CN109021058B - Ocotillol type sapogenin derivative with tumor drug resistance reversal activity and preparation method and application thereof - Google Patents

Ocotillol type sapogenin derivative with tumor drug resistance reversal activity and preparation method and application thereof Download PDF

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CN109021058B
CN109021058B CN201811065082.2A CN201811065082A CN109021058B CN 109021058 B CN109021058 B CN 109021058B CN 201811065082 A CN201811065082 A CN 201811065082A CN 109021058 B CN109021058 B CN 109021058B
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dammarane
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ocotillol
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杨刚强
王洪波
田京伟
任倩文
房效娟
唐晗晗
郭梦琦
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Yantai University
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Abstract

The invention relates to ocotillol type sapogenin derivatives, a pharmaceutical composition containing the ocotillol type sapogenin derivatives, a preparation method of the pharmaceutical composition and application of the pharmaceutical composition in reversing drug resistance of tumors. The ocotillol type sapogenin derivative is obtained on a tumor drug-resistant oral epidermal cancer cell line, and the prepared ocotillol type sapogenin derivative has better drug-resistant reversal activity and is obviously better than the ocotillol type sapogenin. Meanwhile, the product of the invention has good gastrointestinal stability and better drug applicability, which is a characteristic that the compounds disclosed in the prior art do not have.

Description

Ocotillol type sapogenin derivative with tumor drug resistance reversal activity and preparation method and application thereof
Technical Field
The invention relates to the field of organic synthesis and pharmaceutical chemistry, in particular to ocotillol type sapogenin derivatives, a pharmaceutical composition containing the ocotillol type sapogenin derivatives, a preparation method of the pharmaceutical composition and application of the ocotillol type sapogenin derivatives in tumor drug resistance reversal.
Background
Tumor therapy is a worldwide problem, and the emergence of new antitumor drugs makes it promising, however, the concomitant Multidrug Resistance (MDR) of tumor drugs is the biggest obstacle to tumor therapy. The generation of tumor MDR leads the antitumor drug to be unable to stay in the tumor cell, which leads the tumor cell to lose the treatment effect, thus causing the failure of the continuous and effective treatment of the tumor patient, and providing a serious challenge for the current tumor treatment and the drug development thereof. Therefore, the development of novel compounds with brand new structures, low toxicity and high MDR reversal activity is a hotspot of tumor treatment and drug research thereof.
The natural product has the characteristics of various structural types, wide human contact, small toxic and side effects and the like. Ginsenoside is used as effective active component in Ginseng radix, and has wide pharmacological activity and biological effect, including antiinflammatory, immunity regulating, antibacterial, antitumor and antiaging effects. The Ocotillol-type ginsenosides were first found in lichen as a rare saponin of the group of ginsenosides. In recent years, activities of different derivatives of the compounds in resisting myocardial ischemia-reperfusion injury, bacteriostasis and the like have been discovered gradually.
Disclosure of Invention
In order to solve the technical problems, the invention provides a drug with tumor drug resistance reversal activity, and provides ocotillol type sapogenin derivatives and pharmaceutically acceptable salts thereof, which have good tumor drug resistance reversal activity, and also provides a preparation method and application of the derivatives.
The invention aims to solve the technical problem of finding a compound with a new structure type and excellent tumor drug resistance reversal activity, and further providing a pharmaceutical composition which is used for treating gastric cancer, lung cancer, cervical cancer, breast cancer or colon cancer and the like together with a clinical common antitumor drug.
In order to solve the technical problems, the invention provides the following technical scheme:
ocotillol type sapogenin derivatives represented by general formula (I) or (II) and pharmaceutically acceptable salts thereof,
Figure BDA0001797659160000011
wherein R represents (C4-C8) straight chain or branched chain alkyl, R1NHR2、R1O R3、R1(O)CO R3Phenyl, naphthyl, pyrazine rings;
R1represents (C1-C8) straight-chain alkyl or ethoxy chain, CH (NH R)2)R4Phenyl, R2N-tetrahydropyrrole-2-yl;
R2represents hydrogen, Boc, Fmoc;
R3represents hydrogen, t-Bu, benzyl;
R4represents a protecting group and an amino acid side chain group without a protecting group, wherein the protecting group can be Boc, Fmoc, t-Bu, benzyl, methyl and (or) ethyl;
preferably, some of the compounds of the present invention are:
(20S,24R) -epoxy-3 β -N- (2-Boc-aminoacetyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (4-Boc-aminobutyryl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (6-Boc-aminocaproyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (8-Boc-aminooctanoyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (2-aminoacetyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (8-aminocapryl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -hexanoylaminodamane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -octanoylaminodamane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -benzoylaminodammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -nicotinamidodammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β - (4-Boc-aminobenzamidoyl) dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β - (2-hydroxybenzamido) dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- (2-Boc-aminoacetyl) -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- (4-Boc-aminobutyryl) -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- (6-Boc-aminocaproyl) -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- (8-Boc-aminocapryl) -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -hexanoylaminodamane-12 β, 25-diol;
(20S,24S) -epoxy-3 β - (2-hydroxybenzamido) dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (N' -Boc-prolyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -prolylamido dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (N' -Boc-lysyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (N' -Boc-aspartyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (N' -Boc-tyrosyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- { 2- [ 2- (2-Fmoc-aminoethoxy) ethoxy ] acetyl } -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- { 2- [ 2- (2-Fmoc-aminoethoxy) ethoxy ] acetyl } -dammarane-12 β, 25-diol;
the ocotillol type sapogenin derivative and the optical isomer of the compound or the pharmaceutically acceptable solvate thereof.
