IL-2 agonist 2-Thr-dihydroisoquinoline-3S-formyl-AA, and its preparation and anti-tumor application

Abstract

The present invention discloses four types of 3S-2-Thr-dihydroisoquinoline-3-formyl-AA compounds of the following structures (wherein AA is L-Gln residue, L-Asn residue, L-Leu residue and L-Ala residue), discloses the preparation method of the compounds, and further discloses their use in preparing IL-2 agonists and preparing anti-tumor drugs. Experiments have shown that the IL-2 agonist of the present invention has a good anti-tumor effect. Therefore, it is proposed that the present invention provides an effective technical means for anti-tumor.

Description

Technical Field

[0001] The present invention relates to four 3S-2-Thr-tetrahydroisoquinoline-3S-formyl-AA compounds, and relates to their preparation methods and applications in the preparation of IL-2 agonists. Experiments show that the IL-2 agonists of the present invention have good anti-tumor effects. The present invention belongs to the field of biomedicine. Background Art

[0002] According to data released by the World Health Organization (WHO), there are 15 million new malignant tumor patients worldwide each year. Malignant tumors are undoubtedly one of the main causes of disability and death among the working-age population. From the perspective of clinical treatment measures, chemotherapy is still the preferred strategy for malignant tumors. Finding chemotherapeutic agents with low toxicity and good efficacy is one of the important goals of medicinal chemistry.

[0003] The relationship between IL-2, IL-8, IL-10 and TNF-ɑ and the onset of malignant tumors has been clearly described. For example, the IL-2 level of patients with malignant tumors is lower than that of healthy people, and IL-2 can be used alone as an important biological agent for the treatment of malignant tumors; IL-8 is secreted and IL-8 mRNA is expressed in a variety of malignant tumor tissues, while the corresponding normal tissues do not express or express low levels of IL-8; in addition, anti-IL-8 antibodies can inhibit tumor growth in animal models of bladder cancer; IL10 has an inhibitory effect on the growth of malignant tumors; in addition, IL-10 gene deletion increases the risk of malignant tumors; TNF-ɑ can directly kill malignant tumor cells and has no obvious toxicity to normal cells. It is one of the most powerful anti-tumor biological active factors discovered so far. This knowledge shows that the search for chemotherapeutic agents with low toxicity and good efficacy can be transformed into the search for IL-2, IL-8, IL-10 and TNF-ɑ agonists.

[0004] When studying the metabolites of 3S-2-Thr-tetrahydroisoquinoline-3S-formyl-AA, the inventors discovered four 3S-2-Thr-dihydroisoquinoline-3S-formyl-AA compounds (AA represents L-Gln residue, L-Asn residue, L-Leu residue and L-Ala residue). The inventors assumed that 3S-2-Thr-dihydroisoquinoline-3S-formyl-AA may be an agonist of IL-2, IL-8, IL-10 and TNF-ɑ. Therefore, the inventors docked the four compounds with the active sites of IL-2, IL-8, IL-10 and TNF-ɑ. Molecular docking showed that 3S-2-Thr-dihydroisoquinoline-3S-formyl-AA had the lowest docking free energy with IL-10. Activity evaluation showed that 3S-2-Thr-dihydroisoquinoline-3S-formyl-AA effectively upregulated the blood IL-2 concentration. Activity evaluation also shows that these four 3S-2-Thr-dihydroisoquinoline-3S-formyl-AA compounds have good anti-tumor effects. Based on these findings, the applicant proposed the present invention. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide four 2-Thr-dihydroisoquinoline-3-carboxyl-AA compounds (AA is L-Gln residue, L-Asn residue, L-Leu residue and L-Ala residue), and confirm the anti-tumor effect of these four compounds in the preparation of IL-2 agonists. In order to achieve the above purpose, the present invention adopts the following technical means.

[0006] First, the present invention proposes a 2-Thr-dihydroisoquinoline-3-formyl-AA compound having the following structure:

[0007]

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

[0009] Secondly, four 2-Thr-dihydroisoquinoline-3-carboxyl-AA compounds were docked with the active sites of IL-2, IL-8, IL-10 and TNF-ɑ to screen out IL-2 agonists.

