2, 2 '-biquinoline-4, 4'-dicarboxylic acid amino acid derivative as well as preparation method and application thereof

Abstract

The invention provides an amino acid derivative of 2, 2 '-biquinoline-4, 4'-dicarboxylic acid, the structure of the amino acid derivative of 2, 2 '-biquinoline-4, 4'-dicarboxylic acid is shown as the following: # imgabs0, RNH is selected from # imgabs1 # imgabs2 # n selected from 0, 1, 2 and 3, R1 is selected from hydrogen and hydroxyl, R2, R3 and R4 are respectively and independently selected from C1-6 alkyl, and the amino acid derivative of 2, 2 '-biquinoline-4, 4'-dicarboxylic acid has anti-tumor or anti-cancer activity.

Description

Technical Field

[0001] The patent of the present invention relates to the field of medical technology, and in particular to a 2,2'-biquinoline-4,4'-dicarboxylic acid amino acid derivative and its preparation method and application. 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 anti-apoptotic proteins Bcl-2 and Bcl-XL, thereby inducing apoptosis of 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 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, and then replaced the binaphthyl structure with a biquinoline mother core, and considered introducing amino acids at both ends of its structure to reduce toxicity, and designed and synthesized a series of new 2,2'-biquinoline-4,4'-dicarboxylic acid compounds. Disodium 2,2'-biquinoline-4,4'-dicarboxylate itself has no anti-tumor activity, but after being modified with amino acids such as L-phenylalanine methyl ester, L-tyrosine methyl ester and L-histidine methyl ester, it has strong activity in inhibiting tumor cell growth and low toxicity. There has been no related research report so far, which provides a new idea for finding new anti-tumor drugs.

[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 a first aspect of the present invention, the present invention provides an amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid, and the structure of the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid is as follows:

[0005]

[0006] Among them, RNH is selected from

[0007]

[0008] n is selected from 0, 1, 2, 3; R 1 is selected from hydrogen, hydroxyl; R 2 , R 3 , R 4 Each is independently selected from C1-6 alkyl.

[0009] In one or more embodiments of the present invention, n is selected from 0, 1; R 2 , R 3 , R 4 Each is independently selected from C1-3 alkyl.

[0010] In one or more embodiments of the present invention, RNH is selected from

[0011]

[0012] 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 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.

[0013] The structural formulas of the above-mentioned compound 1, compound 2, compound 3, compound 4 and compound 5 are shown below respectively:

[0014]

[0015] In the second aspect of the present invention, the present invention provides a method for preparing the above-mentioned amino acid derivatives of 2,2'-biquinoline-4,4'-dicarboxylic acid. The reaction formula for preparing the amino acid derivatives of 2,2'-biquinoline-4,4'-dicarboxylic acid is as follows:

[0016]

[0017] In one or more embodiments of the present invention, the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid is prepared, comprising the following steps: adding disodium 2,2'-biquinoline-4,4'-dicarboxylate to an organic solvent, then adding L-amino acid methyl ester or its salt, adding 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and after the reaction is completed, quenching the reaction, purifying, and obtaining the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid.

[0018] In one or more embodiments of the present invention, the organic solvent is selected from at least one of dichloromethane, tetrahydrofuran and dimethylformamide; the L-amino acid methyl ester is selected from L-phenylglycine methyl ester, L-tryptophan methyl ester, L-proline methyl ester, L-phenylalanine methyl ester and L-tyrosine methyl ester; the molar ratio of the 2,2'-biquinoline-4,4'-dicarboxylic acid to the L-amino acid methyl ester or its salt is 1:2-3; the molar ratio of the 2,2'-biquinoline-4,4'-dicarboxylic acid to the 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:2-4.

[0019] In the third aspect of the present invention, the present invention provides a pharmaceutical composition comprising the above-mentioned amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid.

