Isothiocyanate derivatives of brefeldin A and their preparation method and application

Abstract

The present invention discloses a class of brefeldin A derivatives, their preparation methods, and applications, belonging to the field of pharmaceutical chemistry technology. Using brefeldin A as a lead compound, the present invention designs and synthesizes a series of isothiocyanate derivatives of brefeldin A. The anti-tumor biological activity of the synthesized brefeldin A derivatives is then tested. Pharmacological tests demonstrate that some of the target derivatives prepared in the present invention exhibit excellent anti-tumor cell proliferation effects and good selectivity against normal cells, and can be used to further prepare anti-tumor drugs.

Description

[0001] This invention application is a divisional application with application number "202310542992.X" and invention name "A class of isothiocyanate derivatives of brefeldin A and their preparation method and application". Technical Field

[0002] The present invention belongs to the field of pharmaceutical chemistry technology and relates to a class of brefeldin A derivatives and their preparation methods and applications, and specifically to a series of brefeldin A derivatives with anti-tumor activity, their preparation methods and their applications in anti-tumor treatment. Background Art

[0003] Brefeldin A is a natural macrolide product isolated from the fermentation broth of Penicillium decumbens and other fungi. Brefeldin A has garnered attention for its diverse biological activities, including antitumor, antibacterial, antiviral, and antimitotic activities. Furthermore, total synthetic routes to increase the yield of Brefeldin A are under investigation. In the field of antitumor drug research, Brefeldin A has been shown to arrest the tumor cell cycle, induce apoptosis, and inhibit tumor cell invasion. However, limited research has been conducted on the application of structural modification of Brefeldin A as a lead compound to antitumor drug development. Structural modification of Brefeldin A to obtain Brefeldin A derivatives with antitumor activity as candidate compounds for clinical application remains of considerable scientific value. Summary of the Invention

[0004] In order to solve the technical problems in the prior art, the purpose of the present invention is to provide a series of brefeldin A derivatives with anti-tumor activity, preparation methods and uses thereof in anti-tumor aspects.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a class of brefeldin A derivatives, whose general structural formulas are shown in Formula 5, Formula 6, and Formula 7:

[0007]

[0008] Wherein, R1 is an aromatic group containing 4 to 6 carbon atoms, or a hydrocarbon group containing 1 to 6 carbon atoms and an oxygen-containing hydrocarbon group; R2 is a hydrocarbon group containing 1 to 6 carbon atoms.

[0009] Based on the above technical solution, further, R1 is an aromatic group containing 5-6 carbon atoms, or a hydrocarbon group and an oxygen-containing hydrocarbon group containing 2-6 carbon atoms; R2 is a hydrocarbon group containing 2-6 carbon atoms.

[0010] Another aspect of the present invention provides a method for preparing a brefeldin A derivative, the preparation route of which is as follows:

[0011]

[0012] Reaction reagents and conditions: (a) 1,1'-thiocarbonyldiimidazole, TEA, rt, 1.5-8h; (d) DMAP, EDCI, rt, 4-10h.

[0013] Based on the above technical solution, further, the preparation method mainly includes one of the following methods:

[0014] Preparation of derivatives 5, 6 and 7:

[0015] Dissolve p-aminobenzoic acid 1 in an organic solvent, add TEA and 1,1'-thiocarbonyldiimidazole, react for 0.5-4 hours, add an organic solvent containing an inorganic acid, and react for another 1-4 hours. Pour the reaction solution into water, filter with suction, wash the filter residue several times with water, and dry to obtain 1.5 g of compound 2.

[0016] Brefeldin A and compound 2 were dissolved in an organic solvent, EDCI and a catalytic amount of DMAP were added, and the reaction was carried out at room temperature for 4 to 12 hours. The resulting product was loaded onto a silica gel column chromatography column and eluted with a PE / EA mixed solution to obtain brefeldin A derivatives 5, 6 and 7.

[0017] Based on the above technical solution, further, the inorganic acid reagent includes hydrochloric acid, sulfuric acid or a mixture of hydrochloric acid and sulfuric acid.

[0018] Based on the above technical solution, further, the organic solvent includes DCM, MeOH, PE, EA, acetone and n-hexane.

[0019] Another aspect of the present invention provides a pharmaceutical composition comprising one or two or more of the above-mentioned brefeldin A isothiocyanate derivatives.

[0020] Based on the above technical solution, further, the pharmaceutical composition contains a therapeutically effective amount of the above-mentioned Brefeldin A derivative and a pharmaceutically acceptable carrier.

[0021] The present invention also provides the use of the above-mentioned brefeldin A isothiocyanate derivative and pharmaceutical composition in the preparation of drugs for treating tumor diseases.

[0022] Furthermore, the tumor includes liver cancer tumor, breast cancer tumor, malignant melanoma tumor, lung cancer tumor and cervical cancer tumor.

