CN114507268A - Toad venom steroid diene derivative and preparation method and application thereof - Google Patents

Toad venom steroid diene derivative and preparation method and application thereof Download PDF

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CN114507268A
CN114507268A CN202011284916.6A CN202011284916A CN114507268A CN 114507268 A CN114507268 A CN 114507268A CN 202011284916 A CN202011284916 A CN 202011284916A CN 114507268 A CN114507268 A CN 114507268A
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吴婉莹
雷敏
周阳
侯晋军
龙华丽
张子佳
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Shanghai Institute of Materia Medica of CAS
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    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
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Abstract

The invention relates to a bufonis diene derivative, a preparation method and application thereof, wherein the bufonis diene derivative has a structure shown in a formula (I):

Description

Bufonis venenum diene derivative, and preparation method and application thereof
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to a bufonis diene derivative and a preparation method and application thereof.
Background
Cancer is a serious threat to human health, as indicated in the global Cancer report issued by International Agency for Research on Cancer (IARC) under the world health organization flag in 2018: in the last year, 1810 ten thousand patients have been diagnosed as cancer patients, 960 ten thousand patients have lost lives due to cancer, and nearly 360 thousand patients have been diagnosed as cancer patients in China.
The toad venom is a rare traditional Chinese medicine in China, wherein the bufostadienone is the main active ingredient of the toad venom steroid, and has remarkable anti-tumor activity, so that the compound has the potential for preparing anti-tumor medicines. However, the most important problems of the bufonis venenum diene compounds are that the bufonis venenum diene compounds have extremely low water solubility and strong toxicity, and the improvement of the water solubility and the toxicity of the bufonis venenum diene compounds is urgently needed.
Disclosure of Invention
Based on the above, a need exists for a bufonis diene derivative, a preparation method and an application thereof. The bufogenin derivative has improved water solubility, reduced toxicity, and retained antitumor activity, and can be used as medicine for preventing and treating malignant tumor.
A bufonisin diene derivative or a pharmaceutically acceptable salt thereof, wherein the bufonisin diene derivative has a structure shown in formula (I):
Figure BDA0002782011710000011
wherein A is- (CR)aRb)n-；
n is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R1is hydroxy, R2Is hydrogen, or R1And R2Together form an epoxy group;
R3is hydrogen or C1-6An alkyl acyloxy group;
R4、R5、R6and R7Each independently is: hydrogen, C1-6Alkyl, 3-8 membered cycloalkyl or 5-8 membered heterocycloalkyl; or R4And R6Together form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl; or R4And R7Taken together to form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl group; or R5And R6Together form a substituted or unsubstituted 5-8 membered cycloalkyl group; or R5And R7Taken together to form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl group;
Raand RbEach independently is: hydrogen, C1-6Alkyl or 3-8 membered cycloalkyl.
The preparation method of the bufonis diene derivative comprises the following steps:
Figure BDA0002782011710000021
a compound of the formula (I-1) and
Figure BDA0002782011710000022
carrying out reaction to obtain the compound shown in the formula (I-3);
carrying out condensation reaction on the compound shown in the formula (I-3) and the compound shown in the formula (I-4) to obtain the compound shown in the formula (I).
A pharmaceutical composition comprising
1) At least one of the above-mentioned bufogenin derivatives and pharmaceutically acceptable salts thereof,
2) at least one of a pharmaceutically acceptable carrier, excipient, adjuvant and diluent.
The application of the bufogenin derivative or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing the medicines for treating or preventing tumors.
The invention creatively utilizes the combination strategy of the antitumor activity and the hydrophilic kushenic acid of the bufonis diene and links the two by a linker to obtain a series of bufonis diene derivatives with novel structures, thereby improving the water solubility, maintaining the activity and reducing the toxicity. Therefore, the bufogenin diene derivative can solve the problem that the bufogenin diene derivative is toxic and drug-like, and has good application prospect.
Detailed Description
In order that the invention may be more fully understood, a more complete description of the invention, and a preferred embodiment of the invention, is now provided. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Interpretation of terms
Unless otherwise indicated, the terms used in the present invention have the following definitions:
when stereoisomers of the compounds exist in the invention, the compounds are understood to include R configuration, S configuration and racemate when not particularly indicated.
