Derivatives of fusidic acid with C-3-site oxazole substitution, C-24-site reduction and C-21-site benzyl ester modification as well as preparation method and application thereof

Abstract

The invention discloses a fusidic acid C-3-site oxazole substituted, C-24-site reduced and C-21-site benzyl ester modified derivative as well as a preparation method and application thereof, and belongs to the technical field of organic chemistry. The structure of the fusidic acid derivative with oxazole substitution at the C-3 site, reduction at the C-24 site and benzyl ester modification at the C-21 site is shown in the specification, wherein R represents 3-methyl benzyl, 3-chlorobenzyl, 2-bromobenzyl, 3-bromobenzyl or 4-cyanobenzyl. The fusidic acid derivative provided by the invention has the beneficial effects that the MDR reverse activity of the fusidic acid derivative substituted at C-3 position by oxazole, reduced at C-24 position and modified by benzyl ester at C-21 position is excellent, the druggability is better, and the fusidic acid derivative can be combined with common antitumor drugs to play a good antitumor role in clinical treatment.

Description

Technical Field

[0001] This invention relates to fusidic acid derivatives, their preparation methods, and applications, specifically to fusidic acid derivatives modified by oxazole substitution at C-3, reduction at C-24, and benzyl ester at C-21, their preparation methods, and their application in the preparation of P-gp inhibitors, belonging to the field of organic chemistry technology. Background Technology

[0002] Malignant tumors are a major global public health problem, seriously affecting human health and socio-economic development. In 2022, my country had approximately 4.8247 million new cases of malignant tumors, with a standard population incidence rate of 208.58 / 100,000, indicating a severe situation for prevention and control. Multidrug resistance (MDR) in tumors refers to the phenomenon where tumor cells, after exposure to an anti-tumor drug, not only develop resistance to that drug but also to multiple drugs with different structures, targets, and mechanisms. MDR is one of the main causes of chemotherapy failure, and 90% of tumor metastases and recurrences are closely related to MDR. Among the many mechanisms of tumor MDR, P-glycoprotein (P-gp) overexpression is one of the most classic and well-studied mechanisms. P-gp is an ATP-dependent transmembrane transport protein that actively pumps multiple anti-tumor drugs out of the cell, significantly reducing intracellular drug concentrations and ultimately leading to chemotherapy failure. Therefore, developing novel P-gp inhibitors to block its efflux function has become a core strategy for reversing P-gp-mediated tumor MDR.

[0003] Currently, research on P-gp inhibitors has gone through three generations of development. Although each generation of inhibitors has been gradually optimized, there are still limitations that have not been overcome: The first generation, represented by verapamil and cyclosporine A, has a certain P-gp inhibitory effect, but its clinical use is limited due to its strong toxic side effects and pharmacokinetic interactions with chemotherapy drugs; the second generation of inhibitors, such as vasoplasta, has improved inhibitory activity compared with the first generation, but still has problems such as insufficient target specificity and easy to lead to compensatory high expression of P-gp; the third generation of inhibitors, such as taribetamiprid and zoquine, has improved target specificity and reduced toxic side effects compared with the first two generations, but due to poor water solubility and low bioavailability, it has not been translated into clinical practice.

[0004] In summary, existing P-gp inhibitors remain unsatisfactory in terms of activity, target specificity, safety, and drug-likeness. Their current structural types are insufficient to meet clinical treatment needs, resulting in the lack of successful market approval for any P-gp inhibitor. Therefore, developing P-gp inhibitors with novel structural types is a crucial research direction in the field of tumor drug resistance. In recent years, natural plants have been extensively studied due to their good biocompatibility, low toxicity, and rich structural diversity, making them an important source for discovering P-gp inhibitors. Searching for effective P-gp inhibitors from natural plants has become a research hotspot.

[0005] Fusidic acid (FA) is a steroidal antibiotic isolated from the fungus *Fusidium coccineum*. Its unique tetracyclic triterpenoid structure provides abundant sites for structural modification. FA inhibits bacterial elongation factor G (EF-G) to block bacterial protein synthesis, exhibiting potent antibacterial activity against Gram-positive bacteria (such as *Staphylococcus aureus*). Clinically, it is commonly used to treat skin and soft tissue infections. In recent years, with in-depth research on FA, the non-antibacterial activities of FA and its derivatives have been gradually explored, especially showing great potential in the field of tumor MDR reversal. Our research group previously conducted systematic studies on key sites of FA (such as C-3, C-11, and C-21) to enhance the antibacterial activity of FA, broaden its antibacterial spectrum, and improve bacterial resistance. Unexpectedly, it was discovered that the FA derivatives with benzyl substitution at the C-21 position exhibited high tumor MDR reversal activity both in vivo and in vitro, significantly superior to verapamil. The mechanism of action has been preliminarily elucidated (see GuoMQ, Ren QW, Wang BH, et al; Discovery and synthesis of 3- and 21-substituted fusidic acid derivatives as reversal agents of P-glycoprotein-mediated multidrug resistance; Eur J Med Chem; 2019, 182: 111668).

