An amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid, its preparation method and application

Abstract

This invention provides a 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative, the structural formula of which is shown below: wherein the RNH is selected from one of the following. The 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative exhibits antitumor activity.

Description

Technical Field

[0001] This invention patent relates to the field of pharmaceutical technology, and in particular to an amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid, its preparation method, and its application. Background Technology

[0002] The treatment of malignant tumors is a major clinical challenge that the global medical community is striving to overcome, making the development of safe and effective anti-tumor drugs particularly urgent. Gossypol is a yellow polyphenolic compound found in the roots, stems, and seeds of cotton (a plant in the Malvaceae family). It is a chiral optical isomer with various biological activities, including spermatogenicity, immunomodulation, and anticancer activity. The anti-tumor activity of gossypol is related to its effect on Bcl family proteins. Gossypol is a BH3 analogue that can bind to the BH3 binding groove of the anti-apoptotic proteins Bcl-2 and Bcl-XL, thereby inducing apoptosis in cancer cells. It has become a hot topic in anti-tumor drug research, but its side effects limit its clinical application. Summary of the Invention

[0003] Considering the association of the formyl group in gossypol with its cytotoxicity, the inventors simplified its structure by removing the formyl group and phenolic hydroxyl group, retaining only the basic 2,2'-binaphthyl-6,6'-dicarboxylic acid skeleton. They then designed and synthesized a series of new 2,2'-binaphthyl-6,6'-dicarboxylic acid compounds by introducing amino acids at both ends of the structure to reduce toxicity. While 2,2'-binaphthyl-6,6'-dicarboxylic acid itself has no antitumor activity, structural modification and antitumor activity screening revealed that 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 exhibit strong inhibitory activity against tumor cell growth with low toxicity; however, no related research has been reported to date.

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

[0005]

[0006] Among them, RNH is selected from

[0007]

[0008] One of them.

[0009] When RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 1; when RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 2; when RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 3; when RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 4. When RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 5. When RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 6. When RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 7, then the amino acid derivative is compound 7. When RNH is... When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 8. When RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 9, the amino acid derivative is... When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 10. When RNH is When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 11.

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

[0011]

[0012]

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

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

[0015] In a third aspect, the present invention provides the use of the above-described amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid or the above-described pharmaceutical preparation in the preparation of an antitumor drug.

[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 a fourth aspect, the present invention provides a method for preparing the above-mentioned 2,2'-binaphthyl-6,6'-dicarboxylic acid amino acid derivative, wherein the derivative 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 by reacting 2,2'-binaphthyl-6,6'-dicarboxylic acid with L-amino acid methyl ester or its hydrochloride is shown below:

[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 includes the following steps:

[0020] 1) Add 2,2-binaphthyl-6,6-dicarboxylic acid, N,N-diisopropylethylamine (DIPEA) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) to the first organic solvent and stir to obtain a mixture;

[0021] 2) Add L-amino acid methyl ester or its hydrochloride and N,N-diisopropylethylamine (DIPEA) to the second organic solvent and stir to obtain the second mixture;

[0022] 3) Add the second mixture obtained in step 2) to the first mixture obtained in step 1), react, filter, add water to the filtrate until a white precipitate is formed, centrifuge, and dry 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 each 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'-tetramethylurea 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 this invention are as follows:

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

[0031] 2. This invention provides the application of the above-mentioned amino acid derivatives of 2,2'-binaphthyl-6,6'-dicarboxylic acid in the preparation of antitumor drugs. The derivatives have stable structures, low cytotoxicity, and exhibit 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 above-mentioned amino acid derivatives of 2,2'-binaphthyl-6,6'-dicarboxylic acid, which is simple in process and easy to industrialize. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. However, the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

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

[0035]

