CN114213501B - C-23 nitrogen-containing heterocyclic derivative of cycloisoxazole ring hederagenin and preparation method thereof - Google Patents

Abstract

The invention discloses an A-ring heteroC-23 nitrogen heterocyclic ring derivative of oxazolidine hederagenin and a preparation method thereof. The invention relates to the field of organic synthesis and pharmaceutical chemistry, in particular to a C-23 nitrogen-containing heterocycle modified derivative of ring A isoxazole ring hederagenin, which has a novel structure, and part of compounds of the invention are as follows: 4- (23-oxyolean-12-en-28-oic acid benzyl ester and [2, 3-)d]Isoxazole) -4-oxo-butyryl- (4-methyl) piperazinamine; 4- (23-oxyolean-12-en-28-oic acid benzyl ester and [2, 3-)d]Isoxazole) -4-oxo-butyryl- (4-ethyl) piperazinamine, and the like. The invention relates to a nitrogen-containing heterocyclic derivative of C-23 position of A-ring isoxazole ring hederagenin of general formula I and application of the nitrogen-containing heterocyclic derivative and medically acceptable salt thereof in preparing tumor drug resistance reversal agents and/or pharmaceutically acceptable carriers for treating mammals, preferably human diseases or symptoms.

Description

C-23 nitrogen-containing heterocyclic derivative of cycloisoxazole ring hederagenin and preparation method thereof

Technical Field

The invention relates to the field of organic synthesis and pharmaceutical chemistry, in particular to a C-23 nitrogen heterocycle modified derivative of ring A isoxazole ring hederagenin and a preparation method thereof.

Technical Field

Malignant tumors seriously harm human health, and according to the latest cancer data of the international agency for research on cancer (IARC): in 2020, 1929 million new cancer cases and 995.8 million death cases are found in the world. The appearance of Multidrug Resistance (MDR) results in failure of chemotherapy in more than 90% of tumor patients, and the efficacy of chemotherapy is significantly reduced. Therefore, the development of novel tumor drug resistance reversal agents is a key focus of attention in the field of medicinal chemistry. Natural products and derivatives thereof of various structural types have been reported to have demonstrated tumor resistance reversal activity, and natural products have become one of the important sources of tumor MDR reversal agents.

Hederagenin (H) is originated from Hederagenin (Hederagenin, H) of Hedera of Araliaceae, and is an oleanane-type pentacyclic triterpene compound. The preliminary study of this subject group found that H derivative H6 has a certain in vivo and in vitro tumor MDR reversal activity, and the mechanism of action has been preliminarily elucidated (see Yang YT, guan DK, lei L, et al H6, a novel chemotherapy derivative in vivo, versions multidrug resistance in vitro and in vivo [ J ]. Toxicology & Applied Pharmacology,2018, 341. Therefore, chemical modification of H enriches the structure types, and is the key point of future work for developing a tumor MDR reversal agent with a novel hederagenin structure.

Disclosure of Invention

The invention aims to provide a C-23 nitrogen heterocyclic ring derivative of hederagenin and a preparation method thereof. The invention aims to solve the technical problems of searching a compound with novel structure and excellent tumor drug resistance reversal activity and further providing a pharmaceutical composition for treating oral epithelial cancer, gastric cancer, lung cancer, cervical cancer, breast cancer or colon cancer and the like by combining with a clinical common antitumor drug.

In order to solve the technical problems, the invention provides the following technical scheme:

a ring-fused isoxazole ring hederagenin C-23-position nitrogen-containing heterocyclic derivatives shown in a general formula I and medically acceptable salts thereof,

wherein,

a compound of the general formula I: x represents a linear or branched non-substituted alkyl group of 1 to 3 carbons;

R ₁ represents N-methylpiperazine, N-ethylpiperazine, morpholine or tetrahydropyrrole.