Compared with ocotillol type sapogenin, the ocotillol type sapogenin derivative shown in the general formulas (I) and (II) and the medically acceptable salt thereof have better tumor drug resistance reversing effect. For example, the IC50 of clinical commonly used drug paclitaxel against tumor-resistant cell strain KBV is 1353.98 ± 303.33nM, and in example 3, under the background of no toxicity to tumor-resistant cell KBV, when the 10 μ M concentration is used together with paclitaxel, the IC50 of paclitaxel is 28.29 ± 2.90nM, and the reversal multiple reaches 48 times, so that the sensitivity of tumor-resistant cell KBV against paclitaxel is significantly improved, and paclitaxel can still generate good antitumor activity against it at very low concentration.
The invention relates to ocotillol type sapogenin derivatives with general formulas (I) and (II) and the application of the ocotillol type sapogenin derivatives in preparing tumor drug resistance reversal agents and/or pharmaceutically acceptable carriers for treating animal diseases or symptoms, preferably human diseases or symptoms.
The ocotillol type sapogenin derivatives with effective dose of general formulas (I) and (II), and the pharmaceutically acceptable salts and the pharmaceutically acceptable carriers thereof are used for being combined with clinical antitumor drugs for treating diseases or symptoms such as gastric cancer, lung cancer, cervical cancer, breast cancer or colon cancer.
Ocotillol-type sapogenin derivatives of general formulae (I) and (II) are prepared synthetically according to the following reaction scheme and description,
Figure BDA0001797659160000031
the method comprises the steps of taking protopanoxadiol as a raw material, carrying out double bond epoxidation and intramolecular nucleophilic attack to prepare (20S,24R) -epoxy dammar -3 beta, 12 beta, 25-triol and (20S,24S) -epoxy dammar -3 beta, 12 beta, 25-triol, oxidizing a 3-hydroxy group to produce ketone, carrying out oximation and stereoselective reduction to produce amine, carrying out an amide reaction to prepare derivatives of general formulas (I) and (II) which do not contain a protecting group and contain a protecting group, and finally treating the derivatives of general formulas (I) and (II) which contain the protecting group by trifluoroacetic acid or hydrochloric acid to prepare the derivatives of general formulas (I) and (II) which are deprotected.
The pharmaceutically acceptable salts of the compounds of the present invention refer to conventional acid addition salts which have the same pharmaceutical efficacy as the compounds and are salts with suitable non-toxic organic or inorganic acids.
The invention also discloses a pharmaceutical composition, which can be added with pharmaceutically acceptable carriers to prepare common pharmaceutical preparations such as tablets, capsules, powder, syrup, liquid, suspending agents and injection, and can be added with common pharmaceutical excipients such as spices, sweeteners, liquid or solid fillers or diluents and the like.
The clinical administration mode of the compound of the invention can adopt oral administration, injection and other modes.
The clinical dosage of the compound of the invention is 0.01 mg-1000 mg/day, and the dosage can deviate from the range according to the severity of the disease condition or different dosage forms.
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FIG. 1 is a graphical representation of the effect of example 3 on the inhibition of KBV cell activity in vivo of paclitaxel.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to these examples.
Example 1
(20S,24R) -epoxy-3 β -N- (2-Boc-aminoacetyl) -dammarane-12 β, 25-diol;
20S-Protopanaxadiol (8.000g,17.36mmol) was dissolved in dichloromethane (160mL), m-CPBA (4.490g, 19.51mmol) was added, and the mixture was stirred at room temperature for 3 h. Diluting with chloroform, washing with water, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography to obtain white solid compound 1[ (20S,24R) -epoxydammara -3 β,12 β, 25-triol ] (5.184g,10.87mmol, 63%) and white solid compound 2[ (20S,24S) -epoxydammara -3 β,12 β, 25-triol ] (3.060g,6.42mmol, 37%).
