A fusidic acid derivative, its preparation method and application

Abstract

The present invention provides a fusidic acid derivative, a preparation method and an application thereof. By connecting 1-carboxy-2-(β-indolyl)ethylamine (or L-tryptophan methyl ester fragment) to the carboxyl group at the C-21 position of fusidic acid, the introduction of 1-carboxy-2-(β-indolyl)ethylamine can promote the interaction of the fusidic acid derivative with the receptor, thereby improving the anti-tumor activity of the fusidic acid derivative, and it has good selectivity for normal cells. Finally, a fusidic acid derivative with a specific structure, excellent anti-tumor activity and good selectivity for normal cells is obtained. The present invention performs structural modification and optimization on the basis of the natural product fusidic acid, improves its physicochemical properties, enhances its pharmacological activity and improves its drug-likeness. The preparation method of the fusidic acid derivative provided by the present invention has the advantages of rich raw material sources, mild reaction conditions, simple operation in the reaction process, and cheap and easily available reagents.

Description

Technical Field

[0001] The present invention relates to the technical field of organic drugs, and particularly relates to a fusidic acid derivative, a preparation method thereof, and an application thereof. Background Art

[0002] In the past few decades, cancer has been one of the most fatal diseases endangering human health and is a major public health problem worldwide. The International Agency for Research on Cancer predicts that the number of new cancer cases globally will reach 28.4 million in 2040. However, so far, there is still no very satisfactory treatment method, and anti-tumor drugs are still one of the most commonly used methods for anti-tumor treatment at present. Therefore, there is an urgent need to develop anti-tumor drugs with novel structures and good curative effects, which highlights the urgency of drug research and development.

[0003] Fusidic acid is a tetracyclic triterpenoid compound, which was first isolated from the fungus Fusidium coccineum in 1960. Fusidic acid has biological activities such as antibacterial, anti-parasitic, anti-tuberculosis, anti-cancer, reversal of tumor multi-drug resistance, anti-inflammatory, and antiviral. Although researchers have conducted extensive research on fusidic acid, there is currently no research on the application of fusidic acid derivatives as anti-tumor drugs in clinical practice. Therefore, it is necessary to further chemically modify it, expand the research in this field, and develop fusidic acid derivative anti-tumor drugs with novel structures and good anti-tumor effects. Summary of the Invention

[0004] The purpose of the present invention is to provide a fusidic acid derivative, a preparation method thereof, and an application thereof. The fusidic acid derivative provided by the present invention has a novel structure and excellent anti-tumor effect.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a fusidic acid derivative having the structural formula shown in Formula I:

[0007]

[0008] The chemical formula of the fusidic acid derivative is C 43 H 60 N2O7.

[0009] The present invention also provides a preparation method of the fusidic acid derivative described in the above technical solution, including the following steps:

[0010] (1) After mixing a solution containing fusidic acid with N,N-diisopropylethylamine and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate, a first substitution reaction is carried out to obtain an intermediate.

[0011] (2) Mix the intermediate obtained in step (1) with DMF, inorganic base and L-tryptophan methyl ester, and then carry out a second substitution reaction to obtain a fusidic acid derivative.

[0012] Preferably, in step (1), the molar ratio of fusidic acid, N,N-diisopropylethylamine and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate in the solution containing fusidic acid is (1.5 - 2.5):(4.2 - 5.5):(2.5 - 3.4).

[0013] Preferably, in step (1), the temperature of the first substitution reaction is 23 - 28 °C, and the time of the first substitution reaction is 10 - 14 h.

[0014] Preferably, after the first substitution reaction in step (1), it further includes: quenching the product of the first substitution reaction with cold water, and then successively carrying out a first extraction and a first evaporation of the solvent to obtain an intermediate.

[0015] Preferably, in step (1), the molar ratio of fusidic acid in the solution containing fusidic acid to the inorganic base and L-tryptophan methyl ester in step (2) is (1.5 - 2.5):(5.2 - 6.2):(3.4 - 4.2).

[0016] Preferably, in step (2), the temperature of the second substitution reaction is 23 - 28 °C, and the time of the second substitution reaction is 6 - 10 h.