Preferably, some of the compounds of the present invention are:

4- (23-oxoolean-12-en-28-oic acid benzylester and [2,3-d ] isoxazole) -4-oxo-butyryl- (4-methyl) piperazinamine;

4- (23-oxoolean-12-en-28-oic acid benzylester and [2,3-d ] isoxazole) -4-oxo-butyryl- (4-ethyl) piperazinamine;

4- (23-oxoolean-12-en-28-oic acid benzylester and [2,3-d ] isoxazole) -4-oxo-butyryl morpholinamide;

4- (23-oxyolean-12-en-28-oic acid benzyl ester and [2,3-d ] isoxazole) -4-oxo-butyrylpyrrolidine.

The preparation route of the A-ring isoxazole ring hederagenin C-23 nitrogen-containing heterocyclic derivative provided by the invention is as follows:

the A-ring isoxazole ring hederagenin C-23-position nitrogen-containing heterocyclic derivative with the general formula I is synthesized by the following method:

a. taking hederagenin as a raw material, and protecting carboxyl by benzyl bromide in the presence of inorganic base;

b. protecting hydroxyl at the C-23 position by using tert-butyldimethylsilyl chloride;

c. oxidizing C-3 hydroxyl of the intermediate product protected by TBS by pyridinium chlorochromate;

d. reacting with ethyl formate under the catalysis of sodium methoxide;

e. reacting with hydroxylamine hydrochloride under the condition of absolute ethanol reflux;

f. reacting with succinic anhydride under the catalysis of DMAP and EDCI;

g. reacting with N-methyl piperazine, N-ethyl piperazine, morpholine and tetrahydropyrrole under the catalysis of HATU and DIEA to obtain a crude product;

h. and purifying the crude product by using a column chromatography method to obtain the target compound.

The C-23 nitrogen heterocyclic ring derivative of hederagenin has tumor drug resistance reversing activity and can be used for preparing tumor drug resistance reversing agents.

The C-23 nitrogen-containing heterocyclic derivative of the cycloisoxazole ring hederagenin A and the optical isomer of the compound or the pharmaceutically acceptable solvate thereof.

The effective amount of the compound of the general formula I or the salt thereof and the pharmaceutically acceptable carrier are used for treating diseases or symptoms such as oral epithelial cancer, gastric cancer, lung cancer, cervical cancer, breast cancer or colon cancer and the like by combining with clinical common antitumor drugs.

The invention relates to an A-ring-fused isoxazole ring hederagenin C-23-position nitrogen-containing heterocyclic derivative of a general formula I and application of the derivative in preparing a tumor drug resistance reversal agent and/or a pharmaceutically acceptable carrier for treating mammals, preferably human diseases or symptoms.

Advantageous effects

Pharmacological tests show that the C-23-position nitrogen-containing heterocyclic derivatives of cycloisoxazole hederagenin of general formula I prepared by introducing nitrogen-containing heterocyclic structures such as N-methyl piperazine, N-ethyl piperazine, morpholine and tetrahydropyrrole into the hederagenin derivatives of cycloisoxazole A by using succinic anhydride have the tumor multidrug resistance reversal activity equivalent to or even better than verapamil, and can obviously improve the sensitivity of drug-resistant KBV cells to paclitaxel. Therefore, the C-23 nitrogen heterocyclic ring derivative of the ring A isoxazole ring hederagenin of the general formula I can be used together with common antitumor drugs to play a good role in antitumor activity.

Detailed Description

The present invention will be described in further detail below by way of examples, but the present invention is not limited to only the following examples.

Example 14- (23-OxyOlean-12-en-28-oic acid benzylo [2,3-D ] isoxazole) -4-oxo-butyryl- (4-methyl) piperazinamine (HYZ-D-JP)

The compound hederagenin (472.0mg, 1.0mmol) is dissolved in N, N-dimethylformamide (15.0 mL), potassium carbonate (300.0mg, 2.1mmol) and bromobenzyl (0.2mL, 1.3mmol) are added, and the mixture is stirred for 6-10h at the temperature of 50 ℃. The reaction mixture was diluted with ethyl acetate (25.0 mL), washed with water three times, washed with saturated brine two times, dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to remove the solvent, and subjected to silica gel column chromatography (V) _{Petroleum ether} :V _{Acetic acid ethyl ester} 1-5).