[0023] The present invention has the following beneficial effects compared to the prior art:

[0024] The present invention uses brefeldin A as a lead compound, designs and synthesizes a series of isothiocyanate derivatives of brefeldin A, and tests the biological activity of the synthesized derivatives in anti-tumor aspects. Pharmacological experiments show that some of the target derivatives prepared by the present invention have excellent anti-proliferation activity against human liver cancer cells, human breast cancer cells, human malignant melanoma cells, human lung cancer cell lines and human cervical cancer cells, and have good selectivity for normal cells, and can be used for further preparation of anti-tumor drugs. DETAILED DESCRIPTION

[0025] The following non-limiting examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0026] The synthetic routes of the derivatives of the present invention are as follows:

[0027]

[0028] Reaction reagents and conditions: (a) 1,1'-thiocarbonyldiimidazole, TEA, rt, 3h; (b) corresponding diol reagents, DMAP, EDCI, rt, 4-12h; (c) corresponding anhydride reagents, TEA, DMAP, rt, 10-24h; (d) DMAP, EDCI, rt, 4-10h.

[0029] Example 1

[0030] The preparation method of Brefeldin A derivatives 5, 6 and 7 mainly comprises the following steps:

[0031]

[0032] (1) p-Aminobenzoic acid 1 (1.2 g, 8.8 mmol) and 1,1'-thiocarbonyldiimidazole (2.1 g, 11.8 mmol) were dissolved in 30 mL of dichloromethane. TEA (1.4 mL, 10.0 mmol) was then added. After 1 h of reaction, 12 mL of a n-hexane solution containing hydrochloric acid and sulfuric acid (3 mL) was added and the reaction continued for 2 h. After TLC monitoring of the reaction completion, the reaction solution was poured into 30 mL of water. After the solid was completely precipitated, the filter cake was filtered and washed with water several times, and then air-dried to obtain 1.5 g of off-white powder compound 2.

[0033] (2) Brefeldin A (56.1 mg, 0.2 mmol) and compound 2 (53.8 mg, 0.3 mmol) were dissolved in 10 mL of dichloromethane, and EDCI (191.7 mg, 1.0 mmol) and a catalytic amount of DMAP (2.4 mg, 0.02 mmol) were added. The mixture was reacted at room temperature for 8 h. After the reaction was completed as monitored by TLC, 40 mL of water was added to the reaction solution, and the mixture was extracted three times with dichloromethane. The organic phases were combined and washed once with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and eluted by silica gel column chromatography with a gradient elution ratio of DCM:MeOH = 400:1 to 100:1 to obtain colorless oily compounds 5, 6, and 7, respectively, with yields of 23%, 12%, and 7%, respectively.

[0034] The characterization data of compound 5 are as follows:

[0035] 1 H NMR (CDCl3, 600MHz), δ: 8.05 (2H, d, J = 8.6Hz, Ar-H), 8.00 (2H, d, J = 8.6Hz, Ar-H), 7.35 (1H, dd ,J=15.7,3.4Hz,H-3),7.28(4H,m,Ar-H),5.79(2H,m,H-2,H-11),5.56(1H,ddd,J=10.4,3.1,1 .7Hz,H-4),5.40(1H,m,H-7),5.30(1H,dd,J=15.1,9.6Hz,H-10),4.88(1H,m,H-15),0.93~2. 67(15H,m,H-5,H-6a,H-6b,H-8a,H-8b,H-9,H-12a,H-12b,H-13a,H-13b,H-14a,H-14b,-CH3);

[0036] 13 C NMR(CDCl3,150MHz), δ:165.5,164.8,164.2,146.6,138.1,138.0,136.2,135.8,135.4,131.6,131.2(×2),131.0( ×2),128.2,127.9,125.8(×2),125.7(×2),118.8,77.1,76.3,72.0,50.1,44.2,40.1,38.5,34.1,31.8,26.5,20.7;

[0037] HR-ESIMS m / z calcd for C 32 H 30 N2NaO6S2[M+Na] +625.1437, found 625.1489.

[0038] The characterization data of compound 6 are as follows:

[0039] 1 H NMR (CDCl3, 600MHz), δ: 8.06 (2H, d, J = 8.6Hz, Ar-H), 7.33 (1H, dd, J = 15.7, 3.3Hz, H-3), 7 .29(2H,d,J=8.6Hz,Ar-H),5.74(2H,m,H-2,H-11),5.56(1H,ddd,J=10.5,3.1,1.9Hz,H-4 ),5.34(1H,dd,J=15.3,9.6Hz,H-10),4.85(1H,m,H-15),4.34(1H,m,H-7),0.90~2.55(1 5H,m,H-5,H-6a,H-6b,H-8a,H-8b,H-9,H-12a,H-12b,H-13a,H-13b,H-14a,H-14b,-CH3);

[0040] 13 C NMR (CDCl3, 150MHz), δ: 165.6, 164.3, 146.9, 138.1, 136.3, 136.1, 131.2 (×2), 130.8, 12 8.0,125.8(×2),118.4,77.3,72.4,71.9,49.7,44.3,43.2,41.1,34.1,31.8,26.6,20.8;

[0041] HR-ESIMS m / z calcd for C 24 H 27 NNaO5S[M+Na] + 464.1502,found 464.1505.