[0010] The third technical means is to propose a method for preparing 2-Thr-dihydroisoquinoline-3-formyl-AA, which comprises the following steps:

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

[0012] 2) preparing 3S-tetrahydroisoquinoline-3-carboxylic acid methyl ester;

[0013] 3) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid methyl ester;

[0014] 4) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid;

[0015] 5) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-AA-OBzl;

[0016] 6) Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxyl-AA;

[0017] 7) Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-AA;

[0018] 8) Preparation of 3S-2-Thr-dihydroisoquinoline-3-carbonyl-AA.

[0019] The fourth technical means is to confirm that the 2-Thr-dihydroisoquinoline-3-carboxyl-AA compound with the above structure can effectively increase the blood IL-2 concentration and is an excellent IL-2 agonist.

[0020] The fifth technical means is to confirm the application of the 2-Thr-dihydroisoquinoline-3-formyl-AA compound of the above structure in the preparation of anti-tumor agents.

[0021] Experiments have shown that the four 2-Thr-dihydroisoquinoline-3-formyl-AA compounds of the structure described in the present invention have the lowest docking free energy with IL-10, can effectively increase the blood IL-2 concentration, and have a good anti-tumor effect. Therefore, the present invention provides an effective technical means for anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The synthetic route of 3S-2-Thr-dihydroisoquinoline-3S-formyl-AA is as follows: i) CH2O, H2SO4; ii) SOCl2, methanol; iii) DCC, HOBt, N-methylmorpholine, Boc-Thr; iv) 0℃, NaOH aqueous solution (2N); v) palladium carbon, continuously passing hydrogen; vi) hydrogen chloride in ethyl acetate solution (4N); viii) dimethylformamide, potassium permanganate. DETAILED DESCRIPTION

[0023] 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.

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

[0025] 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 a uniform mixture. 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 (CHCl3 / CH3OH, 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 (300MHZ, DMSO-d6): δ / ppm=12.89(s,1H),7.41(d,J=7.5Hz,1H),7.31(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,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,J1=8.2Hz,J2=7.2Hz,2H),1.92(s,1H); 13 CNMR (75MHZ, DMSO-d6): δ / ppm=173.2,135.9,130.6,127.5,126.9,126.3,56.6,46.4,35.5,30.2.

[0026] Example 2 Preparation of 3S-tetrahydroisoquinoline-3-carboxylic acid methyl ester (2)

[0027] Cool 50mL of methanol to 0℃, slowly add 10mL of SOCl2 to the cooled methanol, stir for 30 minutes to obtain a cooled methanol-SOCl2 solution system. Add 3S-tetrahydroisoquinoline-3-carboxylic acid (4.2g, 23.7mmol) to the cooled methanol-SOCl2 solution system, stir the reaction mixture at 0℃ for 24 hours, thin layer chromatography (CHCl3 / CH3OH, 5 / 1) shows that 3S-tetrahydroisoquinoline-3-carboxylic acid disappears, and the reaction mixture is concentrated to dryness under reduced pressure. Dissolve the obtained residue in 30mL of methanol, and then concentrate to dryness under reduced pressure. Repeat this operation 3 times, remove the remaining SOCl2 and hydrogen chloride, and obtain 4.3g (95%) of the title compound as a colorless solid. ESI-MS (m / e): 192 [M+H] + ; 1HNMR (300MHZ, DMSO-d6): 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=7.5 Hz, 1H), 4.36 (m, J = 4.5Hz, 2H), 3.88 (t, J = 5.6Hz, 1H), 3.66 (s, 3H), 3.02 (dd, J1 = 8.2Hz, J2 = 7.2Hz, 2H), 1.92 (s, 1H); 13 CNMR (75MHZ, DMSO-d6): δ / ppm=173.8,135.9,130.6,127.5,126.9,126.3,56.6,51.9,46.4,35.5,30.2.

