Amino acid derivative of 2, 2 '-binaphthyl-6, 6'-dicarboxylic acid as well as preparation method and application of amino acid derivative

Abstract

The invention provides a 2, 2 '-binaphthyl-6, 6'-dicarboxylic acid amino acid derivative, the structural formula of the 2, 2 '-binaphthyl-6, 6'-dicarboxylic acid amino acid derivative is as shown in # imgabs0, RNH is selected from one of # imgabs1 # imgabs2 # imgabs3 #, and the 2, 2 '-binaphthyl-6, 6'-dicarboxylic acid amino acid derivative has anti-tumor activity.

Description

Technical Field

[0001] The patent of the present invention relates to the field of medical technology, and in particular to an amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid and a preparation method and application thereof. Background Art

[0002] The treatment of malignant tumors is a major clinical problem that the global medical field is trying hard to overcome, and the development of safe and effective anti-tumor drugs is particularly urgent. Cottonpol is a yellow polyphenol compound contained in the roots, stems and seeds of cotton, a plant of the Malvaceae family. It is a chiral optical isomer with multiple biological activities such as sperm inhibition, immunomodulation, and anti-cancer. The anti-tumor activity of cottonpol is related to its effect on Bcl family proteins. Cottonpol is a BH3 analog that can bind to the BH3 binding groove of the anti-apoptotic proteins Bcl-2 and Bcl-XL, thereby inducing apoptosis of cancer cells, becoming a hot spot in anti-tumor drug research, but its side effects limit its clinical application. Summary of the invention

[0003] Considering that the formyl group of gossypol is related to its cytotoxicity, the inventors simplified its structure, removed the formyl group and phenolic hydroxyl group, and only retained the basic skeleton of the binaphthyl structure of gossypol. Considering the introduction of amino acids at both ends of its structure to reduce toxicity, a series of new 2,2'-binaphthyl-6,6'-dicarboxylic acid compounds were designed and synthesized. 2,2'-binaphthyl-6,6'-dicarboxylic acid itself has no anti-tumor activity, but after structural modification and anti-tumor activity screening, it was found that the conjugates of 2,2'-binaphthyl-6,6'-dicarboxylic acid with L-serine methyl ester, L-isoleucine methyl ester, L-tyrosine methyl ester and L-histidine methyl ester all have strong tumor cell growth inhibition activity and low toxicity. There has been no related research report so far.

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. Therefore, in the first aspect of the present invention, the present invention provides a 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative, and the structural formula of the 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative is as follows:

[0005]

[0006] Among them, RNH is selected from

[0007]

[0008] One of them.

[0009] When RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 1; when RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 2; when RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 3; when RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 4. When RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 5. When RNH is When RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 7. When RNH is When RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 9. When RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 10. When RNH is When the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 11.

[0010] The structural formulas of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, and Compound 11 are shown below:

[0011]

[0012]

[0013] In a second aspect of the present invention, the present invention provides a pharmaceutical preparation, which comprises the amino acid derivative of the above-mentioned 2,2'-binaphthyl-6,6'-dicarboxylic acid.

[0014] In one or more embodiments of the present invention, the pharmaceutical preparation is a tablet, a pill, a capsule, an injection, a suspension, an emulsion or an implant.

[0015] In the third aspect of the present invention, the present invention provides a use of the above-mentioned amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid or the above-mentioned pharmaceutical preparation in the preparation of anti-tumor drugs.

[0016] In one or more embodiments of the present invention, the tumor is selected from at least one of liver cancer, melanoma, lung cancer, pancreatic cancer and breast cancer.