As used herein, "substituted" means substituted with one or more groups. When a plurality of groups are selected from the same series of candidate substituents, they may be the same or different.
The term "optionally" as used herein means that the defined group may or may not be selected from a list of candidate groups.
The term "substituted or unsubstituted" as used herein means that the defined group may or may not be substituted. When a defined group is substituted, it is understood to be optionally substituted with art-acceptable groups including, but not limited to: c1-6Alkyl, cycloalkyl containing 3 to 8 ring atoms, heterocycloalkyl containing 4 to 9 ring atoms, silyl, carbonyl, hydroxy or-NRR', and the above groups may also be further substituted by art-acceptable substituents; it is understood that R and R 'in-NRR' are each independently substituted with art-acceptable groups including, but not limited to, H, C1-6Alkyl, and R 'may form together with the N to which R, R' is attached a 5-10 membered heterocycloalkyl; said C is1-6Alkyl, cycloalkyl containing 3 to 8 ring atoms, heterocycloalkyl containing 4 to 9 ring atoms, optionally further substituted with one or more of the following groups: c1-6Alkyl, cycloalkyl having 3 to 8 ring atoms, heterocyclyl having 3 to 8 ring atoms, halogen, hydroxy, nitro or amino.
In the inventionThe term "alkyl" denotes saturated straight and branched chain alkyl groups of a specified number of atoms, C1-6Alkyl refers to an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl.
"cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbyl substituent. 3-8 membered cycloalkyl is meant to include 3 to 8 carbon atoms. Non-limiting examples include: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like.
"Nitrogen-containing heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbyl substituent containing at least one nitrogen atom. The 5-8 membered nitrogen-containing heterocycloalkyl group is meant to include 5 to 8 atoms (the sum of carbon atoms and nitrogen atoms). Non-limiting examples include: tetrahydropyrrolyl, tetrahydropyrimidinyl, tetrahydropyrazolyl, piperidyl, piperazinyl, hexahydropyrimidine, hexahydropyridazine, cyclohexylimino, 1, 2-homopiperazinyl, 1, 3-homopiperazinyl, 1, 4-homopiperazinyl, 1, 2-diazacarbyl, 1, 3-diazacarbyl, 1, 4-diazacarbyl, 1, 5-diazacarbyl, tetrahydropyridinyl and the like.
"pharmaceutically acceptable salt" refers to a salt of any compound of the indicated structure with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One class of salts is the salts of the compounds of the invention with acids. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc. Another class of salts is that formed from the compounds of the present invention and bases suitable for forming salts include, but are not limited to: alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts, and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.
Application method
The bufonisin diene derivative obtained by the invention can be used for administration to human, and can be used for oral administration, rectal administration, parenteral (intravenous, intramuscular or subcutaneous), bladder perfusion, and local administration (powder, ointment or drop).
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.
In addition to these inert diluents, the above compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
Compositions for parenteral injection or bladder perfusion may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
Dosage forms for topical administration of the compounds of the present invention include ointments, powders, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.
Detailed explanation
One embodiment of the present invention provides a bufonisin diene derivative or a pharmaceutically acceptable salt thereof, having a structure represented by formula (I):
Figure BDA0002782011710000051
wherein, A is- (CR)aRb)n-；
n is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R1is hydroxy, R2Is hydrogen, or R1And R2Together form an epoxy group;
R3is hydrogen or C1-6An alkyl acyloxy group;
R4、R5、R6and R7Each independently is: hydrogen, C1-6Alkyl, 3-8 membered cycloalkyl or 5-8 membered heterocycloalkyl; or R4And R6Taken together to form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl group; or R4And R7Taken together to form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl group; or R5And R6Together form a substituted or unsubstituted 5-8 membered cycloalkyl group; or R5And R7Taken together to form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl group;
Raand RbEach independently is: hydrogen, C1-6Alkyl or 3-8 membered cycloalkyl.