The compound 1 is a compound of formula (I),1H NMR(400MHz,CDCl3)δ3.84(dd,J＝8.8,6.8Hz,1H),3.51(td,J＝10.5,4.6Hz,1H),3.18(dt,J＝9.9,4.5Hz,1H),2.19(td,J＝10.9,3.6Hz,1H),1.28(s,3H),1.27(s,3H),1.14–0.96(m,3H),1.09(s,3H),0.98(s,3H),0.97(s,3H),0.90(s,3H),0.85(s,3H),0.77(s,3H).
the compound (2) is a compound which has a structure shown in the specification,1H NMR(400MHz,CDCl3)δ5.78(s,1H),3.88(dd,J＝10.7,5.3Hz,1H),3.52(td,J＝10.3,4.7Hz,1H),3.20(dd,J＝11.3,4.8Hz,1H),2.25(td,J＝10.5,4.3Hz,1H),1.27(s,3H),1.23(s,3H),1.11(s,3H),1.01(s,3H),0.97(s,3H),0.91(s,3H),0.88(s,3H),0.78(s,3H).
dissolving compound 1(821mg, 1.72mmol) in dichloromethane (20mL), adding PCC (pyridinium chlorochromate, 389mg, 1.80mmol) on ice bath, slowly raising to room temperature, stirring for 15 hours, filtering, concentrating, and performing column chromatography to obtain white solid compound 3[ (20S,24R) -epoxy dammara -12 beta, 25-diol-3-one -12 beta](689mg,1.45mmol,84％)。1H NMR(400MHz,CDCl3)δ3.86(dd,J＝8.8,6.6Hz,1H),3.53(td,J＝10.5,4.5Hz,1H),2.52(ddd,J＝15.7,9.6,7.7Hz,1H),2.42(ddd,J＝15.7,7.7,4.4Hz,1H),2.21(td,J＝10.9,3.7Hz,1H),1.28(s,3H),1.27(s,3H),1.10(s,3H),1.08(s,3H),1.04(s,3H),1.02(s,3H),0.96(s,3H),0.91(s,3H)。
Dissolving compound 3(616mg, 1.295mmol) in pyridine, adding hydroxylamine hydrochloride (722mg,10.34mmol) at room temperature, reacting at 80 ℃ for 1 hour, concentrating, diluting with ethyl acetate, washing with water, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography to obtain white solid compound 4[ (20S,24R) -epoxy dammar -12 beta, 25-diol-3-ketoxime](590mg，1.205mmol，93％)。1H NMR(400MHz,CDCl3)δ5.61(s,1H),3.85(dd,J＝8.9,6.6Hz,1H),3.51(td,J＝10.5,4.6Hz,1H),2.99(ddd,J＝15.5,5.9,4.1Hz,1H),2.27(ddd,J＝15.5,11.4,5.9Hz,1H),2.19(td,J＝10.1,3.0Hz,1H),1.28(s,3H),1.27(s,3H),1.14(s,3H),1.10(s,3H),1.05(s,3H),1.01(s,3H),0.96(s,3H),0.88(s,3H).
Compound 4(800mg,1.63mmol), ammonium acetate (315mg,4.09mmol) and sodium cyanoborohydride (258mg,4.11mmol) were dissolved in isopropanol (45mL), and a 15% titanium trichloride solution (1.6mL) was slowly added dropwise under ice bath, and reacted at room temperature overnight. Adjusting pH of the reaction solution to 10 with sodium hydroxide solution, extracting with ethyl acetate, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and recrystallizing to obtain white solid 5[ (20S,24R) -epoxy dammara -12 beta, 25-diol-3 beta-amine](545mg，1.15mmol，90％)。1H NMR(400MHz,CDCl3)δ3.84(dd,J＝8.4,6.8Hz,1H),3.51(td,J＝10.4,4.3Hz,1H),2.85(dd,J＝10.6,5.8Hz,1H),2.18(td,J＝10.0,3.4Hz,1H),2.09-1.96(m,2H),1.27(s,3H),1.26(s,3H),1.13(s,3H),1.09(s,3H),0.98(s,3H),0.95(s,3H),0.89(s,3H),0.87(s,3H),0.78(d,J＝9.2Hz,1H).