[0017] In the fourth aspect of the present invention, the present invention provides a method for preparing the above-mentioned 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative, which is obtained by reacting 2,2-binaphthyl-6,6-dicarboxylic acid with L-amino acid methyl ester or its hydrochloride. The reaction formula for preparing the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid with L-amino acid methyl ester or its hydrochloride is as follows:

[0018]

[0019] In one or more embodiments of the present invention, the method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative comprises the following steps:

[0020] 1) adding 2,2-binaphthyl-6,6-dicarboxylic acid, N,N-diisopropylethylamine (DIPEA) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) into a first organic solvent respectively, stirring to obtain a mixed solution;

[0021] 2) adding L-amino acid methyl ester or its hydrochloride and N,N-diisopropylethylamine (DIPEA) into a second organic solvent and stirring to obtain a second mixed solution;

[0022] 3) adding the second mixed solution obtained in step 2) to the first mixed solution obtained in step 1), reacting, filtering, adding water to the filtrate until a white precipitate is formed, centrifuging, and drying to obtain the binaphthyl dicarboxylic acid amino acid derivative.

[0023] In one or more embodiments of the present invention, the first organic solvent and the second organic solvent are independently selected from at least one of dichloromethane, tetrahydrofuran and dimethylformamide.

[0024] In one or more embodiments of the present invention, the molar ratio of the 2,2-binaphthyl-6,6-dicarboxylic acid to the L-amino acid methyl ester or its hydrochloride is 1:2-4.

[0025] In one or more embodiments of the present invention, in step 1), the molar ratio of 2,2'-binaphthyl-6,6'-dicarboxylic acid to N,N-diisopropylethylamine is 1:2-4.

[0026] In one or more embodiments of the present invention, in step 1), the molar ratio of 2,2'-binaphthyl-6,6'-dicarboxylic acid to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:2-4.

[0027] In one or more embodiments of the present invention, in step 2), the molar ratio of L-amino acid methyl ester or its hydrochloride to N,N-diisopropylethylamine is 1:0.8-1.2.

[0028] In one or more embodiments of the present invention, the L-amino acid methyl ester is selected from L-serine methyl ester, L-isoleucine methyl ester, L-tyrosine methyl ester, L-histidine methyl ester, L-leucine methyl ester, phenylalanine methyl ester, L-proline methyl ester, L-methionine methyl ester, L-tryptophan methyl ester, L-threonine methyl ester and L-valine methyl ester.

[0029] The beneficial effects of the present invention are:

[0030] 1. The present invention provides an amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid, which can be used to prepare anti-tumor drugs.

[0031] 2. The present invention provides the use of the amino acid derivatives of the above-mentioned 2,2'-binaphthyl-6,6'-dicarboxylic acid in the preparation of anti-tumor drugs, which have stable structures, low cytotoxicity, and show strong inhibitory activity against HepG2, A375, A549, PANC-1 and MCF-7 standard strains in vitro.

[0032] 3. The present invention provides a method for preparing the amino acid derivatives of the above-mentioned 2,2'-binaphthyl-6,6'-dicarboxylic acid, which has a simple process and is easy to industrialize. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific examples, but the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to normal conditions or conditions recommended by the manufacturer. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the recorded content may also be applied to the present invention.

[0034] Example 1 Preparation of the conjugate of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-serine methyl ester (Compound 1)

[0035]

[0036] 300 mg (0.876 mmol) of 2,2'-binaphthyl-6,6'-dicarboxylic acid was placed in a 50 mL eggplant-shaped bottle, 10 ml of DMF was added, and then 0.46 ml (2.628 mmol) of DIPEA and 732.7 mg (1.927 mmol) of HATU were added to the eggplant-shaped bottle, respectively, and stirred at room temperature for 1 hour. At the same time, 408.9 mg (2.628 mmol) of L-serine methyl ester hydrochloride was placed in another 50 mL eggplant-shaped bottle, 10 ml of DMF was added, and then 0.46 ml (2.628 mmol) of DIPEA was added for later use. The amino acid methyl ester reaction solution obtained from the latter was added to the binaphthyl dicarboxylic acid reaction solution obtained from the former, and the reaction was stirred at room temperature for 3 hours, and TLC was used to track until the reaction was completed. Purified water was added dropwise to the reaction solution under magnetic stirring until a white precipitate was formed, and the solution was placed in a 50 ml centrifuge tube for centrifugation. The solid layer was washed three times with purified water, and the liquid layer was discarded. The solid product was purified by column chromatography using petroleum ether / ethyl acetate (1 / 1) as the mobile phase to obtain a white solid with a yield of 79.5% and a melting point of 290.5-291.3°C. 1 H NMR (600MHz, DMSO-d6): δ8.77(d,J=7.3Hz,2H),8.55(s,2H),8.46(s,2H),8.17(d,J=8.7Hz,2H),8.11(s ,2H),8.08(s,2H),7.99(d,J=8.6Hz,2H),5.14(s,2H),4.59(d,J=6.2Hz,2H),3.83(s,4H),3.65(s,6H); 13 CNMR (151MHz, DMSO-d6): δ171.54,166.99,139.08,135.10,131.96,131.74,13 0.20,128.89,128.05,126.51,126.19,125.29,61.50,56.23,52.38; HRMS(ESI + ):m / z calculation forC 30 H 28 N2O8[M+H] + 545.19184,found[M+H] + 545.19318.