In one embodiment, R3Is hydrogen or C1-4An alkyl acyloxy group; further, R3Is hydrogen or acetoxy;
in one embodiment, R1And R2Together form an epoxy group, R3Is acetoxy.
In one embodiment, R1Is hydroxy, R2And R3Are all hydrogen.
In one embodiment, RaAnd RbEach independently is: hydrogen, C1-4Alkyl or 3-6 membered cycloalkyl.
In one embodiment, A is- (CR)aRb)n-; further, RaAnd RbIs H; further, n is 0, 1,2, 3, 4 or 5. It is understood that when n is 0, meaning that A is absent, formula (I) has the following structure:
Figure BDA0002782011710000061
further, R4、R5、R6And R7Each independently is: hydrogen, C1-4Alkyl, 3-6 membered cycloalkyl or 5-6 membered heterocycloalkyl; or R4And R6Taken together to form a substituted or unsubstituted 5, 6 or 7 membered nitrogen containing heterocycloalkyl; or R4And R7Taken together to form a substituted or unsubstituted 5, 6 or 7 membered nitrogen containing heterocycloalkyl; or R5And R6Together form a substituted or unsubstituted 5, 6 or 7 membered cycloalkyl group; or R5And R7Together form a substituted or unsubstituted 5, 6 or 7 membered nitrogen containing heterocycloalkyl.
In one embodiment, R4And R7Is H; in one embodiment, R5And R6Is H; in one embodiment, R4And R6Taken together to form a substituted or unsubstituted 6-membered nitrogen-containing heterocycloalkyl group; in one embodiment, R4And R7Taken together to form a substituted or unsubstituted 6-membered nitrogen-containing heterocycloalkyl group; in one embodiment, R5And R7Taken together to form a substituted or unsubstituted 6-membered nitrogen-containing heterocycloalkyl group;
further, the 5-8 membered cycloalkyl or said 5-8 membered nitrogen containing heterocycloalkyl is further substituted with at least one R selected from: -H, C1-6Alkyl, 3-8 membered cycloalkyl, hydroxy, carboxy, carbonyl or oxo.
Further, the nitrogen-containing heterocycloalkyl group is selected from: piperidinyl, piperazinyl, hexahydropyrimidine, hexahydropyridazine, 1, 2-homopiperazinyl, 1, 3-homopiperazinyl, or 1, 4-homopiperazinyl;
further, cycloalkyl is selected from: cyclopentyl or cyclohexyl;
further, the bufonis diene derivative has a structure shown in a formula (II) or a formula (III):
Figure BDA0002782011710000071
Reselected from H, C1-6Alkyl or 3-8 membered cycloalkyl;
p is 1,2, 3, 4, 5, 6, 7 or 8.
Further, the bufogenin derivative is selected from any one of the following compounds:
Figure BDA0002782011710000072
Figure BDA0002782011710000081
further, the pharmaceutically acceptable salt of the bufonis diene derivative is an organic salt; further, the pharmaceutically acceptable salt of the above-mentioned bufogenin derivative is an oxalate, malonate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, or picrate of the bufogenin derivative; further, the pharmaceutically acceptable salt of the above-mentioned bufogenin derivative is a tartrate salt of a bufogenin derivative.
The invention also provides a preparation method of the bufonis diene derivative, which comprises the following steps:
s101. the compound of formula (I-1) and
Figure BDA0002782011710000082
carrying out reaction to obtain the compound shown in the formula (I-3);
Figure BDA0002782011710000091
further, it is preferable that the step S101 includes the steps of:
s1011, reacting the compound shown in the formula (I-1) with 4-nitro phenyl chloroformate to obtain a compound shown in a formula (I-2);
Figure BDA0002782011710000092
further, step S1011 includes the steps of: mixing a compound with a structure shown in a formula (I-1), 4-nitrophenyl chloroformate, alkali and a solvent, reacting, and carrying out aftertreatment to obtain a compound shown in a formula (I-2);
still further, the base is one or more of triethylamine, pyridine, diisopropylethylamine, and 4- (N, N-dimethyl) aminopyridine; further, the base is pyridine;
still further, the solvent is selected from: one or more of dichloromethane, chloroform, tetrahydrofuran, and acetonitrile; further, the solvent is dichloromethane;
furthermore, the reaction temperature is 15-45 ℃;
furthermore, the post-processing method comprises the following steps: after the reaction is finished, repeatedly washing the mixture by using saturated sodium carbonate or saturated potassium carbonate solution, then washing the mixture by using saturated saline solution, drying and concentrating.