The above-mentioned compound (460.0mg, 0.8mmol) was dissolved in 20.0mL of methylene chloride, and 4-dimethylaminopyridine (122.0mg, 1.0mmol) and t-butyldimethylsilyl chloride (360.0mg, 2.4mmol) were added thereto, and the mixture was stirred at room temperature for 4 to 8 hours. Evaporating to remove dichloromethane and ethyl acetateDiluting the ester (20.0 mL), washing with HCl (5%) until acidic, washing with saturated saline until neutral, drying over anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography (V) _{Petroleum ether} :V _{Ethyl acetate} 1-15) to give a white solid (383.0 mg, 70.0%).

The above compound (380.0 mg,0.6 mmol) was dissolved in 15.0mL of dichloromethane, and fresh pyridinium chlorochromate (300.0 mg,1.3 mmol) was added and stirred at room temperature for 6-10h. Evaporating to remove dichloromethane, diluting with ethyl acetate (20.0 mL), washing with water, washing with saturated saline solution to neutrality, drying with anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography (V) _{Petroleum ether} :V _{Ethyl acetate} =35, 1-20), yielding a white solid (319.0 mg, 84.0%).

The above-mentioned compound (500.0mg, 0.7mmol) and sodium methoxide (400.0mg, 7.4mmol) were dissolved in anhydrous tetrahydrofuran (15.0 mL), and ethyl formate (592.0. Mu.L, 7.4 mmol) was added, followed by stirring at room temperature for 3 hours. After the reaction, ethyl acetate was added for dilution, and the organic layer was washed once with 10% HCl solution, twice with deionized water and twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (V) _{Petroleum ether} :V _{Acetic acid ethyl ester} 1-8) to give a yellow oily liquid (435.5mg, 88.6%).

The above-mentioned compound (500.0mg, 0.7mmol) was dissolved in anhydrous ethanol (30 mL), and hydroxylamine hydrochloride (118mg, 1.7mmol) was added thereto, and the reaction was refluxed for 5 hours. After the reaction is finished, concentrating, diluting with ethyl acetate, washing with deionized water and saturated saline water respectively twice in turn, drying with anhydrous sodium sulfate, filtering, concentrating, and performing silica gel column chromatography (V) _{Petroleum ether} :V _{Ethyl acetate} = 5).

The above compound HYZ (410mg, 0.7 mmol) was dissolved in anhydrous dichloromethane (25.0 mL), and the catalyst DMAP (453.4mg, 3.7 mmol) and succinic anhydride (742.8mg, 7.4mmol) were added to react at room temperature for 2 hours. After the reaction, dichloromethane was added for dilution, and the organic layer was washed once with 5% HCl solution, twice with deionized water and twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (V) _Chloroform :V _Methanol 1-15) to obtain white solid HYZ-D (466.0)mg，90.2％)。

HYZ-D (95mg, 0.1mmol) was dissolved in anhydrous DCM (10.0 ml), HATU (76.0 mg, 0.2mmol) and DIEA (49.7. Mu.L, 0.3 mmol) were added and stirred at room temperature for 0.5h, N-methylpiperazine (22.2. Mu.L, 0.2 mol) was added and the reaction was stirred at room temperature for about 3h. After the reaction, dichloromethane was added for dilution, and the organic layer was washed once with 5% HCl solution and saturated sodium bicarbonate solution, twice with deionized water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (V) _{Petroleum ether} :V _{Ethyl acetate} = 10-1). ¹ H NMR(400MHz,Chloroform-d)δ7.98(s,1H,H-Isoxazole),7.34–7.29(m,5H,5×H-Ar),5.33(t,J＝3.7Hz,1H,H-12),5.10–5.02(m,2H,CH ₂ Ar),4.15(d,J＝1.8Hz,2H,H-23),3.72(t,J＝5.4Hz,2H,N-CH ₂ -Piperazine),3.56(t,J＝5.4Hz,2H,N-CH ₂ -Piperazine),2.91(dd,J＝14.0,4.6Hz,1H,H-18),2.64–2.48(m,8H,4×CH ₂ -Succinic anhydride),2.43–2.38(m,4H,CH ₃ -Piperazine),2.02–1.91(m,4H,CH3-Piperazine,H-13),1.76–1.44(m,12H,CH ₂ ),1.20(s,3H,CH ₃ ),1.13(s,3H,CH ₃ ),0.91(s,3H,CH ₃ ),0.89(s,3H,CH ₃ ),0.85(s,3H,CH ₃ ),0.63(s,3H,CH ₃ ).