[0028] Example 3 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxylic acid methyl ester (3)

[0029] 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] + ; 1HNMR (300MHZ, DMSO-d6): 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-d6): δ / ppm=172.85,171.63,167.88,137.09,129.63,129.13,128.45,12 7.85,127.21,80.02,68.22,67.88,58.14,53.66,46.05,28.57,28.56,28.55,27.49,18.56.

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

[0031] 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] - ; 1HNMR (300MHZ, DMSO-d6): δ / ppm=11.91(s,1H),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,1 H),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-d6): δ / ppm=172.85,171.63,167.88,137.09,129.63,129.13,128.45 ,127.85,127.21,80.02,68.22,67.88,58.14,46.05,28.57,28.56,28.55,27.49,18.56.

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

[0033] 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] + ; 1HNMR (300MHZ, DMSO-d6): 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(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-d6): 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.

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

[0035] 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] + ; 1HNMR (300MHZ, DMSO-d6): δ / ppm=11.92(s,1H),8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J=7.5Hz,1 H),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),5.09(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-d6): δ / ppm=172.65,172.45,172.13,171.68,159.34,134.09,127.59,127.45,127.33,1 27.32,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,28.57,28.56,28.55,28.36,27.49,18.56.

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

[0037] 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] + ; 1HNMR (300MHZ, DMSO-d6): 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),7.33(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t,J=7.5Hz,1H),5.37(s,1H),5. 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-d6): δ / ppm=172.65,172.45,172.13,171.68,134.09,127.59,127.45, 127.33,127.32,126.85,67.56,65.40,55.58,46.34,46.14,31.46,28.36,27.49,18.56.

[0038] Example 8 Preparation of 3S-2-Thr-dihydroisoquinoline-3-formyl-Gln (8a)

[0039] 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Gln (2.03 g, 5 mmol) was slowly mixed with a solution of KMnO4 (1.58 g, 5 mmol) and 200 mL of dimethylformamide at 0°C. The mixing was completed within 30 minutes. The obtained compound was stirred at room temperature for 100 hours, and TLC (CCl3:CH3OH=5:1) showed that 3S-2-Gly-tetrahydroisoquinoline-3-formyl-Ala disappeared. The reaction mixture was concentrated under reduced pressure, and the residue was repeatedly washed with distilled water to obtain 2.02 g (95%) of the title compound as a yellow powder. FT-ICR-MS (m / e): 405.1774 [M+H] + ; 1HNMR (300MHZ, DMSO-d6): 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),7.33(t,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.21(t,J=7.5Hz,1H) ,7.12(s,1H),5.37(s,1H),5.09(t,J=6.3Hz,1H),4.58(m,J=4.5Hz,1H),4.48(d,J=4.5H z,1H),4.45(s,2H),3.16(d,J=6.3Hz,2H),2.01(t,J=4.9Hz,2H),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d6): δ / ppm=172.65,172.45,172.13,171.68,134.09,127.59,127.45, 127.33,127.32,126.85,67.56,65.40,55.58,46.34,46.14,31.46,28.36,27.49,18.56.

[0040] Example 9 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Asn-OBzl (5b)

[0041] 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] + ; 1HNMR (300MHZ, DMSO-d6): 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),6.9 6(t,J=7.5Hz,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=4.5 Hz,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-d6): δ / ppm=172.65,172.45,172.13,171.68,159.34,134.97,134.09,129.63,128.68,128.45,128.41,127.81,1 27.59,127.45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.57,28.56,28.55,28.36,18.56.

[0042] Example 10 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carboxyl-Asn (6b)

[0043] 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] + ; 1HNMR (300MHZ, DMSO-d6): δ / ppm=11.28(s,1H),8.23(s,1H),8.22(s,1H),8.12(s,2H),7.56(d,J=7.5 Hz,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-d6): δ / ppm=172.65,172.45,172.13,171.68,159.34,134.97,128.68,127.81,127.5 9,126.85,80.02,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.57,28.56,28.55,28.36,18.56.