[0037] Example 2 Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-isoleucine methyl ester conjugate (Compound 2)

[0038]

[0039] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-isoleucine methyl ester hydrochloride. Compound 2 is a white solid with a yield of 76.8% and a melting point of 177.5-178.4°C. 1 H NMR (600MHz, DMSO-d6): δ8.78(d,J=7.7Hz,2H),8.52(s,2H),8.46(s,2H),8.17(d,J=8.6Hz,2H),8.10(s,2H),8.08(s,2H),7.97( d,J=7.8Hz,2H),4.41(t,J=7.6Hz,2H),3.65(s,6H),1.99(s,2H),1.56-1.28(m,4H),0.91(d,J=6.8Hz,6H),0.87(t,J=7.4Hz,6H); 13 C NMR (151MHz, DMSO-d6): δ172.76,167.38,138.97,135.08,131.91,131.84,130.19,128. 77,128.14,126.42,126.13,125.51,57.91,52.09,36.15,25.71,15.98,11.34; HRMS(ESI + ): m / z calcd for C 36 H 40 N2O6[M+H] + 597.29591,found597.29663.

[0040] Example 3 Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-tyrosine methyl ester conjugate (Compound 3)

[0041]

[0042] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-tyrosine methyl ester hydrochloride. Compound 3 is a white solid with a yield of 80.6% and a melting point of 237.8-238.6°C. 1H NMR (600MHz, DMSO-d6): δ9.25(s,2H),8.96(d,J=7.6Hz,2H),8.44(s,4H),8.14(d,J=8.6Hz,2H),8.09(s,2H),8.07(s,2H),7.90(d,J=8 .6Hz,2H),7.10(s,2H),7.09(s,2H),6.65(s,2H),6.64(s,2H),4.62(dd,J=13.8,9.0Hz,2H),3.62(s,6H),3.03(dd,J=20.2,7.6Hz,4H); 13 C NMR (151MHz, DMSO-d6): δ172.80,166.92,156.34,139.03,135.06,131.90,131.71,130.47,13 0.20,128.89,128.10,127.99,126.50,126.14,125.19,115.52,55.25,52.37,36.05; HRMS(ESI + ): m / z calcd for C 42 H 36 N2O8[M+H] + 697.25444, found 697.25513.

[0043] Example 4 Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-histidine methyl ester conjugate (Compound 4)

[0044]

[0045] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-histidine methyl ester hydrochloride. Compound 4 is a white solid with a yield of 78.6% and a melting point of 290.8-291.5°C. 1 H NMR (600 MHz, DMSO-d6): δ H8.89(d,J=7.3Hz,2H,2×CONH),8.301(s,2H,2×Ar-H),8.286(s,2H,2×Ar-H),7.9 9(d,J=8.6Hz,2H,2×Ar-H),7.93(m,2H,2×Ar-H),7.93(m,2H,2×Ar-H),7.76(d,J =8.6Hz,2H,2×Ar-H),7.48(s,2H,Imidazole-4-yl-H),6.74(s,2H,Imidazole-4 -yl-H), 4.56 (q, J=7.1Hz, 2H, 2×NCH), 3.45 (s, 6H, 2×OCH3), 2.92 (m, 4H, 2×CH2); 13 C NMR (151 MHz, DMSO-d6):δ C 181.6,175.8,148.1,144.6,144.2,143.10,141.1,140.8,139.3,138.1,137.0,135.6,135.3,134.2,126.03,62.7,61.5,38.0; HRMS (ESI + ):m / z calcd for C 36 H 32 N6O6[M+Na] + 667.2281, found 667.22796.

