic acid disappeared, and the reaction mixture was concentrated to dryness under reduced pressure. The obtained residue was dissolved in 30 mL of methanol and then concentrated to dryness under reduced pressure. This operation was repeated 3 times to remove the remaining SOCl 2 and hydrogen chloride to give 4.3 g (95%) of the title compound as a colorless solid. ESI-MS (m / e): 192 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6 ): δ / ppm=7.41(d,J=7.5Hz,1H),7.31(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t ,J=7.5Hz,1H),4.36(m,J=4.5Hz,2H),3.88(t,J=5.6Hz,1H),3.66(s,3H),3.02(dd,J 1 =8.2Hz,J 2 =7.2Hz,2H),1.92(s,1H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=173.8,135.9,130.6,127.5,126.9,126.3,56.6,51.9,46.4,35.5,30.2.

[0029] Example 3 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid methyl ester (3)

[0030] N-hydroxybenzotriazole (HOBt, 3.04 g, 22.5 mmol) and dicyclohexylcarbodiimide (DCC, 4.71 g, 22.5 mmol) were added to a solution of methyl 3S-tetrahydroisoquinoline-3-carboxylate (4.30 g, 22.5 mmol) and Boc-Thr (4.31 g, 22.5 mmol) in 150 mL of anhydrous tetrahydrofuran at 0°C. 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 that 3S-tetrahydro-β-carboline-3-carboxylic acid disappeared, 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 obtained solution was washed with 5% sodium bicarbonate aqueous 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 8.21 g (93%) of the title compound as a colorless powder. ESI-MS (m / e): 393 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6 ): δ / ppm=8.73(s,1H),7.56(d,J=7.5Hz,1H),7.33(t,J=7.5Hz,1H),7.22( d,J=7.5Hz,1H),7.21(t,J=7.5Hz,1H),5.37(s,1H),5.03(t,J=6.1Hz,1H) ,5.00(m,J=4.5Hz,1H),4.36(d,J=4.5Hz,1H),4.29(s,2H),3.66(s,3H),3 .08(d,J=6.1Hz,2H),1.53(s,3H),1.52(s,3H),1.51(s,3H),1.49(s,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.85,171.63,167.88,137.09,129.63,129.13,128.45,127.85,127. 21,80.02,68.22,67.88,58.14,53.66,46.05,28.57,28.56,28.55,27.49,18.56.

[0031] Example 4 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid (4)

[0032] At 0°C, a solution of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid methyl ester (7.45 g, 19 mmol) and 50 mL of methanol was mixed with 5 mL of aqueous NaOH solution (3N). The reaction mixture was stirred at 0°C for 6 hours. The reaction was terminated when 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid methyl ester disappeared as monitored by thin layer chromatography (ethyl acetate / methanol, 20 / 1). The reaction mixture was adjusted to pH 2 with dilute hydrochloric acid, the filtrate was concentrated to dryness under reduced pressure, and the residue was dissolved with 200 mL of ethyl acetate. The obtained solution was washed 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 6.68 g (93%) of the title compound as a colorless powder. ESI-MS(m / e):377[MH] - ; 1 HNMR (300 MHZ, DMSO-d 6 ): δ / ppm=11.91(s,1H),8.73(s,1H),7.56(d,J=7.5Hz,1H),7.33(t,J=7.5 Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t,J=7.5Hz,1H),5.37(s,1H),5.03(t ,J=6.1Hz,1H),5.00(m,J=4.5Hz,1H),4.36(d,J=4.5Hz,1H),4.29(s,2H), 3.08(d,J=6.1Hz,2H),1.53(s,3H),1.52(s,3H),1.51(s,3H),1.49(s,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.85,171.63,167.88,137.09,129.63,129.13,128.45,127.85,1 27.21,80.02,68.22,67.88,58.14,46.05,28.57,28.56,28.55,27.49,18.56.