[0017] Preferably, after the second substitution reaction in step (2), it further includes: quenching the product of the second substitution reaction with cold water, and then successively carrying out a second extraction, a second evaporation of the solvent and column chromatography to obtain a fusidic acid derivative.

[0018] The present invention also provides the use of the fusidic acid derivative described in the above technical solution in the preparation of anti-tumor drugs.

[0019] The present invention provides a fusidic acid derivative, abbreviated as g4, with the chemical formula C 43 H 60 N2O7 and having the structural formula shown in Formula I as follows:

[0020]

[0021] By connecting 1-carboxy-2-(β-indolyl)ethylamino (or L-tryptophan methyl ester fragment) to the carboxyl group at the C-21 position of fusidic acid, the present invention utilizes the introduction of 1-carboxy-2-(β-indolyl)ethylamino (or L-tryptophan methyl ester fragment) to promote the interaction between the fusidic acid derivative and the receptor, thereby enhancing the anti-tumor activity of the fusidic acid derivative. Moreover, its inhibitory activity against the HCT116 cell line is stronger than that of fusidic acid, and it has good selectivity for normal cells. Finally, a fusidic acid derivative with a specific structure, excellent anti-tumor activity, and good selectivity for normal cells is obtained. The present invention conducts structural modification and optimization on the natural product fusidic acid to improve its physicochemical properties, enhance its pharmacological activity, and increase its drug-likeness. The preparation method of the fusidic acid derivative provided by the present invention has the advantages of rich raw material sources, mild reaction conditions, simple operation process, and cheap and easily available reagents. The results of the examples show that the fusidic acid derivative provided by the present invention exhibits good inhibitory activity against the HCT116 cell line (IC 50 = 7.11 μM), stronger than that of fusidic acid (IC 50 > 30 μM); its IC 50 value in L02 cells is at least 4.22 times higher than the IC 50 value in HCT116 cells, showing good selectivity and can be used in the field of anti-tumor drug preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the structural formula of the fusidic acid derivative prepared in Example 1 of the present invention;

[0023] Figure 2 is the graph showing the effect of the fusidic acid derivative g4 prepared in Example 1 of the present invention on the cell inhibition rate of human colon cancer cells (HCT116). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides a fusidic acid derivative with the structural formula shown in Formula I:

[0025]

[0026] The chemical formula of the fusidic acid derivative is C 43 H 60 N2O7.

[0027] In the present invention, the English name of the fusidic acid derivative is: methyl((Z)-2-((3R,4S,5S,8S,9S,10S,11R,13R,14S,16S)-16-acetoxy-3,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthren-17-ylidene)-6-methylhept-5-enoyl)-L-tryptophanate, abbreviated as g4; the molecular weight of the fusidic acid derivative is: 716.4401.

[0028] The fusidic acid derivative provided by the present invention has a novel structure and excellent anti-tumor effect.

[0029] The present invention also provides a preparation method of the fusidic acid derivative described in the above technical solution, comprising the following steps:

[0030] (1) After mixing a solution containing fusidic acid with N,N-diisopropylethylamine and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate, a first substitution reaction is carried out to obtain an intermediate.

[0031] (2) After mixing the intermediate obtained in the step (1) with DMF, an inorganic base and methyl L-tryptophanate, a substitution reaction is carried out to obtain the fusidic acid derivative.

[0032] In the present invention, unless otherwise specified, the raw materials used are all conventional commercially available products in the art.

[0033] In the present invention, a solution containing fusidic acid is mixed with N,N-diisopropylethylamine and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), and then a first substitution reaction is carried out to obtain an intermediate.

[0034] In the present invention, the molar ratio of fusidic acid, N,N-diisopropylethylamine and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate in the solution containing fusidic acid is preferably (1.5 - 2.5):(4.2 - 5.5):(2.5 - 3.4), more preferably (1.7 - 2.1):(4.6 - 5.0):(2.7 - 3.1). By controlling the amounts of fusidic acid, N,N-diisopropylethylamine and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate within the above ranges, the present invention promotes the full reaction of the three and obtains an intermediate with a high yield.

[0035] In the present invention, the first substitution reaction is preferably carried out under stirring conditions. In the present invention, the temperature of the first substitution reaction is preferably 23 - 28 °C, more preferably 25 °C. In the present invention, the time of the first substitution reaction is 10 - 14 h, more preferably 11 - 13 h. The present invention controls the temperature and time of the first substitution reaction within the above ranges so that the carboxyl group of fusidic acid undergoes a substitution reaction under the action of O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate to obtain an active intermediate.