Compound 5(20mg,0.042mmol) and N-Boc-2-aminoacetic acid (11mg,0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (24mg, 90%).1H NMR(400MHz,CDCl3)δ6.07(d,J＝9.6Hz,1H),3.85(dd,J＝8.9,6.6Hz,1H),3.78(dd,J＝16.5,5.9Hz,1H),3.71(dd,J＝16.5,5.9Hz,1H),3.67-3.60(m,1H),3.49(td,J＝10.5,4.6Hz,1H),2.19(td,J＝10.1,3.2Hz,1H),1.45(s,9H),1.28(s,3H),1.27(s,3H),1.09(s,3H),0.98(s,3H),0.90(s,3H),0.86(s,3H),0.83(s,3H),0.76(s,3H)。
Example 2
(20S,24R) -epoxy-3 β -N- (4-Boc-aminobutyryl) -dammarane-12 β, 25-diol;
compound 5(20mg,0.042mmol) and N-Boc-4-aminobutyric acid (12mg,0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (27mg, 97%).1H NMR(400MHz,CDCl3)δ5.97(d,J＝9.6Hz,1H),4.80(t,J＝6.2Hz,1H),3.85(dd,J＝8.7,6.9Hz,1H),3.65(td,J＝10.7,5.0Hz,1H),3.52(td,J＝10.5,4.6Hz,1H),3.23-3.10(m,2H),2.23-2.16(3H),1.43(s,9H),1.28(s,3H),1.27(s,3H),1.09(s,3H),0.98(s,3H),0.90(s,3H),0.86(s,3H),0.85(s,3H),0.78(s,3H)。
Example 3
(20S,24R) -epoxy-3 β -N- (6-Boc-aminocaproyl) -dammarane-12 β, 25-diol;
compound 5(20mg,0.042mmol) and N-Boc-6-aminocaproic acid (14mg,0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (26mg, 91%).1H NMR(400MHz,CDCl3)δ5.44(d,J＝9.6Hz,1H),4.63(s,1H),3.85(dd,J＝8.9,6.6Hz,1H),3.66(ddd,J＝11.9,10.1,4.12Hz,1H),3.51(td,J＝10.5,4.6Hz,1H),3.13-3.07(m,2H),2.81(s,1H),2.22-2.16(m,3H),1.44(s,9H),1.27(s,3H),1.27(s,3H),1.09(s,3H),0.98(s,3H),0.90(s,3H),0.86(s,3H),0.84(s,3H),0.76(s,3H)。
Example 4
(20S,24R) -epoxy-3 β -N- (8-Boc-aminooctanoyl) -dammarane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and N-Boc-8-aminocaprylic acid (15mg,0.059mmol) were dissolved in anhydrous DMF (0.4mL), stirred and dissolved under argon protection, and HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20 μ L) were added at 0 ℃, reacted at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (26mg, 91%).1H NMR(400MHz,CDCl3)δ5.29(d,J＝10.1Hz,1H),4.53(s,1H),3.85(dd,J＝8.9,6.6Hz,1H),3.66(ddd,J＝12.3,10.1,4.6Hz,1H),3.52(td,J＝10.5,4.6Hz,1H),3.09(dd,J＝7.3,6.4Hz,2H),2.22-2.14(m,3H),1.44(s,9H),1.28(s,3H),1.27(s,3H),1.09(s,3H),0.98(s,3H),0.90(s,3H),0.86(s,3H),0.84(s,3H),0.76(s,3H)。
Example 5
(20S,24R) -epoxy-3 β -N- (2-aminoacetyl) -dammarane-12 β, 25-diol;
example 1(0.03mmol) was dissolved in anhydrous TFA (trifluoroacetic acid, 0.5mL) and reacted at room temperature for 10min, after which the reaction was terminated. Concentration gave the title compound (0.03 mmol).1H NMR(400MHz,CD3OD)δ3.89(t,J＝7.5Hz,1H),3.68(s,2H),3.62(dd,J＝12.6,3.9Hz,1H),3.49(td,J＝10.5,4.6Hz,1H),2.20(td,J＝10.9,3.2Hz,1H),1.27(s,3H),1.22(s,3H),1.13(s,3H),1.03(s,3H),0.95(s,3H),0.91(s,3H),0.86(s,3H),0.82(s,3H)。
Example 6
(20S,24R) -epoxy-3 β -N- (8-aminocapryl) -dammarane-12 β, 25-diol;
example 4(0.03mmol) was dissolved in anhydrous TFA (trifluoroacetic acid, 0.5mL) and reacted at room temperature for 10min, after which the reaction was terminated. Concentration gave the title compound (0.03 mmol).1H NMR(400MHz,CD3OD)δ3.89(t,J＝7.3Hz,1H),3.58(dd,J＝12.3,4.1Hz,1H),3.49(td,J＝10.5,4.6Hz,1H),2.90(t,J＝7.8Hz,2H),2.30-2.17(m,3H),1.27(s,3H),1.22(s,3H),1.13(s,3H),1.02(s,3H),0.94(s,3H),0.91(s,3H),0.83(s,3H),0.80(s,3H)。