S1012: a compound of the formula (I-2) and
Figure BDA0002782011710000093
carrying out reaction to obtain the compound shown in the formula (I-3);
Figure BDA0002782011710000094
further, step S1012 includes the steps of: a compound shown in the formula (I-2),
Figure BDA0002782011710000101
Mixing alkali and solvent, and reactingAfter the reaction is finished, carrying out post-treatment to obtain the compound shown in the formula (I-3);
still further, the base is one or more of triethylamine, pyridine, diisopropylethylamine, and 4- (N, N-dimethyl) aminopyridine; further, the base is triethylamine;
still further, the solvent is selected from: one or more of dichloromethane, chloroform, tetrahydrofuran, and acetonitrile; still further, the solvent is dichloromethane.
S102: carrying out condensation reaction on the compound shown in the formula (I-3) and the compound shown in the formula (I-4) to obtain the compound shown in the formula (I).
Figure BDA0002782011710000102
Further, in step S102, after protecting the N atom in the compound of formula (I-3), a subsequent reaction may be performed, and NH protecting groups commonly used in the art may be used, including but not limited to: -Boc, -Cbz, -Ac, -Ts, -Ms, -Bz, -Bn, -PMB, or schiff base; preferably, the method adopts Boc, so that the yield is higher, and the reaction condition is mild;
further, step S102 includes the steps of:
s1021, preparing the compound shown in the formula (I-4') by carrying out Boc protecting group on the compound shown in the formula (I-4);
Figure BDA0002782011710000103
it is understood that the Boc protecting group-containing starting material is commercially available directly, in which case step S1021 may be omitted, and should not be construed as limiting the invention.
S1022: reacting the compound of formula (I-3) with the compound of formula (I-4') to produce the compound of formula (I');
Figure BDA0002782011710000111
further, step S1022 includes the steps of: mixing a compound with a structure shown in a formula (I-3) and a compound with a structure shown in a formula (I-4'), a condensation reagent, alkali and a solvent, and reacting to obtain a compound shown in a formula (I');
further, the condensation reagent is one or more of EDCI, DCC and HOBt/EDCI; still further, the condensing agent is EDCI;
further, the base is one or more of triethylamine, pyridine, diisopropylethylamine and 4- (N, N-dimethyl) aminopyridine; further, the base is triethylamine
Further, the solvent is selected from: one or more of dichloromethane, chloroform, tetrahydrofuran, and acetonitrile; further, the solvent is dichloromethane;
further, the temperature is 15-45 ℃;
s1023: removing the Boc protecting group of the compound of formula (I') to produce the compound of formula (I);
Figure BDA0002782011710000112
further, step S1023 includes the steps of: mixing a structural compound shown in a formula (I'), acid and a solvent for reaction;
further, the acid is one or more of hydrochloric acid, sulfuric acid, p-toluenesulfonic acid and trifluoroacetic acid; further, the acids are p-toluenesulfonic acid and trifluoroacetic acid; still further, the acid is p-toluenesulfonic acid.
Further, the reaction temperature is 15-45 ℃;
the invention also provides a pharmaceutical composition comprising
1) At least one of the above-mentioned bufogenin derivatives and pharmaceutically acceptable salts thereof;
2) at least one of a pharmaceutically acceptable carrier, excipient, adjuvant and diluent.
The invention also provides the application of the bufonis diene derivative or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing medicaments for treating or preventing tumors.
Further, the tumor is lung cancer, cervical cancer, liver cancer, breast cancer, leukemia, pancreatic cancer, gastric cancer, esophageal cancer, rectal cancer, bladder cancer or renal cancer.
The present invention also provides a method for treating or preventing tumors, which comprises administering a therapeutically effective amount of the above-mentioned bufogenin derivative or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition.