[0006] However, although the aforementioned FA derivatives with benzyl substitution at C-21 exhibit good MDR reversal activity (the survival rate of drug-resistant KBV cells was 18.60±0.37% when used in combination with 100 nM paclitaxel), their chemical structure shows poor stability and a short half-life, making it difficult to meet the needs of clinical treatment. Therefore, their chemical structure still needs to be optimized. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to: reduce the C-24 position of FA, further refine the chemical modifications at the C-3 and C-21 positions to enrich the structural types, and develop compounds with long half-lives while ensuring excellent MDR reversal activity, as well as the preparation method and application of such compounds.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The structure of the fusidic acid C-3 position oxazole-substituted, C-24 position reduced, and C-21 position benzyl ester modified derivative is shown below:

[0009] Wherein, R represents 3-methylbenzyl, 3-chlorobenzyl, 2-bromobenzyl, 3-bromobenzyl or 4-cyanobenzyl.

[0010] Preferably, the fusidic acid C-3 position oxazole-substituted, C-24 position reduced, and C-21 position benzyl ester modified derivative is: 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-ene-21-oic acid m-methyl benzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid-m-chlorobenzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-o-bromobenzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-oic acid benzyl bromide; or 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid p-cyanobenzyl ester.

[0011] The preparation method of the aforementioned fusidic acid C-3 position oxazole substitution, C-24 position reduction, and C-21 position benzyl ester modification derivative includes the following steps: (1) Using fusidic acid as a raw material, the C-24 double bond of fusidic acid was reduced by hydrogen under palladium on carbon catalysis to obtain intermediate product A; (2) Under the catalysis of 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the C-3 hydroxyl group of intermediate A reacts with 4-oxazolamide to give intermediate B; (3) In the presence of an inorganic base, the carboxyl group at position C-21 of intermediate product B reacts with a benzyl bromoanalyte, wherein the benzyl bromoanalyte is 3-methylbenzyl bromide, 3-chlorobenzyl bromide, 2-bromobenzyl bromide, 3-bromobenzyl bromide or 4-cyanobenzyl bromide, to obtain the crude product; (4) The crude product was purified by column chromatography to obtain the target compound.

[0012] Preferably, the preparation method of the aforementioned fusidic acid C-3 position oxazole substitution, C-24 position reduction, and C-21 position benzyl ester modification derivative specifically includes the following steps: (1) Fusidic acid was dissolved in anhydrous ethanol, palladium on carbon was added, the reaction mixture was purged with argon gas and hydrogen gas was introduced, and the mixture was stirred at room temperature. After the reaction was completed, the product was purified to obtain intermediate product A. (2) Intermediate product A, 4-oxazolocarboxylic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in dichloromethane and stirred at room temperature. After the reaction was completed, the product was purified to obtain intermediate product B. (3) Dissolve intermediate product B in acetone, add inorganic base and benzyl bromide analogue, stir at room temperature to obtain crude product; (4) The crude product was concentrated under reduced pressure to remove acetone, then diluted with dichloromethane, and then washed with water, saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to silica gel column chromatography. The target eluent was collected and dried to obtain the target compound.

[0013] The aforementioned fusidic acid derivatives with C-3 oxazole substitution, C-24 reduction, and C-21 benzyl ester modification are used in the preparation of P-gp inhibitors.

[0014] The advantages of this invention are as follows: This invention introduces 3-methylbenzyl, 3-chlorobenzyl, 2-bromobenzyl, 3-bromobenzyl, or 4-cyanobenzyl into the C-21 position of fusidic acid with oxazole substitution at the C-3 position and reduction at the C-24 position, successfully preparing novel fusidic acid C-3 position oxazole-substituted, C-24 position reduced, and C-21 position benzyl ester modified derivatives. Pharmacological tests show that the above-mentioned fusidic acid C-3 position oxazole-substituted, C-24 position reduced, and C-21 position benzyl ester modified derivatives have properties comparable to or even greater than verapamil (e.g., F-EZ-5, p>0.05). Verapamil exhibits superior MDR reversal activity (e.g., F-EZ-1, F-EZ-2, F-EZ-3, F-EZ-4, p<0.001), significantly increasing the sensitivity of drug-resistant KBV cells to paclitaxel. It is a P-gp inhibitor with superior MDR reversal activity. Drug metabolism stability studies show that the above-mentioned fusidic acid C-3 position oxazole-substituted, C-24 position reduced, and C-21 position benzyl ester modified derivative (F-EZ-1) reaches its maximum plasma concentration 2 hours after intraperitoneal injection, with a duration of up to 24 hours and a significantly extended half-life, indicating good drug-likeness. In summary, the fusidic acid C-3 position oxazole-substituted, C-24 position reduced, and C-21 position benzyl ester modified derivatives provided by this invention exhibit excellent MDR reversal activity and good drug-likeness, and can be used in combination with commonly used antitumor drugs to exert a good antitumor effect in clinical treatment. Attached Figure Description