[0036] After the first substitution reaction is completed, the present invention preferably quenches the product of the first substitution reaction with cold water and then successively performs first extraction and first evaporation of the solvent to obtain an intermediate.

[0037] In the present invention, the reagent used for the first extraction is preferably dichloromethane. The present invention has no special limitation on the method of the first evaporation of the solvent, and the solvent in the organic phase obtained by the first extraction can be removed by using a well-known technical solution in the art.

[0038] After obtaining the intermediate, the present invention mixes the intermediate with DMF, an inorganic base, and L-tryptophan methyl ester and then performs a second substitution reaction to obtain a fusidic acid derivative.

[0039] In the present invention, the inorganic base is preferably at least one of sodium carbonate and potassium carbonate.

[0040] In the present invention, the molar ratio of fusidic acid in the solution containing fusidic acid to the inorganic base and L-tryptophan methyl ester in step (2) is preferably (1.5 - 2.5):(5.2 - 6.2):(3.4 - 4.2), more preferably (1.7 - 2.1):(5.5 - 5.9):(3.6 - 4.0).

[0041] In the present invention, the second substitution reaction is preferably carried out under stirring conditions. In the present invention, the temperature of the second substitution reaction is preferably 23 - 28 °C, more preferably 25 °C. In the present invention, the time of the second substitution reaction is 6 - 10 h, more preferably 8 h. The present invention controls the temperature and time of the second substitution reaction within the above ranges so that the benzotriazole fragment part of the active intermediate undergoes a substitution reaction with L-tryptophan methyl ester under alkaline conditions to obtain the target compound g4.

[0042] After the second substitution reaction is completed, the present invention preferably quenches the product of the second substitution reaction with cold water and then successively performs second extraction, second evaporation of the solvent, and column chromatography to obtain a fusidic acid derivative.

[0043] In the present invention, the reagent used for the second extraction is preferably dichloromethane; the number of times of the second extraction is preferably 3 times. The present invention has no special limitation on the method for evaporating the solvent in the second extraction, and the solvent in the organic phase obtained by the second extraction can be removed by using the well-known technical solutions in the art. In the present invention, the method of column chromatography is preferably gradient elution; the reagent used for column chromatography is preferably a mixed solution composed of petroleum ether and ethyl acetate with a volume ratio of (3-6):1.

[0044] The preparation method of the fusidic acid derivative provided by the present invention is simple in operation, mild in reaction conditions, rich in raw material sources, cheap and easy to obtain the reagents used, and suitable for large-scale production.

[0045] The present invention also provides the application of the fusidic acid derivative described in the above technical solution in the preparation of anti-tumor drugs.

[0046] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0047] Example 1

[0048] The preparation method of the fusidic acid derivative is as follows:

[0049] (1) Add N,N-diisopropylethylamine (62 mg, 0.48 mmol) and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (93 mg, 0.29 mmol) to a dichloromethane (5 mL) solution containing fusidic acid (100 mg, 0.19 mmol), mix to obtain a reaction mixture, stir the reaction mixture at 25 °C for the first substitution reaction for 12 h, quench the reaction with cold water, extract the product of the first substitution reaction with dichloromethane for 3 times, collect and combine the organic phases, evaporate the solvent to dryness to obtain an intermediate;

[0050] (2) Dissolve the intermediate obtained in step (1) in a DMF solution, then sequentially add Na2CO3 (60.4 mg, 0.57 mmol) and L-tryptophan methyl ester (0.38 mmol), stir at 25 °C for the second substitution for 8 h, quench the reaction with cold water, extract the product of the second substitution reaction with dichloromethane for 3 times, collect and combine the organic phases, evaporate the solvent in the second step, and then perform column chromatography purification using a silica gel column to obtain 59.9 mg of fusidic acid derivative with a yield of 44%, abbreviated as g4;

[0051] The column chromatography method is gradient elution; the reagent used in the column chromatography is a mixed solution composed of petroleum ether and ethyl acetate with a volume ratio of (3-6):1.