[0020] In one or more embodiments of the present invention, the pharmaceutical composition is in the form of tablets, pills, capsules, injections, suspensions, emulsions or implants.

[0021] In a fourth aspect of the present invention, the present invention provides a use of the above-mentioned amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid and / or the above-mentioned pharmaceutical composition in the preparation of anti-tumor drugs.

[0022] In one or more embodiments of the present invention, the tumor includes at least one of liver cancer, melanoma, lung cancer, pancreatic cancer, and breast cancer.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention provides an amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid, which has anti-tumor activity.

[0025] 2. The present invention provides the use of the amino acid derivatives of the above-mentioned 2,2'-biquinoline-4,4'-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.

[0026] 3. The present invention provides a method for preparing the amino acid derivative of the above-mentioned 2,2'-biquinoline-4,4'-dicarboxylic acid, which has a simple process and is easy to industrialize. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1 Synthesis of L-phenylglycine methyl ester derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid (Compound 1)

[0029]

[0030] 1.00 g (2.6 mmol) of disodium 2,2'-biquinoline-4,4'-dicarboxylate was placed in a 100 mL eggplant-shaped bottle, 30 mL of dry DMF was added, and the mixture was stirred at room temperature for 10 min. 1.25 g (6.2 mmol) of L-phenylglycine methyl ester hydrochloride was added, and the solution changed from the original turbid state to slightly clear. The mixture was stirred for 10 min, and 2.35 g (6.1 mmol) of HATU condensation agent was added. The reaction was continued for 6 h. The reaction progress was monitored by TLC (developing agent was petroleum ether / ethyl acetate: 1 / 1). After the reaction was completed, an equal volume of purified water was added to the reactant to quench the reaction, and the mixture was allowed to stand overnight. The solid layer was filtered, and washed three times with 5-fold diluted ammonia water, and then washed with purified water until neutral. The solid layer was purified by column chromatography with petroleum ether / ethyl acetate (1 / 1) as the mobile phase to obtain a white solid, namely compound 1, with a yield of 50.5% and a melting point of 267.4-267.8 °C. 1 H NMR (600 MHz, DMSO-d 6 )δ9.86(d,J=6.8Hz,2H),8.76(s,2H),8.24(d,J=8.4Hz,2H),8.18(d,J=8.3Hz,2H),7.87(t, J=7.7Hz,2H),7.73(t,J=7.6Hz,2H),7.54-7.36(m,10H),5.79(d,J=6.7Hz,2H),3.74(s,6H); 13 C NMR (151 MHz, DMSO-d 6)δ170.93,167.05,154.17,147.44,143.11,135.45,130.62,129.78,128.7 7,128.56,128.33,128.30,125.44,124.73,116.33,57.09,52.53; HRMS(ESI + ): m / z calcd for C 38 H 30 N 4 O 6 [M+H] + 639.22381, found 639.22803.

[0031] Example 2 Synthesis of L-tryptophan methyl ester derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid (Compound 2)

[0032]

[0033] The synthesis method is the same as that of Example 1, except that L-phenylglycine methyl ester hydrochloride is replaced by L-tryptophan methyl ester hydrochloride. Compound 2 is a brown-yellow solid with a yield of 56.2% and a melting point of 220.8-221.8°C. 13 C NMR (151 MHz, DMSO-d 6 )δ172.18,166.98,154.21,147.46,143.41,136.29,130.58,129.78,128.14,127.13,125.47,1 24.51,123.94,121.12,118.57,118.20,116.08,111.55,109.76,53.75,52.21,26.71; HRMS(ESI + ): m / z calcd for C 44 H 36 N 6 O 6 [M+H] + 745.27690, found 745.27777.