Preparation examples
The invention will be further illustrated with reference to the following specific examples, but the invention is not limited thereto.
In the following preparation examples, the following examples were conducted,1H-NMR was measured with a Varian Mercury AMX 500 instrument. MS was determined with VG ZAB-HS or VG-7070 type and Esquire 3000 plus-01005. All solvents were redistilled before use and the anhydrous solvents used were dried according to standard procedures. All reactions were carried out under argon and followed by TLC unless otherwise stated, and worked up by washing with saturated brine and drying over anhydrous magnesium sulfate. Purification of the product all uses column chromatography on silica gel, 200-300 mesh, GF254Is produced by Qingdao oceanic chemical plants or Nicotin Bo silica gel company.
Example 1: synthesis of Compound A1
Figure BDA0002782011710000121
In a 50mL round-bottom flask, p-nitrophenyl chloroformate (1.206g, 6mmol) was dissolved in 20mL of anhydrous dichloromethane, dried pyridine (0.67mL) was added, bufalin (2mmol) was added with stirring, and the mixture was stirred at room temperature for 6 hours, and after completion of the reaction, the mixture was washed with saturated sodium carbonate 5 to 8 times, then with saturated brine 2 times, and dried over magnesium sulfate. After magnesium sulfate was removed by filtration, 10mL of anhydrous dichloromethane and triethylamine (0.5mL) were added, and ethylenediamine (4mmol) was added and the mixture was stirred at room temperature for 1 hour. Washing with saturated sodium carbonate 5-8 times, washing with saturated salt water 2 times, and drying with magnesium sulfate. After filtration, N-Boc sophora flavescens acid (1.46g, 4mmol), triethylamine (6mmol), EDCI (6mmol, 1.15g) were sequentially added, reacted at room temperature for 3 hours, washed with saturated sodium carbonate 3 times after the reaction, dried over anhydrous sodium sulfate, dissolved in methanol (20mL) after concentration under reduced pressure, added with p-toluenesulfonic acid (6mmol, 1.03mg), stirred at room temperature for 120 hours, and after TLC detection of complete reaction of the raw material, added with ethyl acetate (40mL), washed with saturated sodium carbonate 1 time, washed with water 2 times, washed with saturated brine 2 times, and dried over magnesium sulfate. After magnesium sulfate was removed by filtration and concentrated under reduced pressure, column chromatography was performed (DCM: MeOH: Et)3N30: 1:0.3, v/v/v) to give white powder a1(534mg, 37%).1H NMR(500MHz,DMSO-d6)δ7.95(d,J＝9.4Hz,1H),7.89(s,1H),7.53(s,1H),7.04(t,J＝5.6Hz,1H),6.30(d,J＝9.7Hz,1H),4.83(s,1H),4.27–4.20(m,1H),3.19–2.96(m,6H),2.71(dd,J＝22.4,10.6Hz,2H),2.55(dd,J＝11.9,3.4Hz,1H),2.47(t,J＝7.0Hz,1H),2.09–1.99(m,4H),1.91(t,J＝13.8Hz,1H),1.85–1.74(m,6H),1.69–1.54(m,7H),1.56–1.45(m,7H),1.44–1.27(m,9H),1.26–1.02(m,7H),0.91(s,3H),0.61(s,3H)；ESI-MS(m/z)721.6[M+1]+。
Example 2: synthesis of Compound A2
Figure BDA0002782011710000131
