2,2'-biquinoline-4,4'-dicarboxylic acid amino acid derivatives, and preparation method and application thereof

Abstract

This invention provides an amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid, the structure of which is shown below: wherein RNH is selected from n, which is selected from 0, 1, 2, 3; R1 is selected from hydrogen or hydroxyl; and R2, R3, and R4 are each independently selected from C1-6 alkyl groups. This amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid exhibits antitumor or anticancer activity.

Description

Technical Field

[0001] This invention patent relates to the field of pharmaceutical technology, and in particular to a 2,2'-biquinoline-4,4'-dicarboxylic acid amino acid derivative, 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 binaphthalene structure skeleton of gossypol. They then replaced the binaphthalene structure with a biquinoline core and, considering the introduction of amino acids at both ends of the structure to reduce toxicity, designed and synthesized a series of new 2,2'-biquinoline-4,4'-dicarboxylic acid compounds. Disodium 2,2'-biquinoline-4,4'-dicarboxylic acid itself has no antitumor activity, but after modification with amino acids such as L-phenylalanine methyl ester, L-tyrosine methyl ester, and L-histidine methyl ester, they all exhibit strong inhibitory activity against tumor cell growth with low toxicity. To date, no related research has been reported, providing a new approach for the search for new antitumor drugs.

[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 an amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid, the structure of which is shown below:

[0005]

[0006] Among them, RNH is selected from

[0007]

[0008] n is selected from 0, 1, 2, 3; R1 is selected from hydrogen and hydroxyl; R2, R3, R4 are each independently selected from C1-6 alkyl groups.

[0009] In one or more embodiments of the present invention, n is selected from 0 and 1; R2, R3, and R4 are each independently selected from C1-3 alkyl groups.

[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 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, then the amino acid derivative 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 When the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid is compound 4. When RNH is At that time, the amino acid derivative of the 2,2'-binaphthyl-6,6'-dicarboxylic acid was compound 5.

[0013] The structural formulas of compounds 1, 2, 3, 4, and 5 are shown below:

[0014]

[0015] In a second aspect, the present invention provides a method for preparing the above-mentioned amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid, wherein the reaction formula for preparing the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid is shown below:

[0016]

[0017] In one or more embodiments of the present invention, the preparation of the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid includes the following steps: adding disodium 2,2'-biquinoline-4,4'-dicarboxylic acid to an organic solvent, then adding L-amino acid methyl ester or its salt, adding 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 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 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 2,2'-biquinoline-4,4'-dicarboxylic acid to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1:2-4.

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

[0020] In one or more embodiments of the present invention, the dosage form of the pharmaceutical composition is a tablet, pill, capsule, injection, suspension, emulsion or implant.

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

[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 this invention are as follows:

[0024] 1. This invention provides an amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid, which has antitumor activity.

[0025] 2. This invention provides the application of the above-mentioned amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid in the preparation of antitumor drugs. It has a stable structure, low cytotoxicity, and shows 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 above-mentioned amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid, which is simple in process and easy to industrialize. Detailed Implementation

[0027] 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.

[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 round-bottom flask, and 30 mL of dry DMF was added. The mixture was stirred at room temperature for 10 min, and then 1.25 g (6.2 mmol) of L-phenylglycine methyl ester hydrochloride was added. The solution changed from being turbid to slightly clear. The mixture was stirred for another 10 min, and then 2.35 g (6.1 mmol) of HATU condensing agent was added. The reaction was continued for 6 h. The reaction progress was monitored by TLC (petroleum ether / ethyl acetate: 1 / 1 as the developing solvent). After the reaction was completed, an equal volume of purified water was added to the reactants to quench the reaction, and the mixture was allowed to stand overnight. The solid layer was filtered, and the solid layer was washed three times with ammonia water diluted 5 times, and then washed with purified water until neutral. The solid was purified by column chromatography using petroleum ether / ethyl acetate (1 / 1) as the mobile phase to obtain a white solid, which was compound 1, with a yield of 50.5% and a melting point of 267.4–267.8 °C. 1 H NMR(600MHz,DMSO-d6)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(151MHz,DMSO-d6)δ170.93,167.05,154.17,147.44,143.11,135.45,130.62,129 .78,128.77,128.56,128.33,128.30,125.44,124.73,116.33,57.09,52.53; HRMS(ESI +): m / z calcd forC 38 H 30 N4O6[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 was the same as in Example 1, except that L-phenylglycine methyl ester hydrochloride was replaced with L-tryptophan methyl ester hydrochloride. Compound 2 was a brownish-yellow solid with a yield of 56.2% and a melting point of 220.8–221.8 °C. 13 C NMR(151MHz,DMSO-d6)δ172.18,166.98,154.21,147.46,143.41,136.29,130.58,129.78,128.14,127.13 ,125.47,124.51,123.94,121.12,118.57,118.20,116.08,111.55,109.76,53.75,52.21,26.71; HRMS(ESI + ): m / z calcd for C 44 H 36 N6O6[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 was the same as in Example 1, except that L-phenylglycine methyl ester hydrochloride was replaced with L-proline methyl ester hydrochloride. Compound 3 was a pale yellow solid with a yield of 57.8% and a melting point of 350.5–351.6 °C. 13 C NMR(151MHz,DMSO-d6)δ172.27,165.89,154.64,147.46,143.92,130.93,130.0 5,128.74,124.87,123.72,115.19,58.35,52.25,48.52,29.06,24.52; HRMS(ESI + ): m / z calcd for C 32 H30 N4O6[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 was the same as in Example 1, except that L-phenylglycine methyl ester hydrochloride was replaced with L-phenylalanine methyl ester hydrochloride. Compound 4 was a white solid with a yield of 64.6% and a melting point of 270.5–271.5 °C. 1 H NMR (600MHz, DMSO-d6) δ9.44(s,2H),9.42(s,2H),8.21(s,2H),7.89(d,J=1.6Hz,2H),7.74(s,2 H),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(151MHz,DMSO-d6)δ172.16,167.22,154.62,147.87,143.74,138.13,137.87,131.15,130.27 ,129.74,129.64,128.81,128.59,127.14,126.78,125.86,124.83,55.98,51.85,36.71; HRMS (ESI + ): m / z calcd for C 40 H 34 N4O6[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 was the same as in Example 1, except that L-phenylglycine methyl ester hydrochloride was replaced with L-tyrosine methyl ester hydrochloride. Compound 5 was a yellowish solid with a yield of 61.6% and a melting point of 286.4–286.9 °C. 1H NMR (600MHz, DMSO-d6) 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(151MHz,DMSO-d6)δ172.31,167.23,156.60,154.61,147.88,143.81,131.12,130.69 ,130.14,128.58,127.83,125.93,124.87,116.46,115.61,54.68,52.65,36.01; HRMS(ESI + ): m / z calcd forC 40 H 34 N4O8[M+H] + 699.24494, found699.24640.

