CN114213501A - C-23 nitrogen-containing heterocyclic derivative of A-ring-fused isoxazole ring hederagenin and preparation method thereof - Google Patents

Abstract

The invention discloses a C-23 nitrogen heterocyclic ring derivative of hederagenin of an A-ring isoxazole ring 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 cycloisoxazole ring hederagenin A with a novel structure, wherein partial compounds of the invention are as follows: 4- (23-oxyolean-12-en-28-oic acid benzyl ester [2, 3-)d]Isoxazole) -4-oxo-butyryl- (4-methyl) piperazinamine; 4- (23-oxyolean-12-en-28-oic acid benzyl ester [2, 3-)d]Isoxazole) -4-oxo-butyryl- (4-ethyl) piperazinamine, and the like. The invention relates to a C-23 nitrogen heterocyclic ring derivative of A-ring-fused isoxazole ring hederagenin of a general formula I and application thereof in preparing tumor drug resistance reversal agent and/orThe pharmaceutical carrier is useful for treating a disease or condition in a mammal, preferably a human.

Description

C-23 nitrogen-containing heterocyclic derivative of A-ring-fused isoxazole 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, there are 1929 ten thousand new cancer cases and 995.8 ten thousand death cases worldwide. The appearance of Multidrug Resistance (MDR) causes over 90% of tumor patients to fail chemotherapy, and the curative effect of the chemotherapy drug is obviously reduced. Therefore, the development of novel tumor 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 H6 derivative has certain MDR reversal activity in vivo and in vitro, and the action mechanism has been preliminarily elucidated (see Yang YT, Guan DK, Lei L, et al. H6, a novel heideringenin derivative, variants multi drug resistance in vitro and in vivo [ J ]. diagnosis & Applied Pharmacology,2018,341: 98-105). 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 nitrogenous heterocycle derivative shown in general formula I and medically acceptable salt thereof,

wherein the content of the first and second substances,

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

R₁represents 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-oxoolean-12-en-28-oic acid benzyl ester and [2,3-d ] isoxazole) -4-oxo-butyryl pyrrolidine amine.

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. the intermediate product protected by TBS is oxidized by chloropyridine chromate to obtain C-3 hydroxyl;

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 nitrogen-containing heterocyclic derivative of the hederagenin of the ring A of the general formula I, which is prepared by introducing nitrogen-containing heterocyclic structures such as N-methyl piperazine, N-ethyl piperazine, morpholine and tetrahydropyrrole into the hederagenin derivative of the ring A of the hederagenin by using succinic anhydride, has the tumor multidrug resistance reversal activity which is equivalent to or even better than verapamil, and can obviously increase 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-Oxylon-12-en-28-oic acid benzylo [2,3-D ] isoxazole) -4-oxo-butyryl- (4-methyl) piperazinamine (HYZ-D-JP)

Dissolving compound hederagenin (472.0mg, 1.0mmol) in N, N-dimethylformamide (15.0mL), adding potassium carbonate (300.0mg, 2.1mmol) and benzyl bromide (0.2mL, 1.3mmol), and stirring at 50 deg.C for 6-10 h. Diluting the reaction solution with ethyl acetate (25.0mL), washing with water for three times, washing with saturated salt water for two times, drying with anhydrous sodium sulfate, filtering, evaporating under reduced pressure to remove solvent, and performing silica gel column chromatography (V)_{Petroleum ether}:V_{Ethyl acetate}10:1-5:1) to give a white solid (470.0mg, 83.0%).

The above compound (460.0mg, 0.8mmol) was dissolved in 20.0mL of dichloromethane, 4-dimethylaminopyridine (122.0mg, 1.0mmol) and tert-butyldimethylsilyl chloride (360.0mg, 2.4mmol) were added, and the mixture was stirred at room temperature for 4-8 h. The dichloromethane was evaporated, diluted with ethyl acetate (20.0mL), washed acidic with 5% HCl, washed neutral with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatographed (V)_{Petroleum ether}:V_{Ethyl acetate}30:1-15:1) to give a white solid (383.0mg, 70.0%).

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

The above-mentioned compound (500.0mg,0.7mmol) and sodium methoxide (400.0mg, 7.4mmol) were dissolved in anhydrous tetrahydrofuran (15.0mL), and ethyl formate (592.0. mu.L, 7.4mmol) was added, followed by stirring at room temperature for 3 hours. After the reaction is finished, ethyl acetate is added for dilution, the organic layer is washed once by 10 percent HCl solution and washed twice by deionized water and saturated sodium chloride solution respectively in sequence, and anhydrous sodium sulfate is driedDrying, filtering, concentrating, and performing silica gel column chromatography (V)_{Petroleum ether}:V_{Ethyl acetate}20:1-8:1) to give a yellow oily liquid (435.5mg, 88.6%).