[0015] Figure 1 This is a graph showing the results of the analysis of significant differences in the survival rate of drug-resistant KBV cells under the combined use of each compound and paclitaxel (paclitaxel-containing treatment group), where ns indicates p>0.05 and *** indicates p<0.001. Detailed Implementation

[0016] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0017] I. Structures of fusidic acid derivatives with oxazole substitution at C-3, reduction at C-24, and benzyl ester modification at C-21 The structure of the fusidic acid derivative with oxazole substitution at C-3, reduction at C-24, and benzyl ester modification at C-21 provided by this invention is shown below:

[0018] Wherein, R represents 3-methylbenzyl, 3-chlorobenzyl, 2-bromobenzyl, 3-bromobenzyl or 4-cyanobenzyl.

[0019] The derivatives with partial C-3 oxazole substitution, C-24 reduction, and C-21 benzyl ester modification are: 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-ene-21-oic acid m-methyl benzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid-m-chlorobenzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-o-bromobenzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid m-bromobenzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid p-cyanobenzyl ester.

[0020] II. Preparation methods of fusidic acid derivatives modified by oxazole substitution at C-3, reduction at C-24, and benzyl ester at C-21. The method for preparing the above-mentioned fusidic acid C-3 position oxazole substitution, C-24 position reduction, and C-21 position benzyl ester modification derivative provided by the present invention includes the following steps: (1) Using fusidic acid as a raw material, hydrogen (H2) is used to reduce the double bond at position C-24 of fusidic acid under palladium on carbon (Pd / C) catalysis to obtain intermediate product A; (2) Under the catalysis of 4-dimethylaminopyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), the C-3 hydroxyl group of intermediate A reacts with 4-oxazolamide to give intermediate B; (3) In the presence of an inorganic base, the carboxyl group at position C-21 of intermediate product B reacts with a benzyl bromoanalyte, wherein the benzyl bromoanalyte is 3-methylbenzyl bromide, 3-chlorobenzyl bromide, 2-bromobenzyl bromide, 3-bromobenzyl bromide or 4-cyanobenzyl bromide, to obtain the crude product; (4) The crude product was purified by column chromatography to obtain the target compound.

[0021] Example 1

[0022] Fusidic acid (1.0 g, 1.9 mmol) was dissolved in anhydrous ethanol (50.0 mL), and palladium on carbon (Pd / C, 30.0 mg, 0.3 mmol) was added. The reaction mixture was purged with argon under vacuum and then purged with hydrogen. The mixture was stirred at room temperature (hydrogen reduction of the C-24 double bond of fusidic acid, reaction time 1.5 h). After the reaction was completed, the mixture was subjected to diatomaceous earth filtration (to remove Pd / C), concentration under reduced pressure (to remove some solvent), and silica gel column chromatography (for separation and purification, V...). 石油醚 V 乙酸乙酯 =2:1), collect the target eluted fraction and dry it to obtain intermediate product A (700.0 mg, yield 71.0%).

[0023] Intermediate product A (600.0 mg, 0.8 mmol), reactant 4-oxazolidinyl carboxylic acid (336.0 mg, 2.8 mmol), catalyst 4-dimethylaminopyridine (DMAP, 364.0 mg, 2.8 mmol), and catalyst 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 948.0 mg, 4.8 mmol) were dissolved in dichloromethane (20.0 mL) and stirred at room temperature (the C-3 hydroxyl group of intermediate product A reacts with 4-oxazolidinyl carboxylic acid for 2 h). After the reaction was completed, the reaction solution was diluted with dichloromethane and then subjected to the following steps: washing twice with 5% (w / v) hydrochloric acid, washing twice with water, washing once with saturated brine, drying with anhydrous sodium sulfate, filtration, concentration under reduced pressure, and silica gel column chromatography (V). 石油醚 V 乙酸乙酯 =3:1), collect the target elution fraction and dry it to obtain intermediate product B (508.0 mg, yield 90.7%).

[0024] Intermediate product B (100.0 mg, 0.2 mmol) was dissolved in acetone (3.6 mL), and inorganic base potassium carbonate (53.2 mg, 0.4 mmol) and reactant 3-methylbenzyl bromide (40.0 µL, 0.3 mmol) were added. The mixture was stirred at room temperature (the carboxyl group at C-21 of intermediate product B reacted with 3-methylbenzyl bromide for 4 h) to obtain the crude product.