[0046] Example 5 Preparation of the conjugate of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-leucine methyl ester (Compound 5)

[0047]

[0048] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-leucine methyl ester hydrochloride. Compound 5 is a white solid with a yield of 71.5% and a melting point of 197.1-197.4°C. 1 H NMR (600MHz, DMSO-d6) δ8.95(d,J=7.6Hz,2H),8.55(s,2H),8.47(s,2H),8.18(d,J=8.5Hz,2H),8.11(s,2H),8.10(s,2H),8.03-7.9 8(m,2H),4.57(t,J=4.1Hz,2H),3.65(d,J=1.9Hz,6H),1.85-1.72(m,4H),1.60(s,2H),0.93(d,J=6.6Hz,6H),0.89(d,J=6.5Hz,6H); 13C NMR (151MHz, DMSO-d6) δ173.65,167.05,139.02,135.10,131.95,131.74,130.20,1 28.85,128.09,126.48,125.35,52.38,51.52,36.20,24.91,23.34,21.60; HRMS(ESI + ): m / z calcd for C 36 H 40 N2O6[M+H] + 597.29591,found597.29688.

[0049] Example 6 Preparation of the conjugate of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-phenylalanine methyl ester (Compound 6)

[0050]

[0051] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-phenylalanine methyl ester hydrochloride. Compound 6 is a white solid with a yield of 73.9% and a melting point of 190.3-191.5°C. 1 H NMR (600MHz, DMSO-d6) δ9.09(d,J=7.7Hz,2H),8.47(s,2H),8.45(s,2H),8.15(d,J=8.6Hz,2H),8.10(s,2H),8.08(s,2H),7.92(d,J=8.6Hz,2H), 7.34(s,2H),7.33(s,2H),7.28(d,J=7.4Hz,2H),7.26(s,2H),7.18(t,J= 7.4Hz,2H),4.73(dd,J=7.7,3.0Hz,2H),3.64(s,6H),3.21-3.12(m,4H); 13 C NMR(151MHz,DMSO-d6)δ172.72,166.86,139.04,138.15,135.08,131.91,131.66,130.20,129 .56,128.89,128.74,128.07,126.99,126.52,126.19,125.19,54.91,52.46,36.73; HRMS(ESI + ): m / z calcd for C 42 H 36 N2O6[M+H] + 665.26461,found665.26617.

[0052] Example 7 Preparation of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-proline methyl ester conjugate (Compound 7)

[0053]

[0054] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-proline methyl ester hydrochloride. Compound 7 is a white solid with a yield of 62.4% and a melting point of 198.7-199.6°C. 1 H NMR (600MHz, DMSO-d6) δ8.45(s,2H),8.19(s,2H),8.18(d,J=11.0Hz,2H),8.09(s,2H),8.07(s,2H),7.65(d,J=8.4Hz,2 H), 4.52 (d, J = 3.6Hz, 2H), 3.67 (s, 6H), 3.63 (d, J = 5.9Hz, 4H), 2.61-2.07 (m, 4H), 1.89 (dd, J = 10.4, 4.7Hz, 4H); HRMS (ESI + ): m / z calcd for C 34 H 32 N2O6[M+H] + 565.23331, found 565.23407.