[0033] Example 5 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Gln-OBzl (5a)

[0034] 4.95 g (83%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid (3.78 g, 10 mmol) and Gln-OBzl (2.36 g, 10 mmol) using the method of Example 3 as a colorless powder. ESI-MS (m / z): 597 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6 ): δ / ppm=8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J=7.5Hz,1H),7.33(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t,J= 7.5Hz,1H),7.17(d,J=8.5Hz,1H),7.15(d,J=7.3Hz,1H),7.14(d,J=7.3Hz,1H),7.02(t,J=7.4Hz,1H),6.96(t,J=7.5Hz,1H),6.1 8(s,2H),5.37(s,1H),5.09(t,J=6.3Hz,1H),5.00(t,J=4.9Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.45(s,2H),3 .16(d,J=6.3Hz,2H),2.18(m,J=4.9Hz,2H),2.01(t,J=4.9Hz,2H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,159.34,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.59,127.45 ,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.57,28.56,28.55,28.36,27.49,18.56.

[0035] Example 6 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxyl-Gln (6a)

[0036] Dissolve 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Gln-OBzl (4.77 g, 8 mmol) in 50 mL of methanol, add 240 mg of Pd / C, pass hydrogen, and hydrogenolyze for 48 hours. Filter out Pd / C, concentrate under reduced pressure to remove methanol, and wash the residue with petroleum ether (30 mL×3) to obtain 3.85 g (95%) of the title compound as a colorless powder. ESI-MS (m / z): 507 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6 ): δ / ppm=11.92(s,1H),8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J=7.5Hz,1H),7.33(t,J=7.5 Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t,J=7.5Hz,1H),5.37(s,1H),5.09(t,J=6.3Hz,1H),5.00(t,J= 4.9Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.45(s,2H),3.16(d,J=6.3Hz,2H),2.18(m ,J=4.9Hz,2H),2.01(t,J=4.9Hz,2H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,159.34,134.09,127.59,127.45,127.33,127.32,126 .85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,28.57,28.56,28.55,28.36,27.49,18.56.

[0037] Example 7 Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-carboxyl-Gln (7a)

[0038] Dissolve 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Gln (3.54 g, 7 mmol) in 100 mL of 4N hydrogen chloride in anhydrous ethyl acetate and stir at 0°C for 50 minutes. Add anhydrous ether to the reaction mixture and concentrate under reduced pressure until the hydrogen chloride gas is completely exhausted. 2.71 g (95%) of the title compound is obtained as a colorless powder. FT-ICR-MS (m / e): 407.1931 [M+H]+ ; 1 HNMR (300 MHZ, DMSO-d 6 ): δ / ppm=11.92(s,1H),8.23(s,1H),8.22(s,2H),8.12(s,2H),7.56(d,J=7.5Hz,1H),7.3 3(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t,J=7.5Hz,1H),5.37(s,1H),5.09(t,J=6. 3Hz,1H),5.00(t,J=4.9Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.45(s,2H), 3.16(d,J=6.3Hz,2H), 2.18(m,J=4.9Hz,2H), 2.01(t,J=4.9Hz,2H), 1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,134.09,127.59,127.45,127.33,12 7.32,126.85,67.56,65.40,55.58,46.34,46.14,31.46,28.36,27.49,18.56.

[0039] Example 8 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Asn-OBzl (5b)

[0040] 4.84 g (83%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid (3.78 g, 10 mmol) and Asn-OBzl (2.22 g, 10 mmol) using the method of Example 3 as a colorless powder. ESI-MS (m / z): 583 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6): δ / ppm=8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J=7.5Hz,1H),7.33(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.21 (t,J=7.5Hz,1H),7.17(d,J=8.5Hz,1H),7.15(d,J=7.3Hz,1H),7.14(d,J=7.3Hz,1H),7.02(t,J=7.4Hz,1H),6.96(t,J=7.5 Hz,1H),6.18(s,2H),5.37(s,1H),5.09(t,J=6.3Hz,1H),5.00(t,J=4.9Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H ),4.45(s,2H),3.16(d,J=6.3Hz,2H),2.18(d,J=4.9Hz,2H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,159.34,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.59,127 .45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.57,28.56,28.55,28.36,18.56.