Example 7
(20S,24R) -epoxy-3 β -hexanoylaminodamane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and hexanoic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (19mg, 78%).1H NMR(400MHz,CDCl3)δ5.33(d,J＝10.1Hz,1H),3.85(dd,J＝8.7,6.9Hz,1H),3.67(ddd,J＝12.3,10.1,4.12Hz,1H),3.52(td,J＝10.4,4.7Hz,1H),2.22-2.16(m,3H),2.08-1.83(m,5H),1.71-1.41(m,12H),1.28(s,3H),1.27(s,3H),1.09(s,3H),0.98(s,3H),0.90(s,3H),0.86(s,3H),0.84(s,3H),0.76(s,3H)。
Example 8
(20S,24R) -epoxy-3 β -octanoylaminodamane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and octanoic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ and reacted at room temperature for 10min to complete the reaction. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (23mg, 90%).1H NMR(400MHz,CDCl3)δ5.25(d,J＝10.1Hz,1H),3.85(dd,J＝8.9,6.6Hz,1H),3.67(ddd,J＝12.3,10.1,4.1Hz,1H),3.52(td,J＝10.3,4.6Hz,1H),2.22-2.16(m,1H),2.17(t,J＝7.8Hz,2H),2.09-1.96(m,2H),1.94-1.83(m,3H),1.71-1.41(m,11H),1.28(s,3H),1.27(s,3H),1.09(s,3H),0.98(s,3H),0.90(s,3H),0.86(s,3H),0.84(s,3H),0.76(s,3H)。
Example 9
(20S,24R) -epoxy-3 β -benzoylaminodammarane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and benzoic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (23mg, 94%).1H NMR(400MHz,CDCl3)δ7.76-7.74(m,2H),7.49(tt,J＝7.3,1.8Hz,1H),7.45-7.41(m,2H),5.96(d,J＝9.6Hz,1H),3.92-3.83(m,2H),3.54(td,J＝10.5,4.6Hz,1H),2.20(td,J＝10.1,3.0Hz,1H),2.09-1.83(m,5H),1.76-1.44(m,9H),1.28(s,3H),1.27(s,3H),1.10(s,3H),1.00(s,3H),0.95(s,3H),0.92(s,3H),0.87(s,3H),0.86(s,3H)。
Example 10
(20S,24R) -epoxy-3 β -nicotinamidodammarane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and nicotinic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (20mg, 82%).1H NMR(400MHz,CDCl3)δ8.94(d,J＝1.4Hz,1H),8.72(dd,J＝4.9,1.5Hz,1H),8.10(dt,J＝7.8,1.8Hz,1H),7.39(dd,J＝7.8,5.0Hz,1H),5.96(d,J＝10.1Hz,1H),3.93-3.83(m,2H),3.54(td,J＝10.5,4.6Hz,1H),2.20(td,J＝10.1,3.0Hz,1H),2.09-1.83(m,5H),1.78-1.45(m,10H),1.28(s,3H),1.27(s,3H),1.10(s,3H),1.00(s,3H),0.95(s,3H),0.92(s,3H),0.89(s,3H),0.87(s,3H)。
Example 11
(20S,24R) -epoxy-3 β - (4-Boc-aminobenzamidoyl) dammarane-12 β, 25-diol;
compound 5(20mg,0.042mmol) and 4-Boc-aminobenzoic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) with stirring under argon protectionAfter the mixture was stirred and dissolved, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added thereto at 0 ℃ and reacted at room temperature for 10 minutes to complete the reaction. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (22mg, 75%).1H NMR(400MHz,CDCl3)δ7.69(dd,J＝6.9,1.8Hz,2H),7.43(d,J＝8.7Hz,2H),6.82(s,1H),5.88(d,J＝10.1Hz,1H),3.90-3.83(m,2H),3.53(td,J＝10.5,4.6Hz,1H),2.20(td,J＝10.1,3.0Hz,1H),2.09-1.83(m,5H),1.52(s,9H),1.28(s,3H),1.27(s,3H),1.10(s,3H),0.99(s,3H),0.94(s,3H),0.92(s,3H),0.87(s,3H),0.85(s,3H)。
Example 12