[0042] The characterization data of compound 7 are as follows:

[0043] 1H NMR (CDCl3, 600MHz), δ: 8.00 (2H, dt, J = 8.6, 2.0Hz, Ar-H), 7.37 (1H, dd, J = 15.7, 3.1Hz, H-3), 7.27(2H,dt,J=8.6,2.0Hz,Ar-H),5.94(1H,dd,J=15.7,1.9Hz,H-2),5.75(1H,m,H-11),5.40( 1H,m,H-7),5.24(1H,dd,J=15.2,9.4Hz,H-10),4.88(1H,m,H-15),4.18(1H,m,H-4),0.92~2. 50(15H,m,H-5,H-6a,H-6b,H-8a,H-8b,H-9,H-12a,H-12b,H-13a,H-13b,H-14a,H-14b,-CH3);

[0044] 13 C NMR (CDCl3, 150MHz), δ: 166.0, 164.9, 151.1, 137.9, 135.8, 135.7, 131.2, 131.0 (×2), 12 9.0,125.7(×2),117.9,76.3,75.8,71.7,52.4,44.0,40.2,38.8,34.1,31.8,26.6,20.8;

[0045] HR-ESIMS m / z calcd for C 24 H 27 NNaO5S[M+Na] + 464.1502,found 464.1498.

[0046] Example 2

[0047] This example evaluates the pharmacological activity of the compound prepared in Example 1.

[0048] Experimental equipment and reagents

[0049] Table 1. Instruments, reagents, and cell lines used in the experiments of this example

[0050]

[0051]

[0052] Experimental methods

[0053] Cell growth inhibitory activity assay method

[0054] The CCK-8 assay was used to detect the anti-proliferative activity of the target compounds in six cancer cells (human liver cancer cell lines HepG2 and Bel-7402, human breast cancer cell line MDA-MB-231, human malignant melanoma cell line A375, human lung cancer cell line A549, and human cervical cancer cell line HeLa) and one normal cell line (human normal liver cell line L-02). All cell lines were obtained from KeyGEN Biotech, Nanjing, China. MDA-MB-231, A549, A375, and HeLa cells were cultured in standard DMEM medium, while HepG2, Bel-7402, and L-02 cells were cultured in standard RPMI-1640 medium. After incubation at 37°C in a humidified atmosphere with 5% CO2 for 24 h, logarithmically growing cells were seeded into 96-well plates and incubated at 37°C and 5% CO2 for 24 h. Then, the target compound or positive control was added to the different cell lines at a predetermined concentration and cultured for 48 h. The culture medium was carefully aspirated, and a mixed solution of 90 μL of the corresponding culture medium and 10 μL of CCK-8 was added to each well and incubated for 1 h. The OD value of each well was then measured on a microplate reader at a wavelength of 450 nm to calculate the IC value of the target compound. 50 value.

[0055] Experimental results

[0056] Table 2 Antiproliferative effects of target compounds on different cell lines

[0057]

[0058] a IC 50 : half-inhibitory concentration measured by CCK-8 assay, the above values ​​are the mean ± SD of three independent experiments.

[0059] Pharmacological experiments have shown that the target derivatives prepared by the present invention have excellent anti-proliferation activity against human liver cancer cell lines, human breast cancer cell lines, human malignant melanoma cell lines, human lung cancer cell lines, and human cervical cancer cell lines and have good selectivity for normal cells, and can be used for further preparation of anti-tumor drugs.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of brefeldin A derivatives, characterized in that Its general structural formula is shown in Formula 5, Formula 6, and Formula 7: Formula 5, Formula 6, Formula 7.

2. The method for preparing the brefeldin A derivative according to claim 1, wherein The preparation route of the preparation method is as follows: The preparation methods of derivatives 5, 6 and 7 include: Dissolve p-aminobenzoic acid 1 in an organic solvent, add TEA and 1,1'-thiocarbonyldiimidazole, react for 0.5-4 h, add an organic solvent containing an inorganic acid, and react for another 1-4 h. Pour the reaction solution into water, filter, wash the filter residue with water several times, and dry to obtain 1.5 g of compound 2; Brefeldin A and compound 2 were dissolved in an organic solvent, EDCI and a catalytic amount of DMAP were added, and the reaction was carried out at room temperature for 4-10 h. The resulting product was loaded onto a silica gel column chromatography column and eluted with a PE / EA mixed solution to obtain Brefeldin A derivatives 5, 6 and 7.

3. The preparation method according to claim 2, characterized in that The inorganic acid includes hydrochloric acid, sulfuric acid, or a mixture of hydrochloric acid and sulfuric acid; the organic solvent includes DCM, MeOH, PE, EA, acetone and n-hexane.

4. A pharmaceutical composition, characterized in that It contains one or more of the brefeldin A derivatives according to claim 1.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the brefeldin A derivative and a pharmaceutically acceptable carrier.

6. Use of the brefeldin A derivative according to claim 1 and the pharmaceutical composition according to any one of claims 4 to 5 in the preparation of a medicament for treating tumor diseases, wherein the tumors include liver cancer, breast cancer, malignant melanoma, lung cancer and cervical cancer.

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