Example Synthesis and characterization of benzyl 24- (23-oxoolean-12-en-28-oate [2,3-D ] isoxazole) -4-oxo-butyryl- (4-ethyl) piperazinamine (HYZ + D + YP)

HYZ-D (95mg, 0.1mmol) was dissolved in anhydrous DCM (10.0 ml), HATU (76.0 mg, 0.2mmol) and DIEA (49.7. Mu.L, 0.3 mmol) were added and stirred at room temperature for 0.5h, N-ethylpiperazine (25.5. Mu.L, 0.2 mol) was added and the reaction was stirred at room temperature for about 3h. After the reaction, dichloromethane was added for dilution, and the organic layer was washed once with 5% HCl solution and saturated sodium bicarbonate solution, twice with deionized water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (V) _{Petroleum ether} :V _{Acetic acid ethyl ester} 1-3) to obtain yellow solid HYZ-D-YP (67.4mg, 81.4%). ¹ H NMR(400MHz,Chloroform-d)δ7.99(s,1H,H-Isoxazole),7.36–7.31(m,5H,5×H-Ar),5.34(t,J＝3.7Hz,1H,H-12),5.12–5.04(m,2H,CH ₂ Ar),4.24–4.10(m,2H,H-23),3.64(d,J＝5.4Hz,2H,N-CH ₂ -Piperazine),3.51(d,J＝5.4Hz,2H,N-CH ₂ -Piperazine),2.94(dd,J＝13.7,4.4Hz,1H,H-18),2.64–2.41(m,12H,2×CH ₂ -Succinic anhydride,2×CH ₂ -Piperazine,,CH ₃ -Piperazine,H-13),1.96(dd,J＝14.2,11.4Hz,4H,CH ₂ ),1.77–1.45(m,12H,CH ₂ ),1.22(s,3H,CH ₃ ),1.15(s,3H,CH ₃ ),0.93(s,3H,CH ₃ ),0.91(s,3H,CH ₃ ),0.87(s,3H,CH ₃ ),0.66(s,3H,CH ₃ ).

Example 34 Synthesis and characterization of benzyl [2,3-D ] isoxazole-4-oxo-butyryl-morpholinamide (HYZ + D + ML) -23-oxoolean-12-en-28-oate

HYZ-D (95mg, 0.1mmol) was dissolved in anhydrous DCM (10.0 ml), HATU (76.0 mg,0.2 mmol) and DIEA (49.7. Mu.L, 0.3 mmol) were added and stirred at room temperature for 0.5h, morpholine (17.4. Mu.L, 0.2 mol) was added and the reaction stirred at room temperature for about 3h. After the reaction, dichloromethane was added for dilution, and the organic layer was washed once with 5% HCl solution and saturated sodium bicarbonate solution, twice with deionized water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (V) _{Petroleum ether} :V _{Ethyl acetate} 1-5) to obtain a yellow transparent oily compound HYZ-D-ML (69.2mg, 91.7%). ¹ H NMR(400MHz,Chloroform-d)δ7.98(s,1H,H-Isoxazole),7.33–7.28(m,5H,5×H-Ar),5.34–5.31(m,1H,H-12),5.10–5.01(m,2H,CH ₂ Ar),4.16(q,J＝11.0Hz,2H,H-23),3.66–3.60(m,4H,CH ₂ -morpholine),3.56(q,J＝4.8,3.6Hz,2H,N-CH ₂ -morpholine),3.44(dd,J＝6.0,3.8Hz,2H,N-CH ₂ -morpholine),2.92(dd,J＝13.9,4.5Hz,1H,H-18),2.65–2.47(m,4H CH ₂ -Succinic anhydride),2.41(d,J＝15.2Hz,1H,H-13),2.02–1.91(m,4H,CH ₂ ),1.76–1.42(m,12H,CH ₂ ),1.21(s,3H,CH ₃ ),1.13(s,3H,CH ₃ ),0.91(s,3H,CH ₃ ),0.89(s,3H,CH ₃ ),0.85(s,3H,CH ₃ ),0.64(s,3H,CH ₃ ).