(20S,24R) -epoxy-3 β - (2-hydroxybenzamido) dammarane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and 2-hydroxybenzoic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL), stirred and dissolved under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ and reacted at room temperature for 10min to complete the reaction. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (22mg, 88%).1H NMR(400MHz,CDCl3)δ12.39(s,1H),7.38(td,J＝7.8,1.5Hz,1H),7.33(dd,J＝8.2,1.4Hz,1H),6.98(dd,J＝8.2,0.9Hz,1H),6.84(td,J＝7.5,1.2Hz,1H),6.15(d,J＝9.6Hz,1H),3.90-3.83(m,2H),3.54(td,J＝10.3,4.9Hz,1H),2.20(td,J＝10.1,3.0Hz,1H),1.28(s,3H),1.27(s,3H),1.10(s,3H),1.00(s,3H),0.93(s,3H),0.92(s,3H),0.88(s,6H)。
Example 13
(20S,24S) -epoxy-3 β -N- (2-Boc-aminoacetyl) -dammarane-12 β, 25-diol;
dissolving compound 2(2.500g, 5.24mmol) in dichloromethane (52mL), adding PCC (1.131g, 5.25mmol) on ice bath, slowly raising to room temperature and stirring for 15 hours, filtering, concentrating, and performing column chromatography to obtain white solid compound 6[ (20S,24S) -epoxy dammara -12 beta, 25-diol-3-one](2.121g,4.47mmol,85％)。1H HMR(400MHz,CDCl3)δ3.88(dd,J＝10.7,5.3Hz,1H),3.55(td,J＝10.3,4.6Hz,1H),2.56-2.41(m,2H),2.27(td,J＝10.4,4.3Hz,1H),1.28(s,3H),1.24(s,3H),1.11(s,3H),1.08(s,3H),1.05(s,3H),1.05(s,3H),0.98(s,3H),0.93(s,3H)。
Dissolving compound 6(1.245g, 2.622mmol) in pyridine, adding hydroxylamine hydrochloride (314mg,4.52mmol) at room temperature, reacting at 80 deg.C for 3 hr, concentrating, diluting with ethyl acetate, washing with water, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography to obtain white solid compound 7[ (20S,24S) -epoxy dammar -12 beta, 25-diol-3-ketoxime](1.123g，2.295mmol，87％)。1H HMR(400MHz,CDCl3)δ5.78(s,1H),3.89(dd,J＝10.7,5.3Hz,1H),3.53(td,J＝10.4,4.7Hz,1H),2.98(ddd,J＝15.6,5.7,3.9Hz,1H),2.34-2.22(m,2H),2.06(td,J＝11.1,4.7Hz,1H),1.28(s,3H),1.24(s,3H),1.15(s,3H),1.11(s,3H),1.07(s,3H),1.04(s,3H),0.98(s,3H),0.90(s,3H)。
Compound 7(533mg,1.09mmol), ammonium acetate (210mg,2.72mmol) and sodium cyanoborohydride (172mg,2.74mmol) were dissolved in isopropanol (40mL), and a 15% titanium trichloride solution (1.04mL) was slowly added dropwise under ice bath, and reacted at room temperature overnight. Adjusting pH of the reaction solution to 10 with sodium hydroxide solution, extracting with ethyl acetate, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and recrystallizing to obtain white solid 8[ (20S,24S) -epoxy dammar -12 beta, 25-diol-3 beta-amine](342mg，0.719mmol，66％)。1H HMR(400MHz,CD3OD)δ3.81(dd,J＝10.3,4.8Hz,1H),3.50(td,J＝10.3,4.9Hz,1H),2.91(dd,J＝10.7,5.7Hz,1H),2.22(td,J＝10.1,3.5Hz,1H),1.26(s,3H),1.17(s,3H),1.10(s,3H),1.06(s,6H),0.94(s,6H),0.89(s,3H)。
The compound 8(20mg,0.042mmol) and 2-Boc-glycine (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (24mg, 90%).1H NMR(400MHz,CDCl3)δ6.06(d,J＝9.6Hz,1H),5.19(s,1H),3.88(dd,J＝10.7,5.3Hz,1H),3.78(dd,J＝16.5,5.9Hz,1H),3.72(dd,J＝16.5,5.9Hz,1H),3.68-3.61(m,1H),3.53(td,J＝10.3,4.7Hz,1H),2.25(td,J＝10.3,4.4Hz,1H),1.45(s,9H),1.28(s,3H),1.23(s,3H),1.10(s,3H),1.01(s,3H),0.91(s,3H),0.88(s,3H),0.86(s,3H),0.77(s,3H)。