The procedure was as in the preparation of A1, the starting material being propylenediamine instead of ethylenediamine; by column chromatography (DCM: MeOH: Et)3Gradient elution with N ═ 30:1:0.3, v/v/v) afforded the title compound a2(588mg, 40%).1H NMR(500MHz,DMSO-d6)δ7.93(dd,J＝9.8,2.4Hz,1H),7.78(t,J＝5.3Hz,1H),7.57–7.50(m,1H),6.95(t,J＝5.5Hz,1H),6.28(d,J＝9.7Hz,1H),4.80(s,1H),3.44(s,4H),3.11–2.87(m,6H),2.68(dd,J＝20.0,11.0Hz,2H),2.50–2.39(m,2H),2.07–1.96(m,4H),1.89(t,J＝13.1Hz,1H),1.83–1.72(m,5H),1.64–1.54(m,7H),1.53–1.44(m,8H),1.43–1.37(m,2H),1.35–1.21(m,7H),1.21–1.01(m,6H),0.88(s,3H),0.59(s,3H)；ESI-MS(m/z)735.6[M+1]+。
Example 3: synthesis of Compound A3
Figure BDA0002782011710000132
The preparation of A1 was carried out, for example, by substituting butanediamine for ethylenediamine; by column chromatography (DCM: MeOH: Et)3Gradient elution with N ═ 30:1:0.3, v/v/v) gave the title compound a3(524mg, 35%).1H NMR(500MHz,DMSO-d6)δ7.95(dd,J＝9.7,2.6Hz,1H),7.82(t,J＝5.6Hz,1H),7.53(d,J＝2.4Hz,1H),6.98(t,J＝5.5Hz,1H),6.30(d,J＝9.7Hz,1H),4.86–4.77(m,1H),4.18(s,1H),3.18–3.06(m,3H),3.06–2.91(m,5H),2.76–2.67(m,2H),2.64(dd,J＝12.1,4.1Hz,1H),2.49–2.45(m,1H),2.09–1.98(m,5H),1.96–1.85(m,1H),1.84(dd,J＝11.8,2.9Hz,1H),1.82–1.75(m,4H),1.64–1.42(m,16H),1.39–1.30(m,10H),1.28–1.23(m,6H),0.89(s,3H),0.61(s,3H)；ESI-MS(m/z)749.6[M+1]+。
Example 4: synthesis of Compound A4
Figure BDA0002782011710000141
The procedure was as for the preparation of A1, starting from 4-aminopiperidine instead of ethylenediamine; by column chromatography (DCM: MeOH: Et)3Gradient elution with N ═ 30:1:0.3, v/v/v) afforded target compound a4(700mg, 46%).1H NMR(500MHz,DMSO-d6)δ7.94(dd,J＝9.8,2.4Hz,1H),7.75(d,J＝7.7Hz,1H),7.52(d,J＝1.8Hz,1H),6.29(d,J＝9.7Hz,1H),4.85(s,1H),4.16(s,1H),3.87(d,J＝13.1Hz,2H),3.78–3.70(m,1H),3.02–2.98(m,1H),2.92(t,J＝8.3Hz,2H),2.68(dd,J＝21.3,11.0Hz,2H),2.48–2.38(m,3H),2.13–1.99(m,4H),1.98–1.93(m,2H),1.84–1.73(m,6H),1.65–1.56(m,7H),1.56–1.43(m,8H),1.43–1.29(m,6H),1.28–1.15(m,9H),1.15–1.02(m,3H),0.89(s,3H),0.60(s,3H)；ESI-MS(m/z)761.6[M+1]+。
Example 5: synthesis of Compound A5
Figure BDA0002782011710000142
The preparation method comprises the following steps of preparing A1, substituting cinobufagin for bufalin, substituting 4-aminopiperidine for ethylenediamine, and substituting 5% trifluoroacetic acid in dichloromethane for p-toluenesulfonic acid during removal of Boc; by column chromatography (DCM: MeOH: Et)3Gradient elution with N ═ 30:1:0.3, v/v/v) afforded target compound a5(490mg, 30%).1H NMR(500MHz,DMSO-d6)δ9.19(s,2H),8.02(s,1H),7.87(d,J＝9.9Hz,1H),7.49(d,J＝2.5Hz,1H),6.25(d,J＝9.8Hz,1H),5.49(d,J＝9.2Hz,1H),4.85(s,1H),3.96–3.82(m,2H),3.81–3.67(m,2H),3.23–3.14(m,1H),3.00–2.64(m,6H),2.18–2.04(m,3H),2.02–1.92(m,2H),1.83(s,3H),1.81–1.75(m,3H),1.76–1.61(m,7H),1.61–1.45(m,11H),1.43–1.29(m,6H),1.29–1.10(m,7H),1.06–0.99(m,1H),0.94(s,3H),0.72(s,3H)；ESI-MS(m/z)817.6[M+1]+。
Example 6: preparation of Compound A4 tartrate
Figure BDA0002782011710000151
A4(1mmol) was dissolved in 30mL ethanol, tartaric acid (150mg, 1mmol) was added, the mixture was dissolved at room temperature with stirring and clarified, and the mixture was concentrated under reduced pressure to give A4 tartrate as a white solid.