[0044] Example 11 Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Asn (7b)

[0045] 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] + ; 1HNMR (300MHZ, DMSO-d6): δ / ppm=11.28(s,1H),8.23(s,1H),8.22(s,2H),8.12(s,2H),7.5 6(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),5.3 7(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),1.21(d,J=4.5Hz,3H); 13 CNMR (75MHZ, DMSO-d6): δ / ppm=172.65,172.45,172.13,171.68,134.97,128.68,127 .81,127.59,126.85,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.36,18.56.

[0046] Example 12 Preparation of 3S-2-Thr-dihydroisoquinoline-3-formyl-Asn (8b)

[0047] 1.86 g (95%) of the title compound was obtained from 3S-2-Thr-tetrahydroisoquinoline-3-carbonyl-Asn (1.96 g, 5 mmol) using the method of Example 8 as a yellow powder. FT-ICR-MS (m / e): 391.1618 [M+H] + ; 1 HNMR (300MHZ, DMSO-d6): δ / ppm=11.28(s,1H),8.23(s,1H),8.22(s,2H),8.12(s,2 H),7.56(d,J=7.5Hz,1H),7.15(d,J=7.3Hz,1H),7.14(d,J=7.3Hz,1H),7.11(s,1H ),7.02(t,J=7.4Hz,1H),5.37(s,1H),5.09(t,J=6.3Hz,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),1.21(d,J=4.5Hz,3H); 13CNMR (75MHZ, DMSO-d6): δ / ppm=172.65,172.45,172.13,171.68,134.97,128.68,127 .81,127.59,126.85,67.56,65.40,55.58,46.34,46.14,31.46,30.05,28.36,18.56.

[0048] Example 13 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Leu-OBzl (5c)

[0049] 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 (300MHZ, DMSO-d6): 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),4.89(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); 13CNMR (75MHZ, DMSO-d6): 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, 127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,44.46,3 1.46,30.05,28.57,28.56,28.55,28.36,27.36,22.63,22.42,18.56.

[0050] Example 14 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carbonyl-Leu (6c)

[0051] 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 (300MHZ, DMSO-d6): 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=5.6H z,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); 13CNMR (75MHZ, DMSO-d6): 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.

[0052] Example 15 Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Leu (7c)

[0053] 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 (300MHZ, DMSO-d6): 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=5.6Hz,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-d6): δ / ppm=172.65,172.45,172.13,134.97,129.63,127.45,127.33,12 7.32,126.85,67.56,55.58,46.34,44.46,31.46,30.05,28.36,27.36,22.63,22.42,18.56.

[0054] Example 16 Preparation of 3S-2-Thr-dihydroisoquinoline-3-formyl-Leu (8c)

[0055] 1.85 g (95%) of the title compound was obtained from 3S-2-Thr-tetrahydroisoquinoline-3-carbonyl-Leu (1.96 g, 5 mmol) using the method of Example 8 as a yellow powder. FT-ICR-MS (m / e): 390.2029 [M+H] + ; 1 HNMR (300MHZ, DMSO-d6): 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),7.11(s,1H),7.02(t,J=7.4Hz,1H),5.37(s,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),1.99(m,J=5.6Hz ,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-d6): δ / ppm=172.65,172.45,172.13,134.97,129.63,127.45,127.33,12 7.32,126.85,67.56,55.58,46.34,44.46,31.46,30.05,28.36,27.36,22.63,22.42,18.56.

[0056] Example 17 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-formyl-Ala-OBzl (5d)

[0057] 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] + ; 1HNMR (300MHZ, DMSO-d6): 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),4.58(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-d6): δ / ppm=172.65,172.45,172.13,171.68,134.97,134.09,129.63,128.68,128.45,128.41,127.81,127.5 9,127.45,127.33,127.32,126.85,80.02,67.56,65.40,55.58,46.34,44.46,28.57,28.56,28.55,28.36,22.63,22.42,18.56.

[0058] Example 18 Preparation of 3S-2-(Boc-Thr)-tetrahydroisoquinoline-3-carbonyl-Ala (6d)

[0059] 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] + ; 1HNMR (300MHZ, DMSO-d6): 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 =4.9Hz,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-d6): δ / ppm=172.65,172.45,172.13,171.68,134.97,129.63,127.45,127.33,12 7.32,126.85,80.02,67.56,65.40,55.58,46.34,28.57,28.56,28.55,28.36,22.63,22.42,18.56.