[0025] The crude product was concentrated under reduced pressure to remove acetone, then diluted with dichloromethane, followed by washing with water (twice), washing with saturated brine (once), drying with anhydrous sodium sulfate, filtration, concentration under reduced pressure, and silica gel column chromatography (V). 二氯甲烷 V 甲醇 =120:1), the target eluent was collected and dried to give a white solid, denoted as F-EZ-1 (target compound, 93.2 mg, yield 81.2%).

[0026] F-EZ-1 1 H NMR,13 The 13C NMR data are as follows: 1 1H NMR (400 MHz, CDCl3) δ 8.19 (d, J J = 1.0 Hz, 1H, H-Oxazole), 7.93 (d, J J = 1.0 Hz, 1H, H-Oxazole), 7.25 - 7.20 (m, 1H, H-Ar), 7.16 - 7.10 (m, 3H, 3´H-Ar), 5.87 (d, J J = 8.3 Hz, 1H, H-16), 5.20 - 5.16 (m, 2H, H-CH2Ar), 4.88 (d, J J = 12.0 Hz, 1H, H-3), 4.34 (d, J J = 2.5 Hz, 1H, H-11), 3.03 (d, J J = 11.7 Hz, 1H, H-13), 2.47 - 2.35 (m, 2H, CH2), 2.34 (s, 3H, CH3), 2.26 - 2.14 (m, 4H, 2´CH2), 1.94 (s, 3H, CH3), 1.92 - 1.71 (m, 6H, 3´CH2), 1.65 - 1.43 (m, 5H, 2´CH2, H-OH), 1.40 (s, 3H, CH3), 1.30 (d, J J = 14.2 Hz, 2H, CH2), 1.17 - 1.07 (m, 4H, 2´CH2), 1.01 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.87 (d, J J = 6.7 Hz, 3H, CH3), 0.80 (dd, J J = 6.6, 2.5 Hz, 6H, 2´CH3).

[0027] 13 13C NMR (100 MHz, CDCl3) δ 170.56, 170.19, 160.58, 151.65, 147.51, 143.53, 138.28, 135.74, 133.62, 131.15, 129.30, 129.09, 128.55, 125.66, 75.54, 74.43, 68.26, 66.45, 49.12, 48.8 , 43.84, 39.60, 39.12, 38.69, 37.92, 36.95, 35.93, 35.13, 32.65, 31.04, 29.20, 27.89, 27.64, 24.39, 22.82, 22.66, 22.56, 21.42, 21.03, 20.69, 18.02, 15.83.

[0028] F-EZ-1 is: 3α-(4-oxazol)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-ene-21-oic acid m-methyl benzyl ester.

[0029] Example 2

[0030] Intermediate product B was prepared using fusidic acid as a raw material according to the method in Example 1.

[0031] Intermediate product B (100.0 mg, 0.2 mmol) was dissolved in acetone (3.6 mL), and inorganic base potassium carbonate (53.2 mg, 0.4 mmol) and reactant 3-chlorobenzyl bromide (39.0 µL, 0.3 mmol) were added. The mixture was stirred at room temperature (the carboxyl group at C-21 of intermediate product B reacted with 3-chlorobenzyl bromide for 4 h) to obtain the crude product.

[0032] The crude product was concentrated under reduced pressure to remove acetone, then diluted with dichloromethane, followed by washing with water (twice), washing with saturated brine (once), drying with anhydrous sodium sulfate, filtration, concentration under reduced pressure, and silica gel column chromatography (V). 二氯甲烷 V 甲醇 =120:1), collect the target eluent and dry it to give a white solid, denoted as F-EZ-2 (target compound, 101.6 mg, yield 86.0%).

[0033] F-EZ-2 1 H NMR, 13 The C NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ 8.22 (s, 1H, H-Oxazole), 7.96 (s, 1H, H-Oxazole), 7.41-7.26 (m, 4H, 4´H-Ar), 5.90 (d, J =8.3Hz, 1H, H-16), 5.23-5.18 (m, 2H, H-CH2Ar), 4.90 (d, J =12.4Hz, 1H, H-3), 4.37 (d, J =3.4Hz, 1H, H-11), 3.07 (d, J =12.0Hz, 1H, H-13), 2.50-2.31 (m, 2H, CH2), 2.24 (ddt, J=24.7, 13.1, 4.3Hz, 4H, 2´CH2), 1.96 (s, 3H, CH3), 1.93-1.73 (m, 6H, 3´CH2), 1.70-1.47 (m, 4H, 2´C H2), 1.43 (s, 3H, CH3), 1.36-1.08 (m, 7H, 3´CH2, H-OH), 1.03 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.89 (d, J =6.6Hz, 3H, CH3), 0.82 (dd, J =6.6, 2.9Hz, 6H, 2´CH3).