Both organic and inorganic acid salts of the compounds mentioned in the present invention can be prepared in a similar manner.
Test example 1: in vitro antitumor Activity test
(1) Test materials
Human lung cancer cell A549, human cervical cancer cell HeLa, human liver cancer cell Hep3B, human breast cancer cell MCF-7, human acute lymphoblastic leukemia T lymphocyte CCRF-CEM, human leukemia cell MV-4-11, adenocarcinoma cell PANC-1, human liver cancer cell HepG2 and human liver cancer cell BEL-7402.
The positive control is vinorelbine (vinorelbine) (prepared by a conventional method); the purity is more than 98 percent by HPLC-UV detection. The test compound and the positive control were diluted with physiological saline at a concentration gradient of 300nM, 100nM, 30nM, 10nM, 3.0nM, 1.0nM, 0.3nM, 0.1 nM.
(2) Test method
SRB reduction method:
depending on the cell growth rate, tumor cells in logarithmic growth phase were seeded at 100. mu.L/well in 96-well plates and allowed to grow adherently for 24h, followed by 10. mu.L/well of test compound or positive control. Three multiple holes are arranged for each concentration. And a normal saline solvent control with corresponding concentration and a cell-free zeroing hole are arranged. Tumor cells were incubated at 37 ℃ with 5% CO2Culturing for 72h, pouring out culture solution (RPMI-1640), fixing cells with 10% cold TCA, standing at 4 deg.C for 1h, washing with distilled water for 5 times, and air drying. Then 100. mu.L/well of a 4mg/mL solution of SRB (Sigma) in 1% glacial acetic acid was added, stained for 15 minutes at room temperature, the supernatant removed, washed 5 times with 1% acetic acid and air dried. And finally adding 150 mu L/hole Tris solution, and measuring the value A under the wavelength of 515nm by using a microplate reader. The inhibition rate of tumor cell growth was calculated according to the following formula:
inhibition%
The action concentration of the medicine is as follows: 300nM, 100nM, 30nM, 10nM, 3.0nM, 1.0nM, 0.3nM, 0.1 nM. IC was fitted with GraphPad Prism 450。
TABLE 1 inhibitory Activity of Compound A1-5 on A549, HeLa and Hep3B tumor cells
Figure BDA0002782011710000161
The study on the cell proliferation inhibition activity of the bufogenin derivative A1-A5 on three cell lines shows that the compound of the invention basically maintains the antitumor activity, wherein the activity of the A4 compound is optimal (see Table 1). The A4 strain and 6 human tumor cell lines were selected, and all showed good inhibitory activity (see Table 2).
TABLE 2 inhibitory Activity of Compound A4 on various human tumor cells
Figure BDA0002782011710000162
Compared with the prototype bufalin, the compound provided by the invention has the advantages that the activity is maintained, the toxicity is obviously reduced, and the water solubility is obviously improved. LD of bufalin502.2mg/kg LD of A4 tartrate5050 mg/kg. The water solubility of bufalin is less than 1 mug/mL, the water solubility of A4 tartrate is more than 20mg/mL, and is improved by more than 20000 times.