[0060] Example 19 Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-Ala (7d)

[0061] 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] + ; 1HNMR (300MHZ, DMSO-d6): δ / ppm=12.89(s,1H),8.23(s,1H),8.22(s,2H),7.56(d,J=7.5 Hz,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=4.9Hz,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.

[0062] Example 20 Preparation of 3S-2-Thr-dihydroisoquinoline-3-formyl-Ala (8d)

[0063] 2.03 g (95%) of the title compound was obtained from 3S-2-Thr-tetrahydroisoquinoline-3-carboxyl-Ala (1.75 g, 5 mmol) using the method of Example 8 as a yellow powder. FT-ICR-MS (m / e): 348.1559 [M+H] + ; 1 HNMR (300MHZ, DMSO-d6): δ / ppm=12.89(s,1H),8.23(s,1H),8.22(s,2H),7.56( d,J=7.5Hz,1H),7.15(d,J=7.3Hz,1H),7.14(d,J=7.3Hz,1H),7.11(s,1H),6.9 6(t,J=7.5Hz,1H),5.37(s,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),1.44(d,J=5.6Hz,3H),1.21(d,J=4.5Hz,3H); 13CNMR (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.

[0064] Example 21 Molecular Docking of 3S-2-Thr-dihydroisoquinoline-3-carboxyl-AA

[0065] In order to predict the effects of 3S-2-Thr-dihydroisoquinoline-3-formyl-Gln (8a), 3S-2-Thr-dihydroisoquinoline-3-formyl-Asn (8b), 3S-2-Thr-dihydroisoquinoline-3-formyl-Leu (8c) and 3S-2-Thr-dihydroisoquinoline-3-formyl-Ala (8d) on blood cytokines in tumor mice, this application completed the molecular docking of 8a-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 8a-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 flood-filling algorithm was used to select the cavity 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 8a-d was first searched by randomly selecting the flexibility value of the variable torsion angle when selecting the site for 8a-d, and then the three-dimensional regular grid detection site detection was used 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 8a-d to obtain a comprehensive evaluation result. In the fourth step, the docking free energy of 8a-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 8a-d with IL-2 is significantly lower than that with IL-8, IL-10 and TNF-ɑ. It can be seen that IL-2 is a potential molecular target of 8a-d.

[0066] Table 1 Binding energy of 8a-d to the active sites of IL-2, IL-8, IL-10 and TNF-ɑ

[0067]

[0068] Example 22 Evaluation of the antitumor activity of 3S-2-Thr-dihydroisoquinoline-3-carboxyl-AA

[0069] ICR male mice (weight 20±2g) were kept at rest for 2 days in an environment with a temperature of 25℃, with free access to water and food. Afterwards, the mice were inoculated with S180 ascites tumor. The operation of subcutaneously inoculating S180 ascites tumor in the front right armpit of the mouse is as follows: extract the tumor cell-containing fluid of the vigorously growing S180 ascites tumor under sterile conditions, dilute the obtained tumor cell-containing fluid with two volumes of normal saline, and mix it thoroughly to make a tumor cell suspension. The tumor cell suspension is mixed with trypan blue staining solution (trypan blue staining solution is prepared according to standard operation, with a concentration of 0.2%), and then counted according to the white blood cell counting method. Those stained blue are dead cells, and those not stained are living cells. The tumor cell concentration and survival rate are calculated according to the formula: Tumor cell concentration = number of living cells in 4 squares / 4×10 4 × dilution factor = number of tumor cells / mL. Survival rate of tumor cells = number of live cells / (number of live cells + number of dead cells) × 100%. A tumor cell suspension with a survival rate greater than 90% was prepared by homogenization at 2.0 × 10 7 A suspension of 10 cells / mL was prepared and 0.2 mL of cell suspension was inoculated under the skin of the right axilla of each mouse to create an S180 ascites solid tumor mouse model.