[0043] Example 6: Antitumor experiments of compounds 1-5

[0044] All cell lines used in this experiment were purchased from the Shanghai Institutes for Biological 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. The positive control drug, 5-fluorouracil (5-Fu), was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0045] 1. Cell Culture

[0046] (1) Thawing: Remove the frozen cells from the freezer and place them in a 37°C water bath to thaw completely. Add at least 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 at least 10 times the volume of DMEM high-glucose complete medium and centrifuging to obtain 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 and add medium to a final volume of 7 mL. Place the culture flask in a 37°C, 5% CO2 incubator to passage the cells. Change the medium the next day.

[0047] (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.

[0048] (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.

[0049] 2. To investigate the inhibitory activity of the compound against tumor cells using the MTT assay.

[0050] (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.

[0051] (2) Cell seeding: When the tumor cells reach 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.

[0052] (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.

[0053] The IC50 values ​​of each compound were calculated using Graphpad Prism 9 software. 50The 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 hours, and the IC50 was detected and calculated. 50 The values ​​are shown in Table 1.

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

[0055]

[0056] As shown in Table 1, except for compound 3, the other compounds exhibited antitumor activity against one or even all five cell lines (A375, A549, PANC-1, MCF-7, and HepG2), which was superior to 5-fluorouracil. Compound 2 showed antitumor activity against all five cell lines (A375, A549, PANC-1, MCF-7, and HepG2), and its antitumor activity against four of them (A375, A549, PANC-1, and MCF-7) was superior to 5-fluorouracil. It is noteworthy that compound 4 (IC50)... 50 : 1.66 μmol / L) and compound 5 (IC 50 The concentration of 2,2'-biquinoline-4,4'-dicarboxylic acid (1.25 μmol / L) exhibited strong inhibitory activity against MCF-7 cell line, superior to that of 5-fluorouracil. These results indicate that modifying 2,2'-biquinoline-4,4'-dicarboxylic acid with aromatic amino acids enhances its antitumor activity.

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

[0058] Compound 4 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 2,2'-biquinoline-4,4'-dicarboxylic acid amino acid derivative has low toxicity and can be used for anti-tumor drug research.

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

2. The amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid according to claim 1, characterized in that, RNH is selected from , , , and .

3. A method for preparing an amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid as described in claim 1 or 2, characterized in that, The reaction formula for preparing the amino acid derivative of the 2,2'-biquinoline-4,4'-dicarboxylic acid is shown below: ； 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'-dicarboxylic acid to an organic solvent, then adding L-amino acid methyl ester or its salt, and adding 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. After the reaction is completed, the reaction is quenched, and the mixture is purified to obtain the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid.

4. The method for preparing the amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid according to claim 3, 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 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 2,2'-biquinoline-4,4'-dicarboxylic acid to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1:2-4.

5. A pharmaceutical composition, characterized in that, Amino acid derivatives of 2,2'-biquinoline-4,4'-dicarboxylic acid as described in claim 1 or 2.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is tablet, pill, capsule, injection, suspension, emulsion or implant.

7. The use of an amino acid derivative of 2,2'-biquinoline-4,4'-dicarboxylic acid as described in claim 1 or 2 and / or the pharmaceutical composition as described in claim 5 or 6 in the preparation of an antitumor drug; wherein the tumor is liver cancer, melanoma, lung cancer, pancreatic cancer or breast cancer.

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