Comparative experiment
Figure BDA0002782011710000171
The product B, acetylated at the single end, had no antitumor activity, indicating that acylation would render the compound inactive. Meanwhile, the product C acylated with Boc-protected matrinic acid, although it also retained a nitrogen-containing water-soluble group, was also devoid of antitumor activity.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A bufonisin diene derivative or a pharmaceutically acceptable salt thereof, wherein the bufonisin diene derivative has a structure represented by formula (I):
Figure FDA0002782011700000011
wherein A is- (CR)aRb)n-；
n is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R1is hydroxy, R2Is hydrogen, or R1And R2Together form an epoxy group;
R3is hydrogen or C1-6An alkyl acyloxy group;
R4、R5、R6and R7Each independently is: hydrogen, C1-6Alkyl, 3-8 membered cycloalkyl or 5-8 membered heterocycloalkyl; or R4And R6Taken together to form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl group; or R4And R7Taken together to form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl group; or R5And R6Together form a substituted or unsubstituted 5-8 membered cycloalkyl group; or R5And R7Together form a substituted or unsubstituted 5-8 membered nitrogen containing heterocycloalkyl;
Raand RbEach independently is: hydrogen, C1-6Alkyl or 3-8 membered cycloalkyl.
2. The bufonisin diene derivative or the pharmaceutically acceptable salt thereof according to claim 1, wherein said 5-to 8-membered cycloalkyl group or said 5-to 8-membered nitrogen-containing heterocycloalkyl group is further substituted with at least one R selected from the group consisting of: -H, C1-6Alkyl, 3-8 membered cycloalkyl, hydroxy, carboxy, carbonyl or oxo.
3. The bufonisin diene derivative or the pharmaceutically acceptable salt thereof according to claim 1, wherein the 5-to 8-membered nitrogen-containing heterocycloalkyl group is selected from: piperidinyl, piperazinyl, hexahydropyrimidine, hexahydropyridazine, 1, 2-homopiperazinyl, 1, 3-homopiperazinyl, or 1, 4-homopiperazinyl.
4. The bufonisin diene derivative according to claim 1, which has a structure represented by formula (II) or formula (III):
Figure FDA0002782011700000021
Reselected from H, C1-6Alkyl or 3-8 membered cycloalkyl;
p is 1,2, 3, 4, 5, 6, 7 or 8.
5. The bufonisin diene derivative or the pharmaceutically acceptable salt thereof according to claim 1, which is selected from the group consisting of:
Figure FDA0002782011700000022
Figure FDA0002782011700000031
6. the process for producing a bufogenin derivative according to any one of claims 1 to 5, comprising the steps of:
Figure FDA0002782011700000032
a compound of the formula (I-1) and
Figure FDA0002782011700000041
carrying out reaction to obtain the compound shown in the formula (I-3);
carrying out condensation reaction on the compound shown in the formula (I-3) and the compound shown in the formula (I-4) to obtain the compound shown in the formula (I).
7. The process according to claim 6, wherein the compound of formula (I-1)A compound and
Figure FDA0002782011700000042
the step of performing the reaction comprises the steps of:
Figure FDA0002782011700000043
reacting the compound shown in the formula (I-1) with 4-nitrophenyl chloroformate to obtain a compound shown in a formula (I-2);
a compound of the formula (I-2) and
Figure FDA0002782011700000044
carrying out reaction to obtain the compound shown in the formula (I-3); and/or
The step of subjecting the compound represented by the formula (I-3) and the compound represented by the formula (I-4) to condensation reaction comprises the steps of:
and (3) carrying out a condensation reaction on the protective group on the N atom of the compound shown in the formula (I-4) and the compound shown in the formula (I-3) to remove the protective group, thus preparing the compound shown in the formula (I).
8. A pharmaceutical composition, comprising:
1) at least one of the bufogenin derivatives and pharmaceutically acceptable salts thereof according to any one of claims 1 to 5;
2) at least one of a pharmaceutically acceptable carrier, excipient, adjuvant and diluent.
9. Use of the bufogenin derivative or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, or the pharmaceutical composition according to claim 8 for the preparation of a medicament for the treatment or prevention of a tumor.
10. The use according to claim 9, wherein the tumor is lung cancer, cervical cancer, liver cancer, breast cancer, blood cancer, pancreatic cancer, stomach cancer, esophageal cancer, rectal cancer, bladder cancer or kidney cancer.
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CN110483608A (en) * 2018-05-15 2019-11-22 中国科学院上海药物研究所 Arenobufagin derivative and preparation method thereof, the composition comprising the derivative, and application thereof
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