[0034] Example 3 Synthesis of L-proline methyl ester derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid (Compound 3)

[0035]

[0036] The synthesis method is the same as that of Example 1, except that L-phenylglycine methyl ester hydrochloride is replaced by L-proline methyl ester hydrochloride. Compound 3 is a light yellow solid with a yield of 57.8% and a melting point of 350.5-351.6°C. 13 C NMR (151 MHz, DMSO-d 6 )δ172.27,165.89,154.64,147.46,143.92,130.93,130.05,128.74,124.87,123.72,115.19,58.35,52.25,48.52,29.06,24.52; HRMS (ESI + ): m / z calcd for C 32 H 30 N 4 O 6 [M+H] + 567.22381,found567.22668.

[0037] Example 4 Synthesis of L-phenylalanine methyl ester derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid (Compound 4)

[0038]

[0039] The synthesis method is the same as that of Example 1, except that L-phenylglycine methyl ester hydrochloride is replaced by L-phenylalanine methyl ester hydrochloride. Compound 4 is a white solid with a yield of 64.6% and a melting point of 270.5-271.5°C. 1 H NMR (600 MHz, DMSO-d 6 )δ9.44(s,2H),9.42(s,2H),8.21(s,2H),7.89(d,J=1.6Hz,2H),7.74(s,2H),7.62(d ,J=7.0Hz,2H),7.24(s,10H),4.89(dd,J=4.9,2.8Hz,2H),3.75(s,6H),2.83(s,4H).; 13 C NMR (151 MHz, DMSO-d 6 )δ172.16,167.22,154.62,147.87,143.74,138.13,137.87,131.15,130.27,129.74,1 29.64,128.81,128.59,127.14,126.78,125.86,124.83,55.98,51.85,36.71; HRMS(ESI + ): m / z calcd for C 40 H34 N 4 O 6 [M+H] + 667.25511, found 667.25555.

[0040] Example 5 Synthesis of L-tyrosine methyl ester derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid (Compound 5)

[0041]

[0042] The synthesis method is the same as that of Example 1, except that L-phenylglycine methyl ester hydrochloride is replaced by L-tyrosine methyl ester hydrochloride. Compound 5 is a yellowish solid with a yield of 61.6% and a melting point of 286.4-286.9°C. 1 H NMR (600 MHz, DMSO-d 6 )δ9.41(s,2H),9.39(s,2H),8.65(d,J=3.3Hz,2H),8.26(d,J=8.4Hz,2H),7.89(s,2H),7.76(d,J=8.4Hz,2H),7.64(s, 2H),7.16(s,2H),7.15(s,2H),6.76(s,2H),6.74(s,2H),4.80(dd,J=4.9,2.8Hz,2H),3.74(s,6H),3.14-2.92(m,4H); 13 C NMR (151 MHz, DMSO-d 6 )δ172.31,167.23,156.60,154.61,147.88,143.81,131.12,130.69,130.14,1 28.58,127.83,125.93,124.87,116.46,115.61,54.68,52.65,36.01; HRMS(ESI + ): m / z calcd for C 40 H 34 N 4 O 8 [M+H] + 699.24494,found699.24640.

[0043] Example 6 Antitumor Experiment of Compounds 1-5

[0044] The cell lines used in this experiment were purchased from the Shanghai Institutes for Biological 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.

[0045] 1. Cell culture

[0046] (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 the culture bottle, and add medium to 7 mL. Place the culture bottle in a 37°C, 5% CO 2 Incubate the cells in an incubator for subculture and replace the medium the next day.

[0047] (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. After blowing the cells evenly, add them evenly to the culture bottle, and pass them in 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.

[0048] (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.

[0049] 2. Investigate the inhibitory activity of compounds on tumor cells by MTT assay

[0050] (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.

[0051] (2) Cell inoculation: When the tumor cells grow to 80-90% abundance, the cell suspension is inoculated into a 96-well plate, with approximately 10,000 tumor cells in each well. Incubate at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator, and after the cells adhered to the wall, complete culture medium containing the drug to be tested in the above series of concentrations was given and incubated for 72 hours.