[0036] 300 mg (0.876 mmol) of 2,2'-binaphthyl-6,6'-dicarboxylic acid was placed in a 50 mL round-bottom flask, and 10 mL of DMF was added. Then, 0.46 mL (2.628 mmol) of DIPEA and 732.7 mg (1.927 mmol) of HATU were added to the round-bottom flask, and the mixture was stirred at room temperature for 1 h. Simultaneously, 408.9 mg (2.628 mmol) of L-serine methyl ester hydrochloride was placed in another 50 mL round-bottom flask, and 10 mL of DMF was added. Then, 0.46 mL (2.628 mmol) of DIPEA was added. The amino acid methyl ester reaction solution obtained from the latter was added to the binaphthyl-6,6'-dicarboxylic acid reaction solution obtained from the former. The mixture was stirred at room temperature for 3 h, and the reaction was monitored by TLC until the reaction was completed. Under magnetic stirring, purified water was added dropwise to the reaction solution until a white precipitate formed. The precipitate was then centrifuged in a 50 ml centrifuge tube. 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 calcd 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 was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-isoleucine methyl ester hydrochloride. Compound 2 was 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 was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-tyrosine methyl ester hydrochloride. Compound 3 was 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 was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-histamine methyl ester hydrochloride. Compound 4 was a white solid with a yield of 78.6% and a melting point of 290.8–291.5 °C. 1 H NMR (600MHz, 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 (151MHz, 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 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-leucine methyl ester conjugate (compound 5)

[0047]

[0048] The synthesis method was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-leucine methyl ester hydrochloride. Compound 5 was 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 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-phenylalanine methyl ester conjugate (compound 6)

[0050]

[0051] The synthesis method was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-phenylalanine methyl ester hydrochloride. Compound 6 was 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 was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-proline methyl ester hydrochloride. Compound 7 was 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 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-methionine methyl ester conjugate (compound 8)

[0056]

[0057] The synthesis method was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-methionine methyl ester hydrochloride. Compound 8 was 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, found633.20972.

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

[0059]

[0060] The synthesis method was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-tryptophan methyl ester hydrochloride. Compound 9 was 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 calcd forC 46 H 38 N4O6[M+H]+ 743.28641, found 743.28766.

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

[0062]

[0063] The synthesis method was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with L-threonine methyl ester hydrochloride. Compound 10 was 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 2,2'-binaphthyl-6,6'-dicarboxylic acid and L-valine methyl ester conjugate (compound 11)

[0065]

[0066] The synthesis method was the same as in Example 1, except that L-serine methyl ester hydrochloride was replaced with LL-valine methyl ester hydrochloride. Compound 11 was 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 activity of compounds 1-11

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

[0069] 2. Cell Culture

[0070] (1) Thawing: Remove the frozen cells from the freezer and place them in a 37°C water bath to thaw completely. Add more than 10 times the volume of DMEM high-glucose complete medium, centrifuge, discard the liquid layer, and obtain a cell pellet. Repeat the process of adding more than 10 times the volume of DMEM high-glucose complete medium, centrifuging, and obtaining a cell pellet again. Add 2 mL of DMEM high-glucose complete medium to disperse the cells into single cells. Transfer the medium containing the single cells to a culture flask, add medium to 7 mL, and place the culture flask in a 37°C, 5% CO2 incubator to passage the cells. Change the medium the next day.

[0071] (2) Culture and passage: Replace the culture medium with fresh medium according to the cell growth status. When the cell confluence reaches 80%, discard the culture medium, wash twice with PBS, and add 1 mL of 0.25% trypsin to immerse all the cells at the bottom of the flask in the solution. Discard the trypsin. Place the culture flask back in the incubator for 1-2 minutes of digestion, and then stop digestion with complete culture medium. After the cells are evenly distributed by pipetting, add them to the culture flasks and passage them at a ratio of 1:3 to 1:5. When the cells grow to 85%-90% abundance, confluence them for passage. Add the cells to the culture plate, add culture medium to the appropriate volume, and wait for the cells to enter the logarithmic growth phase.

[0072] (3) Cryopreservation: Replace the culture medium with fresh one night before. Disperse the cells into single cells, centrifuge, discard the supernatant, prepare the cell cryopreservation solution, and adjust the cell density to 1×10⁶ cells / year. 6 Cells / mL. The cell solution was placed in a 4°C freezer for 30 min, then in a -20°C freezer for 30 min, and finally in a -80°C freezer for long-term cryopreservation.