The above-mentioned compound (500.0mg,0.7mmol) was dissolved in anhydrous ethanol (30mL), and hydroxylamine hydrochloride (118mg, 1.7mmol) was added thereto, followed by reflux reaction 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}5:1) to give HYZ as a white solid (375.8mg, 91.7%).

The above compound HYZ (410mg, 0.7mmol) was dissolved in anhydrous dichloromethane (25.0mL), and the catalyst DMAP (453.4mg, 3.7mmol) and succinic anhydride (742.8mg, 7.4mmol) were added to react at room temperature for 2 hours. After the reaction is finished, dichloromethane is added for dilution, the organic layer is washed once by 5 percent HCl solution and washed twice by deionized water and saturated sodium chloride solution respectively in turn, dried by anhydrous sodium sulfate, filtered, concentrated and chromatographed by silica gel column (V)_Chloroform:V_Methanol50:1-15:1) to give HYZ-D as a white solid (466.0mg, 90.2%).

HYZ-D (95mg, 0.1mmol) was dissolved in anhydrous DCM (10.0ml), HATU (76.0mg, 0.2mmol) and DIEA (49.7. mu.L, 0.3mmol) were added and stirred at room temperature for 0.5h, N-methylpiperazine (22.2. mu.L, 0.2mol) was added and the reaction was stirred at room temperature for about 3 h. After the reaction is finished, dichloromethane is added for dilution, the organic layer is washed once by 5 percent HCl solution and saturated sodium bicarbonate solution in sequence, washed twice by deionized water and saturated sodium chloride solution, dried by anhydrous sodium sulfate, filtered, concentrated and chromatographed by silica gel (V)_{Petroleum ether}:V_{Ethyl acetate}10:1-5:1) to yield HYZ-D-JP as a yellow solid (62.5mg, 81.4%).¹H 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,CH₂Ar),4.15(d,J＝1.8Hz,2H,H-23),3.72(t,J＝5.4Hz,2H,N-CH₂-Piperazine),3.56(t,J＝5.4Hz,2H,N-CH₂-Piperazine),2.91(dd,J＝14.0,4.6Hz,1H,H-18),2.64–2.48(m,8H,4×CH₂-Succinic anhydride),2.43–2.38(m,4H,CH₃-Piperazine),2.02–1.91(m,4H,CH3-Piperazine,H-13),1.76–1.44(m,12H,CH₂),1.20(s,3H,CH₃),1.13(s,3H,CH₃),0.91(s,3H,CH₃),0.89(s,3H,CH₃),0.85(s,3H,CH₃),0.63(s,3H,CH₃).

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

HYZ-D (95mg, 0.1mmol) was dissolved in anhydrous DCM (10.0ml), HATU (76.0mg, 0.2mmol) and DIEA (49.7. mu.L, 0.3mmol) were added and stirred at room temperature for 0.5h, N-ethylpiperazine (25.5. mu.L, 0.2mol) was added and the reaction stirred at room temperature for about 3 h. After the reaction is finished, dichloromethane is added for dilution, the organic layer is washed once by 5 percent HCl solution and saturated sodium bicarbonate solution in sequence, washed twice by deionized water and saturated sodium chloride solution, dried by anhydrous sodium sulfate, filtered, concentrated and chromatographed by silica gel (V)_{Petroleum ether}:V_{Ethyl acetate}10:1-3:1) to give HYZ-D-YP as a yellow solid (67.4mg, 81.4%).¹H 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,CH₂Ar),4.24–4.10(m,2H,H-23),3.64(d,J＝5.4Hz,2H,N-CH₂-Piperazine),3.51(d,J＝5.4Hz,2H,N-CH₂-Piperazine),2.94(dd,J＝13.7,4.4Hz,1H,H-18),2.64–2.41(m,12H,2×CH₂-Succinic anhydride,2×CH₂-Piperazine,,CH₃-Piperazine,H-13),1.96(dd,J＝14.2,11.4Hz,4H,CH₂),1.77–1.45(m,12H,CH₂),1.22(s,3H,CH₃),1.15(s,3H,CH₃),0.93(s,3H,CH₃),0.91(s,3H,CH₃),0.87(s,3H,CH₃),0.66(s,3H,CH₃).