[0055] Example 8 Preparation of the conjugate of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-methionine methyl ester (Compound 8)

[0056]

[0057] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-methionine methyl ester hydrochloride. Compound 8 is a white solid with a yield of 75.0% and a melting point of 196.5-197.2°C. 1 H NMR (600MHz, DMSO-d6) δ8.99(d,J=7.4Hz,2H),8.54(s,2H),8.48(s,2H),8.18(s,2H),8.12(d,J=1.9Hz,2H),8.11(d,J=1.8Hz,2H) ,8.00(s,2H),4.64(d,J=5.0Hz,2H),3.66(d,J=1.8Hz,6H),2.66-2.57(m,4H),2.10(dd,J=8.3,5.8Hz,4H),2.06(d,J=1.7Hz,6H); 13C NMR (151MHz, DMSO-d6) δ173.00,167.21,139.03,135.11,131.94,131.71,130.22,12 8.88,128.13,126.50,126.21,125.35,52.51,52.25,30.52,30.35,14.99; HRMS(ESI + ): m / z calcd for C 34 H 36 N2O6S2[M+H] + 633.20875, found 633.20972.

[0058] Example 9 Preparation of the conjugate of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-tryptophan methyl ester (Compound 9)

[0059]

[0060] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-tryptophan methyl ester hydrochloride. Compound 9 is a yellow solid with a yield of 61.6% and a melting point of 209.8-210.5°C. 1 H NMR (600MHz, DMSO-d6) δ10.89 (s, 2H), 9.05 (d, J = 7.5Hz, 2H), 8.49 (s, 2H), 8. 46(s,2H),8.14(s,2H),8.10(s,2H),8.08(s,2H),7.95(d,J=8.7Hz,2H),7.61 (d,J=7.9Hz,2H),7.33(d,J=7.8Hz,2H),7.27(s,2H),7.06(s,2H),7.00(s,2H ),4.76(t,J=7.9Hz,2H),3.65(d,J=3.0Hz,6H),3.32(dd,J=14.3,7.5Hz,4H); 13 C NMR (151MHz, DMSO-d6) δ173.05,166.91,139.06,136.60,135.08,131.94,131.77,130.18,128.85,128.09,127 .59,126.51,126.20,125.28,124.20,121.48,118.92,118.53,111.97,110.45,54.49,52.42,27.19; HRMS(ESI + ): m / z calculation for C 46 H 38 N4O6[M+H]+ 743.28641, found 743.28766.

[0061] Example 10 Preparation of the conjugate of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-threonine methyl ester (Compound 10)

[0062]

[0063] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by L-threonine methyl ester hydrochloride. Compound 10 is a white solid with a yield of 77.8% and a melting point of 214.0-215.1°C. 1 H NMR (600MHz, DMSO-d6) δ8.58(s,2H),8.50(s,2H),8.48(s,2H),8.20(s,2H),8.12(s,2H),8.01(s,2H),5.05(dd,J =7.4,1.9Hz,2H),4.56(d,J=4.6Hz,2H),4.22(d,J=5.9Hz,2H),3.66(d,J=2.0Hz,6H),1.19-1.17(m,6H); HRMS(ESI + ): m / z calcd for C 32 H 32 N2O8[M+H] + 573.22314, found 573.22437.

[0064] Example 11 Preparation of the conjugate of 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-valine methyl ester (Compound 11)

[0065]

[0066] The synthesis method is the same as that of Example 1, except that L-serine methyl ester hydrochloride is replaced by LL-valine methyl ester hydrochloride. Compound 11 is a white solid with a yield of 71.3% and a melting point of 254.7-255.3°C. 1 H NMR (600MHz, DMSO-d6) δ8.83(d,J=7.7Hz,2H),8.55(s,2H),8.48(s,2H),8.18(d,J=8.6Hz,2H),8.11(d,J=2.4Hz,2H),8.10(s,2 H),8.00-7.96(m,2H),4.36-4.33(m,2H),3.66(d,J=1.7Hz,6H),2.21(d,J=6.8Hz,2H),1.02-1.00(m,6H),0.95(d,J=6.5Hz,6H);13 C NMR (151MHz, DMSO-d6) δ172.79,167.43,138.97,135.08,131.91,131.85,130.20,12 8.77,128.20,126.43,126.17,125.55,59.24,52.15,30.02,19.64,19.61; HRMS(ESI + ): m / z calcdfor C 34 H 36 N2O6[M+H] + 569.26461,found569.26587.