[0041] Example 9 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxyl-Asn (6b)

[0042] 4.42 g (95%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carbonyl-Asn-OBzl (4.31 g, 8 mmol) using the method of Example 6 as a colorless powder. ESI-MS (m / z): 493 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6): δ / ppm=11.28(s,1H),8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J=7.5Hz,1H),7.15(d, J=7.3Hz,1H),7.14(d,J=7.3Hz,1H),7.02(t,J=7.4Hz,1H),5.37(s,1H),5.09(t,J=6.3Hz,1H) ,5.00(t,J=4.9Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.45(s,2H),3.16(d,J=6 .3Hz,2H),2.18(d,J=4.9Hz,2H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,159.34,134.97,128.68,127.81,127.59,126.85, 80.02,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.57,28.56,28.55,28.36,18.56.

[0043] Example 10 Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Asn (7b)

[0044] 2.31 g (95%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carbonyl-Asn (3.13 g, 7 mmol) using the method of Example 7 as a colorless powder. FT-ICR-MS (m / e): 393.1774 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6): δ / ppm=11.28(s,1H),8.23(s,1H),8.22(s,2H),8.12(s,2H),7.56(d,J=7.5Hz,1H) ,7.15(d,J=7.3Hz,1H),7.14(d,J=7.3Hz,1H),7.02(t,J=7.4Hz,1H),5.37(s,1H),5. 09(t,J=6.3Hz,1H),5.00(t,J=4.9Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1 H),4.45(s,2H),3.16(d,J=6.3Hz,2H),2.18(d,J=4.9Hz,2H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,159.34,134.97,128.68,127.81,12 7.59,126.85,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.36,18.56.

[0045] Example 11 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Leu-OBzl (5c)

[0046] 4.99 g (86%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid (3.78 g, 10 mmol) and Leu-OBzl (2.22 g, 10 mmol) using the method of Example 3 as a colorless powder. ESI-MS (m / z): 582 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6): δ / ppm=8.23(s,1H),8.22(s,1H),7.56(d,J=7.5Hz,1H),7.33(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t,J=7.5Hz,1H),7.17(d,J =8.5Hz,1H),7.15(d,J=7.3Hz,1H),7.14(d,J=7.3Hz,1H),7.02(t,J=7.4Hz,1H),6.96(t,J=7.5Hz,1H),6.18(s,2H),5.37(s,1H),5.00( t,J=4.9Hz,1H),4.89(t,J=4.8Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.45(s,2H),2.18(d,J=4.9Hz,2H),1.99(m,J=5.6H z,2H),1.98(m,J=5.6Hz,1H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J=4.5Hz,3H),1.04(d,J=4.7Hz,3H),1.02(d,J=4.7Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.59,127.45,127.33,12 7.32,126.85,80.02,67.56,65.40,55.58,46.34,44.46,31.46,30.05,28.57,28.56,28.55,28.36,27.36,22.63,22.42,18.56.

[0047] Example 12 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carbonyl-Leu (6c)

[0048] 3.73 g (95%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carbonyl-Leu-OBzl (4.65 g, 8 mmol) using the method of Example 6 as a colorless powder. ESI-MS (m / z): 492 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6): δ / ppm=11.13(s,1H),8.23(s,1H),8.22(s,1H),7.56(d,J=7.5Hz,1H),7.15(d,J=7.3Hz,1H),7.14(d,J=7 .3Hz,1H),7.02(t,J=7.4Hz,1H),5.37(s,1H),5.00(t,J=4.9Hz,1H),4.89(t,J=4.8Hz,1H),4.58(m,J=4.5Hz ,1H),4.48(d,J=4.5Hz,1H),4.45(s,2H),2.18(d,J=4.9Hz,2H),1.99(m,J=5.6Hz,2H),1.98(m,J=5.6Hz,1H) ,1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J=4.5Hz,3H),1.04(d,J=4.7Hz,3H),1.02(d,J=4.7Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,134.97,129.63,127.45,127.33,127.32,126.85,80.02, 67.56,55.58,46.34,44.46,31.46,30.05,28.57,28.56,28.55,28.36,27.36,22.63,22.42,18.56.