[0034] 13 C NMR (100MHz, CDCl3) δ 170.52, 169.89, 160.57, 151.67, 148.29, 143.55, 137.83, 134.47, 133.56, 130.76, 129.94, 128.47, 126.53, 74.37, 68.20, 65.39, 49.07, 48.82, 43.97 , 39.57, 39.10, 38.64, 37.88, 36.92, 35.88, 35.09, 32.61, 30.99, 29.15, 27.86, 27.69, 27.42, 24.39, 22.81, 22.64, 22.54, 21.02, 20.67, 18.02, 15.82.

[0035] F-EZ-2 is: 3α-(4-oxazol)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid-m-chlorobenzyl ester.

[0036] Example 3

[0037] Intermediate product B was prepared using fusidic acid as a raw material according to the method in Example 1.

[0038] Intermediate product B (100.0 mg, 0.2 mmol) was dissolved in acetone (3.6 mL), and inorganic base potassium carbonate (53.2 mg, 0.4 mmol) and reactant 2-bromobenzyl bromide (39.0 µL, 0.3 mmol) were added. The mixture was stirred at room temperature (the carboxyl group at C-21 of intermediate product B reacted with 2-bromobenzyl bromide for 4 h) to obtain the crude product.

[0039] The crude product was concentrated under reduced pressure to remove acetone, then diluted with dichloromethane, followed by washing with water (twice), washing with saturated brine (once), drying with anhydrous sodium sulfate, filtration, concentration under reduced pressure, and silica gel column chromatography (V). 二氯甲烷 V 甲醇 =130:1), the target eluent was collected and dried to give a white solid, denoted as F-EZ-3 (target compound, 96.8 mg, yield 77.4%).

[0040] F-EZ-3 1 H NMR, 13 The C NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ 8.19 (d, J =1.0Hz, 1H, H-Oxazole), 7.93 (d, J =1.1Hz, 1H, H-Oxazole), 7.56 (dd, J =8.0, 1.2Hz, 1H, H-Ar), 7.42 (dd, J =7.7, 1.8Hz, 1H, H-Ar), 7.30 (td, J =7.5, 1.3Hz, 1H, H-Ar), 7.18 (td, J =7.7, 1.8Hz, 1H, H-Ar), 5.88 (d, J =8.3Hz, 1H, H-16), 5.33 (d, J =12.8Hz, 1H, H-CH2Ar), 5.19 (d, J =2.7Hz, 1H, H-CH2Ar), 4.97 (d, J =12.9Hz, 1H, H-3), 4.34 (d, J =2.5Hz, 1H, H-11), 3.04 (d, J =11.4Hz, 1H, H-13), 2.47-2.31 (m, 2H, CH2), 2.21 (dddd, J =20.6, 18.4, 9.6, 4.3Hz, 4H, 2´CH2), 1.97 (s, 3H, CH3), 1.92-1.73 (m, 6H, 3´CH2) , 1.66-1.51 (m, 4H, 2´CH2), 1.41 (s, 3H, CH3), 1.33-1.24 (m, 4H, 2´CH2), 1.13 (dt, J =8.5, 7.0Hz, 3H, CH2, H-OH), 1.01 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.87 (d,J =6.7Hz, 3H, CH3), 0.80 (dd, J =6.6, 2.2Hz, 6H, 2´CH3).

[0041] 13 C NMR (100MHz, CDCl3) δ 170.53, 169.84, 160.61, 151.65, 148.12, 143.54, 135.24, 133.67, 132.95, 130.95, 130.43, 129.91, 127.63, 123.78, 74.45, 68.31, 65.95, 49.15, 48.87 , 43.96, 39.64, 39.15, 38.79, 37.97, 36.99, 35.94, 35.14, 32.70, 31.09, 29.25, 27.97, 27.78, 24.45, 22.83, 22.69, 22.64, 21.11, 20.70, 18.08, 15.86.

[0042] F-EZ-3 is: 3α-(4-oxazol)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-o-bromobenzyl ester.

[0043] Example 4

[0044] Intermediate product B was prepared using fusidic acid as a raw material according to the method in Example 1.

[0045] Intermediate product B (100.0 mg, 0.2 mmol) was dissolved in acetone (3.6 mL), and inorganic base potassium carbonate (53.2 mg, 0.4 mmol) and reactant 3-bromobenzyl bromide (39.0 µL, 0.3 mmol) were added. The mixture was stirred at room temperature (the carboxyl group at C-21 of intermediate product B reacted with 3-bromobenzyl bromide for 4 h) to obtain the crude product.