Example 14
(20S,24S) -epoxy-3 β -N- (4-Boc-aminobutyryl) -dammarane-12 β, 25-diol;
the compound 8(20mg,0.042mmol) and 4-Boc-aminobutyric acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (23mg, 90%).1H NMR(400MHz,CDCl3)δ6.06(d,J＝9.6Hz,1H),5.19(s,1H),3.88(dd,J＝10.7,5.3Hz,1H),3.78(dd,J＝16.5,5.9Hz,1H),3.72(dd,J＝16.5,5.9Hz,1H),3.68-3.61(m,1H),3.53(td,J＝10.3,4.7Hz,1H),2.25(td,J＝10.3,4.4Hz,1H),1.45(s,9H),1.28(s,3H),1.23(s,3H),1.10(s,3H),1.01(s,3H),0.91(s,3H),0.88(s,3H),0.86(s,3H),0.77(s,3H)。
Example 15
(20S,24S) -epoxy-3 β -N- (6-Boc-aminocaproyl) -dammarane-12 β, 25-diol;
the compound 8(20mg,0.042mmol) and 6-Boc-aminocaproic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (26mg, 90%).1H NMR(400MHz,CDCl3)δ5.92(s,1H),5.40(d,J＝9.6Hz,1H),4.67(s,1H),3.94(dd,J＝10.7,5.3Hz,1H),3.74(ddd,J＝12.3,10.1,4.6Hz,1H),3.61(td,J＝10.4,4.7Hz,1H),3.28-2.99(m,2H),2.32(td,J＝10.3,4.3Hz,1H),2.25(t,J＝7.5Hz,2H),2.21-1.88(m,7H),1.88-1.68(m,5H),1.67-1.53(m,8H),1.50(s,9H),1.34(s,3H),1.30(s,3H),1.17(s,3H),1.08(s,3H),0.98(s,3H),0.94(s,3H),0.93(s,3H),0.83(s,3H)。
Example 16
(20S,24S) -epoxy-3 β -N- (8-Boc-aminocapryl) -dammarane-12 β, 25-diol;
the compound 8(20mg,0.042mmol) and 8-Boc-aminocaprylic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (30mg, 100%).1H NMR(400MHz,CDCl3)δ5.31(d,J＝10.1Hz,1H),4.54(s,1H),3.88(dd,J＝11.0,5.5Hz,1H),3.67(ddd,J＝12.3,10.1,4.57Hz,1H),3.53(td,J＝10.3,4.7Hz,1H),3.12-3.07(m,2H),2.25(td,J＝10.3,4.0Hz,1H),2.17(t,J＝7.5Hz,2H),1.44(s,9H),1.28(s,3H),1.23(s,3H),1.10(s,3H),1.01(s,3H),0.92(s,3H),0.87(s,3H),0.87(s,3H),0.77(s,3H)。
Example 17
(20S,24S) -epoxy-3 β -hexanoylaminodamane-12 β, 25-diol;
the compound 8(20mg,0.042mmol) and hexanoic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (20mg, 83%).1H NMR(400MHz,CDCl3)δ5.31(d,J＝10.1Hz,1H),4.54(s,1H),3.88(dd,J＝11.0,5.5Hz,1H),3.67(ddd,J＝12.3,10.1,4.57Hz,1H),3.53(td,J＝10.3,4.7Hz,1H),3.12-3.07(m,2H),2.25(td,J＝10.3,4.0Hz,1H),2.17(t,J＝7.5Hz,2H),1.44(s,9H),1.28(s,3H),1.23(s,3H),1.10(s,3H),1.01(s,3H),0.92(s,3H),0.87(s,3H),0.87(s,3H),0.77(s,3H)。
Example 18
(20S,24S) -epoxy-3 β - (2-hydroxybenzamido) dammarane-12 β, 25-diol;
the compound 8(20mg,0.042mmol) and 2-hydroxybenzoic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL), stirred and dissolved under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ and reacted at room temperature for 10min to complete the reaction. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (23mg, 92%).1H NMR(400MHz,CDCl3)δ12.39(s,1H),7.39(t,J＝8.5Hz,1H),7.33(d,J＝7.7Hz,1H),6.99(d,J＝8.2Hz,1H),6.85(t,J＝7.4Hz,1H),6.16(d,J＝9.9Hz,1H),3.91-3.85(m,2H),3.56(td,J＝10.2,4.8Hz,1H),2.27(td,J＝10.2,4.2Hz,1H),1.28(s,3H),1.24(s,3H),1.11(s,3H),1.03(s,3H),0.94(s,3H),0.93(s,3H),0.92(s,3H),0.89(s,3H)。
Example 19
(20S,24R) -epoxy-3 β -N- (N' -Boc-prolyl) -dammarane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and N-Boc-proline (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (25mg,0.037mmol, 88%).1H NMR(400MHz,CDCl3)δ4.31-4.26(m,1H),3.85(dd,J＝8.7,6.9Hz,1H),3.68-3.56(m,1H),3.52(td,J＝10.5,4.6Hz,1H),3.46-3.32(m,2H),2.19(td,J＝10.1,3.0Hz,1H),2.08-1.83(m,6H),1.46(s,9H),1.27(s,3H),1.27(s,3H),1.09(s,3H),0.98(s,3H),0.90(s,3H),0.88(s,3H),0.84(s,3H),0.76(s,3H)。