[0049] Example 13 Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Leu (7c)

[0050] 2.61 g (95%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carbonyl-Leu (3.44 g, 7 mmol) using the method of Example 7 as a colorless powder. FT-ICR-MS (m / e): 392.2185 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6): δ / ppm=11.13(s,1H),8.23(s,1H),8.12(s,2H),7.56(d,J=7.5Hz,1H),7.15(d,J=7.3Hz,1H),7. 14(d,J=7.3Hz,1H),7.02(t,J=7.4Hz,1H),5.37(s,1H),5.00(t,J=4.9Hz,1H),4.89(t,J=4.8Hz,1H ),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.45(s,2H),2.18(d,J=4.9Hz,2H),1.99(m,J=5.6 Hz,2H),1.98(m,J=5.6Hz,1H),1.21(d,J=4.5Hz,3H),1.04(d,J=4.7Hz,3H),1.02(d,J=4.7Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,134.97,129.63,127.45,127.33,127.32,126. 85,67.56,55.58,46.34,44.46,31.46,30.05,28.36,27.36,22.63,22.42,18.56.

[0051] Example 14 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Ala-OBzl (5d)

[0052] 4.64 g (86%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid (3.78 g, 10 mmol) and Ala-OBzl (1.79 g, 10 mmol) using the method of Example 3 as a colorless powder. ESI-MS (m / z): 540 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6): δ / ppm=8.23(s,1H),8.22(s,1H),7.56(d,J=7.5Hz,1H),7.33(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t,J=7.5H z,1H),7.17(d,J=8.5Hz,1H),7.15(d,J=7.3Hz,1H),7.14(d,J=7.3Hz,1H),7.02(t,J=7.4Hz,1H),6.96(t,J=7.5Hz,1H) ,6.18(s,2H),5.37(s,1H),5.00(t,J=4.9Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.16(m,J=5.6Hz,1H),4 .45(s,2H),2.18(d,J=4.9Hz,2H),1.44(d,J=5.6Hz,3H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.59,127.45, 127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,44.46,28.57,28.56,28.55,28.36,22.63,22.42,18.56.

[0053] Example 15 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carbonyl-Ala (6d)

[0054] 3.41 g (95%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxyl-Ala-OBzl (4.31 g, 8 mmol) using the method of Example 6 as a colorless powder. ESI-MS (m / z): 450 [M+H] + ; 1 HNMR (300 MHZ, DMSO-d 6): δ / ppm=12.89(s,1H),8.23(s,1H),8.22(s,1H),7.56(d,J=7.5Hz,1H),7.15(d,J=7.3Hz, 1H),7.14(d,J=7.3Hz,1H),6.96(t,J=7.5Hz,1H),5.37(s,1H),5.00(t,J=4.9Hz,1H),4.58( m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.16(m,J=5.6Hz,1H),4.45(s,2H),2.18(d,J=4.9H z,2H),1.44(d,J=5.6Hz,3H),1.43(s,3H),1.42(s,3H),1.41(s,3H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d 6 ): δ / ppm=172.65,172.45,172.13,171.68,134.97,129.63,127.45,127.33,127.32,126. 85,80.02,67.56,65.40,55.58,46.34,28.57,28.56,28.55,28.36,22.63,22.42,18.56.

[0055] Example 16 Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Ala (7d)

[0056] 2.32 g (95%) of the title compound was obtained from 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxyl-Ala (3.14 g, 7 mmol) using the method of Example 7 as a colorless powder. FT-ICR-MS (m / e): 350.1716 [M+H] + ;12.89(s,1H),8.23(s,1H),8.22(s,2H),7.56(d,J=7.5Hz,1H),7.15(d,J=7.3 Hz,1H),7.14(d,J=7.3Hz,1H),6.96(t,J=7.5Hz,1H),5.37(s,1H),5.00(t,J=4. 9Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5Hz,1H),4.16(m,J=5.6Hz,1H),4 .45(s,2H),2.18(d,J=4.9Hz,2H),1.44(d,J=5.6Hz,3H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d6 ): δ / ppm=172.65,172.45,172.13,134.97,129.63,127.45,127.33,127.32,126.85,67.56,65.40,55.58,46.34,28.36,22.63,22.42,18.56.