[0046] The crude product was concentrated under reduced pressure to remove acetone, then diluted with dichloromethane, followed by washing with water (twice), washing with saturated brine (twice), drying with anhydrous sodium sulfate, filtration, concentration under reduced pressure, and silica gel column chromatography (V). 二氯甲烷 V 甲醇 =130:1), the target eluent was collected and dried to give a white solid, denoted as F-EZ-4 (target compound, 105.7 mg, yield 84.5%).

[0047] F-EZ-4 1 H NMR, 13 The C NMR data are as follows: 1 H NMR(400MHz,CDCl3)δ 8.17(d, J =1.0Hz,1H,H-Oxazole),7.91(d, J =1.0Hz,1H,H-Oxazole),7.47(t, J =1.8Hz,1H,H-Ar),7.40(dt, J =7.8,1.6Hz,1H,H-Ar),7.25-7.23(m,1H,H-Ar),7.18(t, J =7.7Hz,1H,H-Ar),5.86(d, J =8.3Hz,1H,H-16),5.17-5.13(m,2H,H-CH2Ar),4.85(d, J =12.4Hz,1H,H-3),4.32(d, J =2.5Hz,1H,H-11),3.02(d, J =11.0Hz,1H,H-13),2.36(ddd, J =21.9,8.4,4.0Hz,2H,CH2),2.23-2.11(m,4H,2´CH2),1.91(s,3H,CH3),1.89-1.71(m,6H,3´CH2),1.63-1.51(m,4H,2´CH2),1.38(s,3H,CH3),1.30-1.24(m,3H,CH2,H-OH),1.22(s,3H,CH3),1.10(tt, J =6.8,1.6Hz,4H,2´CH2),0.98(s,3H,CH3),0.90(s,3H,CH3),0.78(dd, J =6.6,3.1Hz,6H,2´CH3)。

[0048] 13C NMR (100MHz, CDCl3) δ 170.48, 169.83, 160.55, 151.66, 148.31, 143.54, 138.13, 133.58, 131.40, 13 1.38, 130.77, 130.20, 127.01, 122.63, 75.52, 74.37, 68.20, 65.31, 49.10, 48 .83, 43.98, 39.58, 39.12, 38.65, 37.88, 36.92, 35.91, 35.12, 32.60, 31.00, 29.75, 27.86, 27.68, 24.37, 22.81, 22.65, 22.55, 21.00, 20.68, 18.02, 15.81.

[0049] F-EZ-4 is: 3α-(4-oxazol)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid m-bromobenzyl ester.

[0050] Example 5

[0051] Intermediate product B was prepared using fusidic acid as a raw material according to the method in Example 1.

[0052] Intermediate product B (100.0 mg, 0.2 mmol) was dissolved in acetone (3.6 mL), and inorganic base potassium carbonate (53.2 mg, 0.4 mmol) and reactant 4-cyanobenzyl bromide (38 µL, 0.3 mmol) were added. The mixture was stirred at room temperature (the carboxyl group at C-21 of intermediate product B reacted with 4-cyanobenzyl bromide for 4 h) to obtain the crude product.

[0053] The crude product was concentrated under reduced pressure to remove acetone, then diluted with dichloromethane, followed by washing with water (twice), washing with saturated brine (once), drying with anhydrous sodium sulfate, filtration, concentration under reduced pressure, and silica gel column chromatography (V). 二氯甲烷 V 甲醇 =100:1), collect the target eluent and dry it to give a white solid, denoted as F-EZ-5 (target compound, 82.2 mg, yield 70.5%).

[0054] F-EZ-5 1 H NMR, 13 The C NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ 8.17 (d, J =1.0Hz, 1H, H-Oxazole), 7.91 (d,J =1.0Hz,1H,H-Oxazole),7.62(d, J =8.3Hz,2H,2´H-Ar),7.44(d, J =8.3Hz,2H,2´H-Ar),5.86(d, J =8.3Hz,1H,H-16),5.24(d, J =12.9Hz,1H,H-CH2Ar),5.16(d, J =2.7Hz,1H,H-3),4.91(d, J =13.0Hz,1H,H-CH2Ar),4.33(d, J =2.5Hz,1H,H-11),3.04(d, J =11.2Hz,1H,H-13),2.48-2.29(m,2H,CH2),2.25-2.12(m,4H,2´CH2),1.91(s,3H,CH3),1.89-1.69(m,6H,3´CH2),1.64-1.51(m,4H,2´CH2),1.39(s,3H,CH3),1.30-1.22(m,3H,CH2,H-OH),1.14-1.04(m,4H,2´CH2),0.99(s,3H,CH3),0.91(s,3H,CH3),0.85(d, J =6.7Hz,3H,CH3),0.77(dd, J =6.6,3.5Hz,6H,2´CH3)。

[0055] 13 C NMR(100MHz,CDCl3)δ 170.43,169.71,160.53,151.63,148.76,143.51,141.10,133.56,132.42,130.46,128.74,118.57,75.49,74.37,68.11,65.16,49.06,48.83,44.07,39.56,39.10,38.60,37.86,36.90,35.86,35.07,32.59,30.98,29.12,27.82,27.67,24.36,22.78,22.60,22.51,21.01,20.63,18.02,15.79。

[0056] F-EZ-5 is: 3α-(4-oxazol)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid p-cyanobenzyl ester.