[0052] Figure 1 This is the structural formula of the fusidic acid derivative prepared in Example 1 of the present invention.

[0053] The nuclear magnetic resonance hydrogen spectrum data of the fusidic acid derivative g4 prepared in Example 1 was detected using a nuclear magnetic resonance instrument, and the molecular weight data was detected by high-resolution mass spectrometry as follows:

[0054] White powder; m.p. 86-88°C; Yield: 44%. 1 H-NMR(300MHz,CDCl3)δ:8.20(s,1H,indole-NH),7.62(d,J = 9Hz,1H,-CO-NH-),7.39(d,J = 9Hz,1H,Ar-H),7.24-7.11(m,3H,Ar-H),5.94(d,J = 6Hz,1H,Ar-H),5.68(d,J = 9Hz,1H,C 16 -H),5.01(t,J = 6Hz,1H,C 24 -H),4.85(t,J = 6Hz,1H,-CH-COO-),4.33(s,1H,C 11 -H),3.77(s,1H,C3-H),3.65(s,3H,-COO-CH3),3.40(dd,J1 = 15Hz,J2 = 6Hz,1H,indole-CH2-),3.20(dd,J1 = 15Hz,J2 = 6Hz,1H,indole-CH2-),2.94(d,J = 12Hz,1H,C 13 -H),2.48-2.38(m,1H),2.30-2.23(m,2H),2.18-2.07(m,4H),1.95(s,3H),1.88-1.72(m,4H),1.62-1.52(m,12H),1.38(s,3H),1.32(d,J = 15Hz,1H),1.19-1.09(m,2H),0.98-0.85(s,9H). 13C-NMR(75MHz, CDCl3) δ: 172.56, 171.25, 170.91, 142.17, 136.25, 135.09, 132.34, 127.50, 123.34, 123.20, 122.23, 119.61, 118.56, 111.34, 109.71, 74.05, 71.42, 68.28, 52.55, 52.26, 49.23, 48.61, 43.21, 39.48, 39.22, 37.01, 36.24, 36.10, 35.64, 32.34, 30.22, 29.96, 29.61, 27.85, 27.43, 25.65, 24.06, 22.80, 20.98, 20.80, 17.88, 17.71, 15.98. ESI-HRMS calcd for C 43 H 61 N2O7 + ([M+H] + ): 717.44733; found: 717.44452.

[0055] Investigation 1. Effect of g4 prepared in Example 1 on the inhibition rate of human colon cancer cells (HCT116)

[0056] To detect the effect of g4 prepared in Example 1 on the inhibition rate of human colon cancer cells (HCT116), an MTT assay was performed. DMSO was used as the blank control, and 5-fluorouracil (5-FU) and fusidic acid were used as positive controls. HCT116 cells were treated with 3.725 μM, 7.5 μM, 15 μM, and 30 μM of g4 for 48 h; then MTT was added and the cells were cultured for an additional 4 h; the medium was removed, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 min in the dark. The OD value at 492 nm was measured using a microplate reader; finally, the inhibition rate and half-maximal inhibitory concentration (IC 50 ) were calculated with the control group as the reference, and the inhibition rate (%) = (1 - δsample / δblank) × 100%, and the IC 50 value was obtained by plotting the concentration-inhibition rate curve. The specific results are shown in Table 1.

[0057] The effect of the fusidic acid derivative g4 prepared in Example 1 on the cell inhibition rate of human colon cancer cells (HCT116) is shown in Figure 2 the figure below.

[0058] From the experimental results (Table 1 and Figure 2)It can be seen that after the fusidic acid derivative g4 prepared in Example 1 was administered, the inhibition rate of HCT116 cells increased significantly. Moreover, as the administered concentration of the fusidic acid derivative g4 increased to 3.725 μM, 7.5 μM, 15 μM, and 30 μM respectively, the corresponding inhibition rates of HCT116 cells were 25.80%, 54.51%, 76.55%, and 94.91% respectively, also increasing in sequence. Importantly, the inhibitory activity of g4 against the HCT116 cell line (IC 50 = 7.11 μM) was stronger than that of fusidic acid (IC 50 > 30 μM).