[0057] Example 17 Molecular docking of 3S-2-Thr-tetrahydroisoquinoline-3-carboxyl-AA

[0058] In order to predict the effects of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Gln (7a), 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Asn (7b), 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Leu (7c) and 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Ala (7d) on inflammatory factors in the blood of inflammatory mice, this application completed the molecular docking of 7a-d and the active sites of IL-2, IL-8, IL-10 and TNF-ɑ. IL-2, IL-8, IL-10 and TNF-ɑ are all from the European protein library. During docking, LigandFit of DiscoveryStudio was used to dock 7a-d to the active sites of IL-2, IL-8, IL-10 and TNF-ɑ. The docking went through four steps. In the first step, the cavity was selected using the flood-filling algorithm in order to select and determine the active sites of IL-2, IL-8, IL-10 and TNF-ɑ as the docking area. In the second step, the conformation of 7a-d was searched by randomly selecting the flexibility value of the variable torsion angle when selecting the site for 7a-d, and then the site detection was performed using a three-dimensional regular grid to estimate the energy required for docking the active sites of IL-2, IL-8, IL-10 and TNF-ɑ. In the third step, the scores of Coulomb force, van der Waals force, binding energy, interatomic distance, hydrogen bond energy, steric interaction, lipophilic interaction, solvation effect and entropy effect of IL-2, IL-8, IL-10 and TNF-ɑ were compared with 7a-d to obtain a comprehensive evaluation result. In the fourth step, the docking free energy of 7a-d was calculated. The data in Table 1 show that when docked to the active sites of IL-2, IL-8, IL-10 and TNF-ɑ, the binding free energy of 7a-d with IL-2 is significantly lower than that with IL-8, IL-10 and TNF-ɑ. It can be seen that IL-12 is a potential molecular target of 7a-d.

[0059] Table 1 Binding energy of 7a-d to the active sites of IL-2, IL-8, IL-10 and TNF-ɑ

[0060]

[0061] Example 18 Evaluation of the anti-inflammatory activity of 3S-2-Thr-tetrahydroisoquinoline-3-carboxyl-AA

[0062] Mouse ear swelling caused by xylene is generally recognized as an acute inflammation model. The present invention determines the anti-inflammatory activity of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-AA on the mouse ear swelling model caused by xylene. Aspirin is a positive drug for treating acute inflammation. The present invention selects aspirin as a positive control. ICR male mice (weight 35±2g) were rested in an environment with a temperature of 25°C for 2 days, with free access to water and food. Afterwards, they were randomly divided into groups. Mice were orally administered with normal saline (dose of 10 mL / kg, 10 mice), or aspirin (dose of 1110 μmol / kg, 10 mice), or 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Gln (7a, 10 nmol / kg, 10 mice), or 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Asn (7b, 10 nmol / kg, 10 mice), or 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Leu (7c, 10 nmol / kg, 10 mice), or 3S-2-Thr-tetrahydroisoquinoline-3-formyl-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 mouse. Two hours later, the mouse was anesthetized with ether and killed by breaking the cervical vertebrae. Blood was collected, and then the left and right ears were cut off. A 7 mm diameter puncher was used to take circular ear pieces at the same position of the two ears, and the weight difference between the two ears was taken as the degree of swelling. That is, the degree of swelling = the weight of the left ear piece – the weight of the right ear piece.

[0063] The data in Table 2 show that 7a-d effectively inhibits xylene-induced inflammation at an oral dose of 10 nmol / kg (p<0.01 compared with 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 is 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 an outstanding technical effect.