[0052] (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%. The IC of each compound was calculated using Graphpad Prism 9 software. 50 , and the average value was taken after repeating the experiment three times.

[0053] The IC of each compound was calculated using Graphpad prism9 software. 50 The experiment was repeated three times and the average value was taken. HepG2, A375, A549, PANC-1 and MCF-7 cell lines were treated with test compounds 1-5 for 72 h, and the IC 50 The results are shown in Table 1.

[0054] Table 1 Antitumor activity of compounds 1-5

[0055]

[0056] From Table 1, it can be seen that except for compound 3, the other compounds have certain antitumor activity against one or even five of the five cell lines, including A375, A549, PANC-1, MCF-7 and HepG2, which is better than 5-fluorouracil. Among them, compound 2 has antitumor activity against five cell lines, including A375, A549, PANC-1, MCF-7 and HepG2, and its antitumor activity against four cell lines, including A375, A549, PANC-1 and MCF-7, is better than 5-fluorouracil. It is worth noting that compound 4 (IC 50 :1.66μmol / L) and compound 5 (IC 50 :1.25μmol / L) have strong inhibitory activity against MCF-7 cell line, which is better than 5-fluorouracil. The above results show that the modification of 2,2'-biquinoline-4,4'-dicarboxylic acid with aromatic amino acids can enhance its anti-tumor activity.

[0057] Example 7 Acute toxicity test of 2,2'-biquinoline-4,4'-dicarboxylic acid amino acid derivatives

[0058] Compound 4 was selected for acute toxicity experiment. 18-22 g mice 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 2,2'-biquinoline-4,4'-dicarboxylic acid amino acid derivative has low toxicity and can be used in anti-tumor drug research.

[0059] 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. An amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid, characterized in that: The structure of the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid is shown below: Among them, RNH is selected from n is selected from 0, 1, 2, 3; R1 is selected from hydrogen, hydroxyl; R2, R3, R4 are independently selected from C1-6 alkyl.

2. The amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid according to claim 1, characterized in that: n is selected from 0 and 1; R2, R3 and R4 are independently selected from C1-3 alkyl.

3. The amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid according to claim 1, characterized in that: RNH is selected from 4. A method for preparing an amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid according to any one of claims 1 to 3, characterized in that: The reaction formula for preparing the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid is as follows:

5. The method for preparing the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid according to claim 4, characterized in that: The preparation of the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid comprises the following steps: adding disodium 2,2'-biquinoline-4,4'-dicarboxylate to an organic solvent, then adding L-amino acid methyl ester or its salt, and adding 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and after the reaction is completed, quenching the reaction, purifying, and obtaining the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid.

6. The method for preparing the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid according to claim 4, characterized in that: The organic solvent is selected from at least one of dichloromethane, tetrahydrofuran and dimethylformamide; the L-amino acid methyl ester is selected from L-phenylglycine methyl ester, L-tryptophan methyl ester, L-proline methyl ester, L-phenylalanine methyl ester and L-tyrosine methyl ester; the molar ratio of the 2,2'-biquinoline-4,4'-dicarboxylic acid to the L-amino acid methyl ester or its salt is 1:2-3; the molar ratio of the 2,2'-biquinoline-4,4'-dicarboxylic acid to the 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is 1:2-4.

7. A pharmaceutical composition, characterized in that The amino acid derivative comprising the 2,2'-biquinoline-4,4'-dicarboxylic acid according to any one of claims 1 to 3.

8. The pharmaceutical composition according to claim 7, characterized in that The dosage form of the pharmaceutical composition is tablet, pill, capsule, injection, suspension, emulsion or implant.

9. Use of the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid according to any one of 1 to 3 and / or the pharmaceutical composition according to claim 7 or 8 in the preparation of antitumor drugs.

10. The use according to claim 9, characterized in that: The tumor includes at least one of liver cancer, melanoma, lung cancer, pancreatic cancer, and breast cancer.

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