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

HYZ-D (95mg, 0.1mmol) was dissolved in anhydrous DCM (10.0ml), HATU (76.0mg, 0.2mmol) and DIEA (49.7. mu.L, 0.3mmol) were added and stirred at room temperature for 0.5h, morpholine (17.4. mu.L, 0.2mol) was added and the reaction stirred at room temperature for about 3 h. After the reaction was completed, dichloromethane was added to dilute the reaction solution, and the organic layer was successively diluted with 5% HCl solutionWashing with saturated sodium bicarbonate solution, washing with deionized water and saturated sodium chloride solution twice, drying with anhydrous sodium sulfate, filtering, concentrating, and performing silica gel column chromatography (V)_{Petroleum ether}:V_{Ethyl acetate}15:1-5:1) gave HYZ-D-ML (69.2mg, 91.7%) as a yellow clear oil.¹H 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,CH₂Ar),4.16(q,J＝11.0Hz,2H,H-23),3.66–3.60(m,4H,CH₂-morpholine),3.56(q,J＝4.8,3.6Hz,2H,N-CH₂-morpholine),3.44(dd,J＝6.0,3.8Hz,2H,N-CH₂-morpholine),2.92(dd,J＝13.9,4.5Hz,1H,H-18),2.65–2.47(m,4H CH₂-Succinic anhydride),2.41(d,J＝15.2Hz,1H,H-13),2.02–1.91(m,4H,CH₂),1.76–1.42(m,12H,CH₂),1.21(s,3H,CH₃),1.13(s,3H,CH₃),0.91(s,3H,CH₃),0.89(s,3H,CH₃),0.85(s,3H,CH₃),0.64(s,3H,CH₃).

Example Synthesis and characterization of benzyl [2,3-D ] isoxazole-4-oxo-butyrylpyrrolidine- (23-oxoolean-12-en-28-oate)

HYZ-D (95mg, 0.1mmol) was dissolved in anhydrous DCM (10.0ml), HATU (76.0mg, 0.2mmol) and DIEA (49.7. mu.L, 0.3mmol) were added and stirred at room temperature for 0.5h, then pyrrolidine (16.8. mu.L, 0.2mol) was added and the reaction stirred at room temperature for about 3 h. After the reaction is finished, dichloromethane is added for dilution, the organic layer is washed once by 5 percent HCl solution and saturated sodium bicarbonate solution in sequence, washed twice by deionized water and saturated sodium chloride solution, dried by anhydrous sodium sulfate, filtered, concentrated and chromatographed by silica gel (V)_{Petroleum ether}:V_{Ethyl acetate}15:1-5:1) gave HYZ-D-4H (65.4mg, 88.6%) as a yellow, clear oil.¹HNMR(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,CH₂Ar),4.20–4.08(m,2H,H-23),3.42–3.33(m,4H,N-CH₂-Pyrrolidine),2.90(dd,J＝14.0,4.8Hz,1H H-18),2.71–2.42(m,4H,CH₂-Succinic anhydride),2.39(m,1H,H-13),2.01–1.89(m,6H,CH₂,CH₂-Pyrrolidine),1.81(q,J＝6.7Hz,2H,CH₂-Pyrrolidine),1.74–1.40(m,12H,CH₂),1.18(s,3H,CH₃),1.12(s,3H,CH₃),0.90(s,3H,CH₃),0.87(s,3H,CH₃),0.84(s,3H,CH₃),0.62(s,3H,CH₃).

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. After adding 100. mu.L of complete medium containing 0.5mg/mL MTT per well, culturing was continued for 4 hours, and the supernatant was discarded, 150. mu.L of LDMSO was added per well to dissolve MTT formazan precipitate, and the mixture was shaken and mixed by a micro shaker, and then the Optical Density (OD) was measured with a microplate reader at a reference wavelength of 450nm and a 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 equation₅₀。

Cell survival rate (%). The average OD value of the administered group/the average OD value of the control group X100%

IC₅₀(control group mean OD value-administration group mean OD value)/control group mean OD value. times.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 in 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 obviously differs from the structural modification of the hederagenin in 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 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.

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 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;

R₁represents N-methylpiperazine, N-ethylpiperazine, morpholine or tetrahydropyrrole.

2. The C-23 nitrogen-containing heterocycle modified derivative of A-ring-fused isoxazole ring hederagenin 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-oxoolean-12-en-28-oic acid benzyl ester and [2,3-d ] isoxazole) -4-oxo-butyryl pyrrolidine amine.

3. The preparation method of the C-23 nitrogen-containing heterocycle modified 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 using tert-butyldimethylsilyl chloride;

c. the intermediate product protected by TBS is oxidized by chloropyridine chromate to obtain C-3 hydroxyl;

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.