[0059] Investigation 2. Effect of g4 on cytotoxicity

[0060] To detect the effect of g4 on the cytotoxicity of human normal cells, an MTT assay was performed. DMSO was used as the blank control, and 5-fluorouracil (5-FU) was used as the positive control. Human normal liver cells (L02) were treated with 3.725 μM, 7.5 μM, 15 μM, and 30 μM of g4 for 48 h; then MTT was added and the cells were cultured for another 4 h; the culture medium was removed, 150 μL of DMSO was added to each well, and the mixture was shaken for 10 min in the dark. The OD value at 492 nm was measured using an enzyme-linked immunosorbent assay reader; finally, the inhibition rate, half-maximal inhibitory concentration (IC 50 ), and selectivity index (SI) were calculated with reference to the control group. Moreover, the inhibition rate (%) = (1 - δsample / δblank) × 100%, the IC 50 value was calculated by plotting the concentration-inhibition rate curve, and SI = L02 IC 50 / HCT116 IC 50 . The specific results are shown in Table 1.

[0061] Table 1 Pharmacological activity data of g4, fusidic acid, and 5-FU prepared in Example 1

[0062]

[0063] As can be seen from Table 1, the selectivity of g4 prepared in Example 1 for HCT116 cells was at least 4.22 times higher than that for normal L02 cells, exceeding 0.77 times that of 5-FU, indicating that the compound g4 not only exhibited strong anti-proliferative activity against HCT116 cells but also had good selectivity.

[0064] In summary, the present invention provides a fusidic acid derivative with a novel structure. By connecting 1-carboxy-2-(β-indolyl)ethylamine (or L-tryptophan methyl ester fragment) to the carboxyl group at the C-21 position of fusidic acid for the first time, the fusidic acid derivative exhibits good inhibitory activity against the HCT116 cell line (IC 50 = 7.11 μM), which is stronger than that of fusidic acid (IC50 > 30 μM); and the fusidic acid derivative has an IC 50 value in L02 cells that is at least 4.22 times higher than the IC 50 value in HCT116 cells, showing good selectivity. The present invention for the first time discovers that the introduction of L-tryptophan methyl ester can improve the anti-tumor activity of fusidic acid and can be used in the field of anti-tumor drug preparation.

[0065] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fusidic acid derivative having the structural formula shown in Formula I: The chemical formula of the fusidic acid derivative is C 43 H 60 N2O7.

2. The preparation method of the fusidic acid derivative according to claim 1, comprising the following steps: (1) Mix the fusidic acid-containing solution with N,N-diisopropylethylamine and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate, and then carry out the first substitution reaction to obtain an intermediate; (2) Mix the intermediate obtained in the step (1) with DMF, an inorganic base, and L-tryptophan methyl ester, and then carry out the second substitution reaction to obtain a fusidic acid derivative.

3. The preparation method according to claim 2, characterized in that In the fusidic acid-containing solution in the step (1), the molar ratio of fusidic acid, N,N-diisopropylethylamine, and O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate is (1.5 - 2.5):(4.2 - 5.5):(2.5 - 3.4).

4. The preparation method according to claim 2, characterized in that In the step (1), the temperature of the first substitution reaction is 23 - 28 °C, and the time of the first substitution reaction is 10 - 14 h.

5. The preparation method according to claim 2, characterized in that After the completion of the first substitution reaction in the step (1), it further includes: quenching the product of the first substitution reaction with cold water, and then successively carrying out the first extraction and the first evaporation of the solvent to obtain the intermediate.

6. The preparation method according to claim 2, characterized in that In the fusidic acid-containing solution in the step (1), the molar ratio of fusidic acid to the inorganic base and L-tryptophan methyl ester in the step (2) is (1.5 - 2.5):(5.2 - 6.2):(3.4 - 4.2).

7. The preparation method according to claim 2, characterized in that In the step (2), the temperature of the second substitution reaction is 23 - 28 °C, and the time of the second substitution reaction is 6 - 10 h.

8. The preparation method according to claim 2, characterized in that After the completion of the second substitution reaction in the step (2), it further includes: quenching the product of the second substitution reaction with cold water, and then successively carrying out the second extraction, the second evaporation of the solvent, and column chromatography to obtain the fusidic acid derivative.

9. The application of the fusidic acid derivative according to claim 1 in the preparation of anti-tumor drugs.

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