[0067] Example 12 In vitro antitumor effects of compounds 1-11

[0068] 1. Cells: The cell lines used in this experiment were purchased from the Shanghai Institute of Life Sciences, Chinese Academy of Sciences, including: human hepatoma cell line HepG2, human malignant melanoma cell line A375, human non-small cell lung cancer cell line A549, human pancreatic cancer cell line PANC-1 and human breast cancer cell line MCF-7. Positive control drug: 5-fluorouracil (Fluorouracil, 5-Fu), purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0069] 2. Cell culture

[0070] (1) Recovery: Take out the frozen cells from the refrigerator, place them in a 37°C water bath to completely thaw, then add 10 times more DMEM high-glucose complete medium, centrifuge, discard the liquid layer, obtain the cell pellet, repeat adding 10 times more DMEM high-glucose complete medium, centrifuge, and obtain the cell pellet again. Add 2 mL of DMEM high-glucose complete medium to blow the cells into single cells, transfer the medium containing single cells to a culture bottle, add medium to 7 mL, place the culture bottle in a 37°C, 5% CO2 incubator, and passage the cells. The next day, change the medium.

[0071] (2) Culture and passaging: Replace the culture medium with fresh one according to the growth status of the cells. When the cell confluence reaches 80%, discard the culture medium, wash twice with PBS, add 1 mL of 0.25% trypsin, immerse the cells at the bottom of the bottle in the solution, and discard the trypsin. Return the culture bottle to the incubator and digest for 1 to 2 minutes, then terminate the digestion with complete culture medium. Beat the cells evenly and add them evenly to the culture bottle. Passage them in bottles at a ratio of 1:3 to 1:5. When the cells grow to 85% to 90% abundance, they are confluent and passaged. Add the cells to the culture plate, add culture medium to the corresponding volume, and wait until the cells enter the logarithmic growth phase.

[0072] (3) Cryopreservation: Replace the culture medium with fresh one the night before. Blow the cells apart into single cells, centrifuge, discard the supernatant, prepare the cell freezing solution, and adjust the cell density to 1×10 6 The cell solution was placed in a 4°C refrigerator for 30 min, then in a -20°C refrigerator for 30 min, and finally in a -80°C low-temperature refrigerator for long-term freezing.

[0073] 3. MTT assay to detect the effect of compounds on tumor cell activity

[0074] (1) Preparation of compounds: Dissolve the compound in DMSO to prepare a stock solution with a concentration of 25 mg / mL, and dilute it with complete culture medium to a series of concentrations of 50 μmol / L, 15 μmol / L, 5 μmol / L, 1.5 μmol / L, 0.5 μmol / L, 0.15 μmol / L, 0.05 μmol / L, and 0.015 μmol / L.

[0075] (2) Cell inoculation: When the tumor cells grow to 80-90% abundance, the cell suspension is inoculated into a 96-well plate, with about 10,000 tumor cells in each well. The cells are cultured in a 37°C, 5% CO2 constant temperature incubator. After the cells adhere to the wall, the above-mentioned series of concentrations of complete medium containing the drug to be tested are respectively given and incubated for 72 hours.

[0076] (3) Add 10 μl of MTT solution to each well and incubate in a cell culture incubator in the dark for 4 h. Discard the supernatant, add 100 μl of DMSO to each well, and shake for 10 min. Detect the absorbance of each well at 570 nm using an ELISA reader. Inhibition rate = (OD control group - OD drug group) / OD control group × 100%. Calculate the IC of each compound using graphpad prism9 software 50 , the experiment was repeated three times and the average value was taken. The results are shown in Table 1.

[0077] Table 1 Antitumor activity of compounds 1-11

[0078]

[0079] *IC 50 The calculation method is: plot the compound concentration as the horizontal axis and the inhibition rate as the vertical axis to obtain the compound concentration when the inhibition rate is 50%. 5-Fu: 5-fluorouracil.