Example Synthesis and characterization of benzyl [2,3-D ] isoxazole-4-oxo-butyryl-pyrrolidinamine (HYZ + D + 4H) -44- (23-oxoolean-12-en-28-oate

HYZ-D (95mg, 0.1mmol) was dissolved in anhydrous DCM (10.0 ml), HATU (76.0 mg,0.2 mmol) and DIEA (49.7. Mu.L, 0.3 mmol) were added and stirred at room temperature for 0.5h, then tetrahydropyrrole (16.8. Mu.L, 0.2 mol) was added and the reaction was stirred at room temperature for about 3h. After the reaction was completed, dichloromethane was added for dilution, the organic layer was washed once with 5% HCl solution and saturated sodium bicarbonate solution in this order, twice with deionized water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography (V) _{Petroleum ether} :V _{Ethyl acetate} 1-5) to obtain a yellow transparent oily compound HYZ-D-4H (65.4mg, 88.6%). ¹ HNMR(400MHz,Chloroform-d)δ7.96(s,1H,H-Isoxazole),7.32–7.27(m,5H,5×H-Ar),5.30(t,J＝3.6Hz,1H,H-12),5.08–5.00(m,2H,CH ₂ Ar),4.20–4.08(m,2H,H-23),3.42–3.33(m,4H,N-CH ₂ -Pyrrolidine),2.90(dd,J＝14.0,4.8Hz,1H H-18),2.71–2.42(m,4H,CH ₂ -Succinic anhydride),2.39(m,1H,H-13),2.01–1.89(m,6H,CH ₂ ,CH ₂ -Pyrrolidine),1.81(q,J＝6.7Hz,2H,CH ₂ -Pyrrolidine),1.74–1.40(m,12H,CH ₂ ),1.18(s,3H,CH ₃ ),1.12(s,3H,CH ₃ ),0.90(s,3H,CH ₃ ),0.87(s,3H,CH ₃ ),0.84(s,3H,CH ₃ ),0.62(s,3H,CH ₃ ).

The following are the pharmacological tests and data for some of the compounds of the invention.

1 experimental method: examples 1-4 detection of the survival Rate of the antitumor drug paclitaxel in KBV resistant Strain cells

KBV cells in logarithmic phase are digested with 0.25% pancreatin to prepare single cell suspension with certain concentration. Based on the difference in cell growth rate, the cells were seeded at 4000 wells in a 96-well plate, and 100. Mu.L of cell suspension was added to each well. After 24h, complete medium was added at various concentrations of compound and 100nM of paclitaxel and corresponding solvent control. mu.L of DMSO (final DMSO concentration < 0.1%) was added to each well, 3 wells were placed in each group, incubation was continued at 37 ℃ for 72h, and the supernatant was discarded. Adding 100 μ L complete medium containing 0.5mg/mL MTT into each well, culturing for 4 hr, discarding supernatant, adding 150 μ L LDMSO into each well to dissolve MTT formazan precipitate, and micro-shakingAfter the mixture is oscillated and mixed uniformly, an enzyme-labeling instrument measures the Optical Density (OD) value under the conditions of reference wavelength of 450nm and detection wavelength of 570 nm. Using tumor cells treated by solvent control as a control group, and calculating the survival rate of different tumor cells under the action of each compound by using the following formula; tumor cells treated by solvent control are used as a control group, the inhibition rate of the compound on the tumor cells is calculated by the following formula, and the IC is calculated according to the middle effect equation ₅₀ 。

Cell viability (%) = average OD value in administration group/average OD value in control group × 100%

IC ₅₀ = (control group mean OD value-administration group mean OD value)/control group mean OD value × 100%

2, experimental results:

examples 1-4 cell viability when administered alone and in combination is shown in table 1.