[0064] Table 2 Anti-inflammatory activity of 3S-2-Thr-tetrahydroisoquinoline-3-carboxyl-AA

[0065]

[0066]

[0067] a) Compared with normal saline and aspirin, p<0.01; n=10.

[0068] Example 19 Evaluation of the effect of 3S-2-Thr-tetrahydroisoquinoline-3-carboxyl-AA on inflammatory factors

[0069] The blood of inflammatory mice was added with 3.8% (g / 100 mL) sodium citrate in physiological saline solution (v / v=9:1) at 4°C and centrifuged at 3000g for 15 minutes. The supernatant was then aspirated as a sample for IL-2, IL-8, IL-10 and TNF-ɑ enzyme-linked immunosorbent assay.

[0070] When the ELISA method was used to quantitatively determine IL-2, IL-8, IL-10, and TNF-ɑ in plasma samples, the microplate was coated with purified IL-2, IL-8, IL-10, and TNF-ɑ antibodies. The operation was carried out according to the method of the ELISA kit, that is, the standard or the plasma samples of inflammatory mice treated with saline and the plasma samples of inflammatory mice treated with 7a-d were added to the microwells coated with monoclonal antibodies in sequence, and biotinylated IL-2, IL-8, IL-10, and TNF-ɑ antibodies and horseradish peroxidase-labeled avidin were added. Then, the microplate was thoroughly washed and the substrate TMB was added for color development. During color development, the blue color induced by the action of peroxidase was first seen, and then the blue color turned into yellow under the action of acid. The depth of yellow was positively correlated with the concentration of IL-2, IL-8, IL-10, and TNF-ɑ in the sample. The absorbance (OD) of the standard was measured by an enzyme reader at a wavelength of 450nm, and a standard curve was drawn. The absorbance (OD) of plasma samples from saline-treated inflammatory mice and 7a-d-treated inflammatory mice was measured by an ELISA reader at a wavelength of 450 nm, and the concentrations of IL-2, IL-8, IL-10 and TNF-ɑ in plasma samples from saline-treated inflammatory mice and 7a-d-treated inflammatory mice were calculated using the standard curve. Each sample was repeated 6 times. The data in Table 3 show that 7a-d significantly increased the concentration of plasma IL-2 in inflammatory mice (p<0.01 compared with saline). The data in Table 3 also show that 7a-d had no significant effect on the concentrations of plasma IL-8, IL-10 and TNF-ɑ in inflammatory mice (p>0.05 compared with saline). It can be seen that IL-2 in mouse plasma is indeed the molecular target of 7a-d to inhibit inflammatory response.

[0071] Table 3 Effects of 7a-d on plasma IL-2, IL-8, IL-10 and TNF-ɑ

[0072]

[0073] a) p<0.01 for the ratio of 7a-d to normal saline; b) p>0.05 for the ratio of 7a-d to normal saline; c) p>0.05 for the ratio of 7a-d to normal saline; d) p>0.05 for the ratio of 7a-d to normal saline; n=10.

Claims

1. Four 3S-2-Thr-tetrahydroisoquinoline-3-carboxyl-AA compounds of the following structure: wherein AA is selected from a L-Gln residue, a L-Asn residue, a L-Leu residue or a L-Ala residue.

2. The method for preparing the four 3S-2-Thr-tetrahydroisoquinoline-3-formyl-AA compounds according to claim 1, wherein: The method comprises the following steps: 1) preparing 3S-tetrahydroisoquinoline-3-carboxylic acid; 2) preparing 3S-tetrahydroisoquinoline-3-carboxylic acid methyl ester; 3) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid methyl ester; 4) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid; 5) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-AA-OBzl; 6) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3S-formyl-AA; 7) Preparation of 3S-2-Thr-tetrahydroisoquinoline-3S-formyl-AA.

3. Use of the four 3S-2-Thr-tetrahydroisoquinoline-3S-formyl-AA compounds according to claim 1 in the preparation of anti-inflammatory drugs.

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