[0057] III. Pharmacological Activities of Fusidic Acid Derivatives with C-3 Oxazole Substitution, C-24 Reduction, and C-21 Benzyl Ester Modification Drug-resistant KBV cells in logarithmic growth phase were digested with trypsin at a concentration of 0.25% (w / v) and then prepared into a solution with a concentration of 4 × 10⁻⁶. 4 A single-cell suspension of cells per mL.

[0058] The prepared single-cell suspension was seeded at 4000 cells / well in a 96-well plate (100 μL of single-cell suspension was added to each well) and incubated at 37°C for 24 h.

[0059] After culture, in the paclitaxel-containing group, 50 μL of the test compound (verapamil, F-EZ-1, F-EZ-2, F-EZ-3, F-EZ-4, or F-EZ-5, all dissolved in dimethyl sulfoxide (DMSO)) and 50 μL of paclitaxel solution were added to each well. The final concentration of the test compound was 10 μM, and the final concentration of paclitaxel was 100 nM. In the paclitaxel-free group, 50 μL of the test compound (verapamil, F-EZ-1, F-EZ-2, F-EZ-3, F-EZ-4, or F-EZ-5, all dissolved in DMSO) and 50 μL of complete culture medium were added to each well. The final concentration of the test compound was 10 μM. In the control group, 100 μL of paclitaxel solution was added to each well. DMSO solution, with a final concentration of DMSO <0.1% (v / v), was used. A blank control group (with only 100 μL of complete culture medium in each well, and no cells) was set up to remove the background. Each group had 3 parallel wells and was cultured at 37°C for 72 h.

[0060] After the culture was completed, the supernatant was discarded, and 100 μL of complete medium containing 0.5 mg / mL thiazolyl blue (MTT) was added to each well of each group. The culture was continued at 37°C for 4 h.

[0061] After the culture was completed, the supernatant was discarded, and 150 μL of DMSO was added to each well of each group to dissolve the MTT formazan precipitate. The mixture was then shaken and mixed with a micro-shaker. The optical density (OD) was then measured using a microplate reader at a reference wavelength of 450 nm and a detection wavelength of 570 nm.

[0062] The following formula was used to calculate the survival rate (%) of resistant KBV cells under the action of each compound alone and in combination with paclitaxel: Cell viability (%) = (mean OD value of the treatment group / mean OD value of the control group) × 100% The calculated survival rates (%) of resistant KBV cells under the treatment of each compound alone (in the group without paclitaxel) and in combination with paclitaxel (in the group containing paclitaxel) are shown in Table 1. The results of the analysis of significant differences in the survival rates of resistant KBV cells under the treatment of each compound in combination with paclitaxel (in the group containing paclitaxel) are shown in Table 1. Figure 1 .

[0063] Table 1. Calculation results of drug-resistant KBV cell survival rate (%) in the treatment groups

[0064] From Table 1 and Figure 1 It can be seen that the compounds (F-EZ-1, F-EZ-2, F-EZ-3, F-EZ-4 and F-EZ-5) prepared in Examples 1 to 5 all have good tumor NDR reversal activity, and all show MDR reversal activity comparable to or even better than that of the positive control drug verapamil at the same dose (e.g., F-EZ-5, p>0.05) (e.g., F-EZ-1, F-EZ-2, F-EZ-3, F-EZ-4, p<0.001), and can significantly increase the sensitivity of drug-resistant KBV cells to paclitaxel.

[0065] In summary, the fusidic acid derivatives provided by this invention, which are oxazole-substituted at C-3, reduced at C-24, and benzyl ester-modified at C-21 (F-EZ-1, F-EZ-2, F-EZ-3, F-EZ-4, and F-EZ-5), can be used in combination with the commonly used antitumor drug paclitaxel to exert good antitumor activity.

[0066] IV. Drug metabolic stability of fusidic acid derivatives with C-3 oxazole substitution, C-24 reduction, and C-21 benzyl ester modification. Three female SD rats (weighing 200-220g) were used for pharmacokinetic analysis. They were fasted and deprived of water for 12 hours before administration.

[0067] The F-EZ-1 prepared in Example 1 was used with a mixture of castor oil and physiological saline (V 蓖麻油 V 生理盐水 A sample solution with a concentration of 3.1 mg / mL was prepared by mixing a ratio of 1:2.