TABLE 1 cell viability in examples 1-4 taken alone and in combination

Examples 1-4 viability assay of anti-tumor drug paclitaxel on KBV resistant strain cells.

The survival rate evaluation results of KBV drug-resistant strain cells of the derivatives show that the examples 1-4 have better tumor drug resistance reversal activity and can obviously increase the sensitivity of the drug-resistant KBV cells to paclitaxel, wherein the activity of the example 2 is superior to that of an equal-dose positive control drug verapamil, and the examples 1, 3 and 4 show the tumor multidrug resistance reversal activity equivalent to that of verapamil.

The experiments show that the invention introduces N-methyl piperazine, N-ethyl piperazine, morpholine, tetrahydropyrrole and other nitrogen-containing heterocyclic structures into the hederagenin derivative of the A-ring isoxazole by using succinic anhydride to obtain a new chemical entity. Moreover, compared with hederagenin, the structural modification of the invention is obviously different from the structural modification of the hederagenin, so that the direct anti-tumor activity of the hederagenin disappears, and the tumor multidrug resistance reversal activity of the hederagenin is superior to or equal to that of verapamil. Therefore, the C-23 nitrogen-containing heterocyclic derivatives of the ring A isoxazole ring hederagenin of the general formula I can be used together with common antitumor drugs to play a good antitumor activity.

Although the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the details of the embodiments, and various equivalent modifications can be made within the technical spirit of the present invention, and the scope of the present invention is also within the scope of the present invention. It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition. In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims (3)

1. A ring-fused isoxazole ring hederagenin C-23-position nitrogen-containing heterocyclic derivatives shown in a general formula I,

wherein, the general formula I: x represents a linear or branched non-substituted alkyl group of 1 to 3 carbons;

R ₁ represents

2. The C-23 nitrogen-containing heterocyclic derivative of cycloisoxazolo ring hederagenin A shown in the general formula I according to claim 1 is characterized in that the derivative is:

4- (23-oxoolean-12-en-28-oic acid benzylester and [2,3-d ] isoxazole) -4-oxo-butyryl- (4-methyl) piperazinamine;

4- (23-oxoolean-12-en-28-oic acid benzylester and [2,3-d ] isoxazole) -4-oxo-butyryl- (4-ethyl) piperazinamine;

4- (23-oxoolean-12-en-28-oic acid benzylester and [2,3-d ] isoxazole) -4-oxo-butyryl morpholinamide;

4- (23-oxyolean-12-en-28-oic acid benzyl ester and [2,3-d ] isoxazole) -4-oxo-butyrylpyrrolidine.

3. The preparation method of the C-23 nitrogen heterocyclic ring derivative of the A-ring-fused isoxazole ring hederagenin shown in the general formula I in claim 1 is characterized by comprising the following steps:

a. taking hederagenin as a raw material, and protecting carboxyl by benzyl bromide in the presence of inorganic base;

b. protecting hydroxyl at the C-23 position by tert-butyldimethylsilyl chloride;

c. oxidizing C-3 hydroxyl of the intermediate product protected by TBS by pyridinium chlorochromate;

d. reacting with ethyl formate under the catalysis of sodium methoxide;

e. reacting with hydroxylamine hydrochloride under the condition of absolute ethanol reflux;

f. reacting with succinic anhydride under the catalysis of DMAP and EDCI;

g. reacting with N-methyl piperazine, N-ethyl piperazine, morpholine and tetrahydropyrrole under the catalysis of HATU and DIEA to obtain a crude product;

h. and purifying the crude product by using a column chromatography method to obtain the target compound.