[0070] Mice were randomly divided into groups 6 days after inoculation with S180 tumor cell suspension. Mice were either orally treated with normal saline (dose of 0.2 mL / mouse / day, once a day, for 11 consecutive days, 12 mice) or intraperitoneally injected with doxorubicin (dose of 2 μmol / kg / day, once a day, for 11 consecutive days, 12 mice) or orally treated with 3S-2-Thr-dihydroisoquinoline-3-carboxyl-AA (8a-d, dose of 10 nmol / kg / day, once a day, for 11 consecutive days, 12 mice each). The day after the last treatment, mice were weighed, anesthetized with ether, blood was collected, and the mice were killed by cervical dislocation. The tumor growth site in the right axilla of the mouse was fixed with forceps, the skin was cut open, the tumor was exposed, bluntly dissected, and weighed. The tumor weight was used to represent the antitumor activity of 3S-2-Thr-dihydroisoquinoline-3-carboxyl-AA (8a-d).

[0071] The data in Table 2 show that 8a-d effectively inhibited tumor growth at an oral dose of 10 nmol / kg / day (p<0.01 compared with saline). The data in Table 2 also show that there is no significant difference between the tumor growth inhibition activity of 8a-d at an oral dose of 10 nmol / kg / day and the tumor growth inhibition activity of doxorubicin at an intraperitoneal dose of 2 μmol / kg / day (p>0.05 compared with doxorubicin). It can be seen that the four 3S-2-Thr-dihydroisoquinoline-3-carboxyl-AA compounds represented by 8a-d have outstanding technical effects on inhibiting tumor growth.

[0072] Table 2 Antitumor activity of 3S-2-Thr-dihydroisoquinoline-3-carboxyl-AA

[0073] Therapeutic agents dose Tumor weight, mean ± SD mg Normal saline 10mL / kg 3.24±0.31 Adriamycin 2 μmol / kg / day 1.33±0.21 8a 10nmol / kg / day <![CDATA[1.32±0.18 a ]]> 8b 10nmol / kg / day <![CDATA[1.35±0.19 a ]]> 8c 10nmol / kg / day <![CDATA[1.32±0.15 a ]]> 8d 10nmol / kg / day <![CDATA[1.31±0.21 a ]]>

[0074] a) P<0.01 compared with saline, P>0.05 compared with doxorubicin; n=12.

[0075] Example 23 Evaluation of the Effect of 3S-2-Thr-Dihydroisoquinoline-3-carboxyl-AA on Cytokines

[0076] The blood of tumor-bearing mice was added with 3.8% (g / 100 mL) sodium citrate in physiological saline (v / v=9:1) at 4°C and centrifuged at 3000 g 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.

[0077] 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 plasma samples of tumor mice treated with normal saline and plasma samples of tumor mice treated with 8a-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. When 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 tumor mice treated with saline and 8a-d 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 tumor mice treated with saline and 8a-d were calculated by standard curves. Each sample was repeated 6 times. The data in Table 3 show that 8a-d significantly increased the concentration of plasma IL-2 in tumor mice (p<0.01 compared with saline). The data in Table 3 also show that 8a-d had no significant effect on the concentrations of plasma IL-8, IL-10 and TNF-ɑ in tumor mice (p>0.05 compared with saline). It can be seen that IL-2 in mouse plasma is indeed the molecular target of 8a-d to inhibit tumor growth.

[0078] Table 3 Effects of 8a-d on plasma IL-2, IL-8, IL-10 and TNF-ɑ

[0079]

[0080] a) 8a-d vs. saline, p<0.01; b) 8a-d vs. saline, p>0.05; c) 8a-d vs. saline, p>0.05; d) 8a-d vs. saline, p>0.05; n=10.

Claims

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

2. The method for preparing the four 3S-2-Thr-dihydroisoquinoline-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-3-carboxyl-AA; 7) Preparation of 3S-2-Thr-tetrahydroisoquinoline-3-formyl-AA; 8) Preparation of 3S-2-Thr-dihydroisoquinoline-3-carbonyl-AA.

3. Use of the 3S-2-Thr-dihydroisoquinoline-3-formyl-AA compound according to claim 1 in the preparation of a drug for inhibiting S180 ascites tumor.

Images