[0068] Administer via intraperitoneal injection at a dose of 7 mg / kg.

[0069] Blood samples of 1 mL were collected via the orbital sinus at 5 min, 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug administration. The samples were placed in centrifuge tubes containing heparin sodium, incubated at 5°C for 30 min, and then centrifuged (5°C, 3000 rpm, 20 min) to separate plasma. Plasma samples were stored at -80°C. Post-processing was performed using an extraction method. 50 μL of plasma sample was added to 200 μL of ethyl acetate, shaken for 5 min, and the organic phase was collected. After nitrogen purging, the sample was reconstituted with 500 μL of internal standard methanol (reserpine 40 ng / mL), centrifuged (4°C, 12000 rpm, 5 min), and the blood concentration of F-EZ-1 was detected by LC-MS / MS (AB SCIEX Triple Quad 4500, SCIEX, USA).

[0070] The results of blood drug concentration detection of F-EZ-1 after intraperitoneal injection are shown in Table 2.

[0071] Table 2. Blood drug concentration test results of F-EZ-1

[0072] Table 2 shows that the blood concentration of F-EZ-1 reached its maximum 2 hours after intraperitoneal injection (C). max =1225.67ng / mL), and the duration is as long as 24h, with a significantly improved half-life and good drug properties.

[0073] In summary, the fusidic acid derivatives modified with oxazole at C-3, reduction at C-24, and benzyl ester at C-21 provided by this invention exhibit excellent MDR reversal activity and good drug-like properties. They can be used in combination with commonly used antitumor drugs and exert a good antitumor effect in clinical treatment.

[0074] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A derivative of fusidic acid modified by oxazole substitution at C-3, reduction at C-24, and benzyl ester at C-21, characterized in that, The structures of the derivatives of fusidic acid with oxazole substitution at C-3, reduction at C-24, and benzyl ester modification at C-21 are shown below: ； Wherein, R represents 3-methylbenzyl, 3-chlorobenzyl, 2-bromobenzyl, 3-bromobenzyl or 4-cyanobenzyl.

2. The fusidic acid C-3 position oxazole-substituted, C-24 position reduced, and C-21 position benzyl ester modified derivative according to claim 1, characterized in that, The fusidic acid derivative with oxazole substitution at C-3, reduction at C-24, and benzyl ester modification at C-21 is: 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-ene-21-oic acid m-methyl benzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid-m-chlorobenzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-o-bromobenzyl ester; 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-oic acid benzyl bromide; or 3α-(4-oxazolyl)-formyloxy-16β-acetoxy-11α-hydroxy-4α,8α,14β-trimethyl-18-nor-5α,10β-cholest-(17Z)-17(20)-en-21-acid p-cyanobenzyl ester.

3. The method for preparing the fusidic acid C-3 position oxazole substitution, C-24 position reduction, and C-21 position benzyl ester modification derivative according to claim 1, characterized in that, Includes the following steps: (1) Using fusidic acid as a raw material, the C-24 double bond of fusidic acid was reduced by hydrogen under palladium on carbon catalysis to obtain intermediate product A; (2) Under the catalysis of 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the C-3 hydroxyl group of intermediate A reacts with 4-oxazolamide to give intermediate B; (3) In the presence of an inorganic base, the carboxyl group at position C-21 of intermediate product B reacts with a benzyl bromoanalyte, wherein the benzyl bromoanalyte is 3-methylbenzyl bromide, 3-chlorobenzyl bromide, 2-bromobenzyl bromide, 3-bromobenzyl bromide or 4-cyanobenzyl bromide, to obtain the crude product; (4) The crude product was purified by column chromatography to obtain the target compound.

4. The method for preparing the fusidic acid C-3 position oxazole substitution, C-24 position reduction, and C-21 position benzyl ester modification derivative according to claim 3, characterized in that, Specifically, the following steps are included: (1) Fusidic acid was dissolved in anhydrous ethanol, palladium on carbon was added, the reaction mixture was purged with argon gas and hydrogen gas was introduced, and the mixture was stirred at room temperature. After the reaction was completed, the product was purified to obtain intermediate product A. (2) Intermediate product A, 4-oxazolocarboxylic acid, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in dichloromethane and stirred at room temperature. After the reaction was completed, the product was purified to obtain intermediate product B. (3) Dissolve intermediate product B in acetone, add inorganic base and benzyl bromide analogue, stir at room temperature to obtain crude product; (4) The crude product was concentrated under reduced pressure to remove acetone, then diluted with dichloromethane, and then washed with water, saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to silica gel column chromatography. The target eluent was collected and dried to obtain the target compound.

5. The use of the fusidic acid C-3 position oxazole-substituted, C-24 position reduced, and C-21 position benzyl ester modified derivative of claim 2 in the preparation of P-gp inhibitors.

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