[0073] 3. MTT assay for the effect of compounds on tumor cell viability

[0074] (1) Preparation of the compound: 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 seeding: When the tumor cells grow to 80-90% abundance, the cell suspension is seeded into 96-well plates, with approximately 10,000 tumor cells per well. The cells are cultured in a 37°C, 5% CO2 incubator. After the cells adhere to the plate, they are incubated with the above-mentioned series of concentrations of complete culture medium containing the drug to be tested for 72 hours.

[0076] (3) Add 10 μl of MTT solution to each well and incubate in the dark for 4 h. Discard the supernatant, add 100 μl of DMSO to each well, and shake for 10 min. Measure the absorbance of each well at 570 nm using a microplate reader. Inhibition rate = (OD control group - OD treatment group) / OD control group × 100%. Calculate the IC50 of each compound using GraphPad Prism 9 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 as follows: plot the compound concentration on the x-axis and the inhibition rate on the y-axis to obtain the compound concentration at a 50% inhibition rate. 5-Fu: 5-fluorouracil.

[0080] As shown in Table 1, except for compound 9, the other new compounds exhibited antitumor activity against one or even all five of the five cell lines: A375, A549, PANC-1, MCF-7, and HepG2. Compound 2 showed antitumor activity against the A375 cell line (IC549, PANC-1, MCF-7, and HepG2). 50=2.92 μmol / L) and PANC-1 cell line (IC50) 50 Compound 6 exhibited the best inhibitory activity against A549 cell line (IC50 = 10.75 μmol / L), and also showed the best inhibitory activity against the A549 cell line (IC50 = 10.75 μmol / L). 50 =6.58 μmol / L) and MCF-7 cell line (IC50) 50 Compound 6 (IC50, 2.46 μmol / L) showed the best inhibitory activity, superior to 5-fluorouracil. Furthermore, compounds 4, 5, and 10 all exhibited antitumor activity against five cell lines: A375, A549, PANC-1, MCF-7, and HepG2, with significantly better antitumor activity against A375, A549, PANC-1, and MCF-7 than against 5-fluorouracil. Notably, compound 6 showed relatively low activity against A549 cell line (IC50, 2.46 μmol / L). 50 =6.58 μmol / L), MCF-7 cell line (IC50) 50 =2.46 μmol / L) and A375 cell line (IC50) 50 (5.08 μmol / L) also showed strong inhibitory activity, superior to 5-fluorouracil. These results indicate that modification of 2,2'-binaphthyl-6,6'-dicarboxylic acid with L-amino acid methyl ester enhances its antitumor activity.

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

[0082] Compound 6 was selected for acute toxicity testing. Mice weighing 18-22 grams were randomly divided into a saline group, a low-dose group (200 mg / kg), and a high-dose group (400 mg / kg), with 10 mice in each group. After 7 consecutive days of administration, there were no significant changes in body weight or hair, and no deaths occurred, indicating that the novel amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid has low toxicity and can be used for antitumor drug research.

[0083] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, all of which should be included within the protection scope of the present invention.

Claims

1. The application of an amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid in the preparation of antitumor drugs, characterized in that, The tumor is selected from at least one of liver cancer, melanoma, lung cancer, pancreatic cancer, and breast cancer, and the structural formula of the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is shown below: ； Among them, RNH is selected from , , , , , , , , , and One of them.

2. The application of a pharmaceutical formulation in the preparation of an antitumor drug, characterized in that, The tumor is selected from at least one of liver cancer, melanoma, lung cancer, pancreatic cancer, and breast cancer, and the pharmaceutical preparation comprises an amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid; the structural formula of the amino acid derivative of 2,2'-binaphthyl-6,6'-dicarboxylic acid is shown below: ； Among them, RNH is selected from , , , , , , , , , and One of them.

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

4. The application according to claim 2, characterized in that, The pharmaceutical preparation is a suspension or emulsion.

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