[0080] From Table 1, it can be seen that, except for compound 9, the other new compounds have certain antitumor activity against one or even five of the five cell lines, including A375, A549, PANC-1, MCF-7 and HepG2. Compound 2 has a certain antitumor activity against A375 cell line (IC 50=2.92 μmol / L) and PANC-1 cell line (IC 50 =10.75 μmol / L) had the best inhibitory activity, and compound 6 had the best inhibitory activity against A549 cell line (IC 50 =6.58 μmol / L) and MCF-7 cell line (IC 50 =2.46μmol / L) showed the best inhibitory activity, which was better than 5-fluorouracil. In addition, compounds 4, 5 and 10 had antitumor activity against five cell lines, including A375, A549, PANC-1, MCF-7 and HepG2, and their antitumor activities against four cell lines, A375, A549, PANC-1 and MCF-7, were better than 5-fluorouracil. It is worth noting that compound 6 had an antitumor activity against the A549 cell line (IC 50 =6.58 μmol / L), MCF-7 cell line (IC 50 =2.46 μmol / L) and A375 cell line (IC 50 =5.08μmol / L) also have strong inhibitory activity, which is better than 5-fluorouracil. The above results show that the modification of 2,2'-binaphthyl-6,6'-dicarboxylic acid with L-amino acid methyl ester can enhance its antitumor activity.

[0081] Example 13 Acute toxicity test of 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivatives

[0082] Compound 6 was selected for acute toxicity experiment. Mice weighing 18-22 grams were randomly divided into normal saline group, low-dose group 200 mg / kg, and high-dose group 400 mg / kg, with 10 mice in each group. After the mice were given the drug for 7 consecutive days, there was no significant change in body weight and hair, and no death occurred, indicating that the new amino acid derivatives of 2,2'-binaphthyl-6,6'-dicarboxylic acid have low toxicity and can be used in anti-tumor drug research.

[0083] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, which shall all be included in the scope of protection of the present invention.

Claims

1. A 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative, characterized in that: The structural formula of the 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative is as follows: Among them, RNH is selected from One of them.

2. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid according to claim 1.

3. The pharmaceutical preparation according to claim 2, characterized in that The pharmaceutical preparation is a tablet, a pill, a capsule, an injection, a suspension, an emulsion or an implant.

4. Use of the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid according to claim 1 or the pharmaceutical preparation according to claim 2 in the preparation of anti-tumor drugs.

5. The use according to claim 4, characterized in that: The tumor is selected from at least one of liver cancer, melanoma, lung cancer, pancreatic cancer and breast cancer.

6. A method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative according to claim 1, characterized in that: The reaction formula for preparing the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid by reacting 2,2-binaphthyl-6,6-dicarboxylic acid with L-amino acid methyl ester or its hydrochloride is as follows:

7. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative according to claim 6, characterized in that: The steps include: 1) adding 2,2-binaphthyl-6,6-dicarboxylic acid, N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate to a first organic solvent respectively, stirring to obtain a mixed solution; 2) adding L-amino acid methyl ester or its hydrochloride and N,N-diisopropylethylamine to a second organic solvent and stirring to obtain a second mixed solution; 3) adding the second mixed solution obtained in step 2) to the first mixed solution obtained in step 1), reacting, filtering, adding water to the filtrate until a white precipitate is formed, centrifuging, and drying to obtain the binaphthyl dicarboxylic acid amino acid derivative.

8. The method for preparing the 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative according to claim 7, characterized in that: The first organic solvent and the second organic solvent are independently selected from at least one of dichloromethane, tetrahydrofuran and dimethylformamide.

9. The method for preparing the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid according to claim 6, characterized in that: The molar ratio of the 2,2-binaphthyl-6,6-dicarboxylic acid to the L-amino acid methyl ester or its hydrochloride is 1:2-4.

10. The method for preparing 2,2-binaphthyl-6,6-dicarboxylic acid according to claim 6, wherein the L-amino acid methyl ester is selected from L-serine methyl ester, L-isoleucine methyl ester, L-tyrosine methyl ester, L-histidine methyl ester, L-leucine methyl ester, phenylalanine methyl ester, L-proline methyl ester, L-methionine methyl ester, L-tryptophan methyl ester, L-threonine methyl ester and L-valine methyl ester.

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