Example 20
(20S,24R) -epoxy-3 β -prolylamido dammarane-12 β, 25-diol;
example 18(0.03mmol) was dissolved in anhydrous TFA (trifluoroacetic acid, 0.5mL) and reacted at room temperature for 10min before terminating the reaction. Concentration gave the title compound (0.03 mmol).1H NMR(400MHz,CD3OD)δ4.26(dd,J＝8.2,6.4Hz,1H),3.89(t,J＝7.3Hz,1H),3.63(dd,J＝12.8,4.1Hz,1H),3.49(td,J＝10.5,4.6Hz,1H),3.42-3.32(m,2H),2.47-2.40(m,1H),2.20(td,J＝10.1,3.0Hz,1H),1.27(s,3H),1.22(s,3H),1.13(s,3H),1.03(s,3H),0.95(s,3H),0.92(s,3H),0.83(s,6H)。
Example 21
(20S,24R) -epoxy-3 β -N- (N' -Boc-lysyl) -dammarane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and N-Boc-lysine (0.059mmol) were dissolved in anhydrous DMF (0.4mL), stirred and dissolved under argon protection, and HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20 μ L) were added at 0 ℃, reacted at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (30mg,0.037mmol, 89%).1H NMR(400MHz,CDCl3)δ6.08(d,J＝9.6Hz,1H),5.22(s,1H),4.64(s,1H),3.99(dd,J＝12.6,7.5Hz,1H),3.84(dd,J＝8.5,6.6Hz,1H),3.61(td,J＝10.6,4.7Hz,1H),3.51(td,J＝10.5,4.6Hz,1H),3.14-3.07(m,2H),2.22-2.16(m,1H),2.08-1.80(m,6H),1.44(s,18H),1.27(s,3H),1.27(s,3H),1.09(s,3H),0.98(s,3H),0.90(s,3H),0.85(s,3H),0.84(s,3H),0.75(s,3H)。
Example 22
(20S,24R) -epoxy-3 β -N- (N' -Boc-aspartyl) -dammarane-12 β, 25-diol;
compound 5(20mg,0.042mmol) and N-Boc-aspartic acid (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. Extracting with ethyl acetate, washing with saturated sodium bicarbonate solution, drying with anhydrous sodium sulfate, and concentratingAnd (4) shrinking. Column chromatography gave the title compound (25mg,0.033mmol, 80%).1H NMR(400MHz,CDCl3)δ6.46(d,J＝9.6Hz,1H),5.73(d,J＝6.9Hz,1H),4.43-4.38(m,1H),3.84(dd,J＝8.9,6.6Hz,1H),3.58(ddd,J＝11.4,10.1,5.0Hz,1H),3.51(td,J＝10.5,4.6Hz,1H),2.85(dd,J＝17.0,4.7Hz,1H),2.61(dd,J＝16.9,6.9Hz,1H),2.19(td,J＝10.1,3.2Hz,1H),2.11-1.82(m,5H),1.45(s,9H),1.44(s,9H),1.27(s,3H),1.27(s,3H),1.09(s,3H),0.97(s,3H),0.90(s,3H),0.84(s,3H),0.83(s,3H),0.76(s,3H)。
Example 23
(20S,24R) -epoxy-3 β -N- (N' -Boc-tyrosyl) -dammarane-12 β, 25-diol;
the compound 5(20mg,0.042mmol) and N-Boc-tyrosine (0.059mmol) were dissolved in anhydrous DMF (0.4mL) and stirred under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 24mg,0.076mmol) and triethylamine (20. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (28mg,0.035mmol, 84%).1H NMR(400MHz,CDCl3)δ7.11(d,J＝8.2Hz,2H),6.91(d,J＝8.7Hz,2H),5.77(d,J＝9.6Hz,1H),5.01(s,1H),4.27(q,J＝7.2Hz,1H),3.84(dd,J＝8.7,6.4Hz,1H),3.60-3.47(m,2H),3.08(dd,J＝13.7,5.9Hz,1H),2.99(dd,J＝14.2,7.8Hz,1H),2.19(td,J＝10.1,3.0Hz,1H),2.10-1.82(m,5H),1.68-1.63(m,2H),1.42(s,9H),1.32(s,9H),1.27(s,3H),1.27(s,3H),1.09(s,3H),0.96(s,3H),0.89(s,3H),0.78(s,3H),0.70(s,3H),0.55(s,3H)。
Example 24
(20S,24R) -epoxy-3 β -N- { 2- [ 2- (2-Fmoc-aminoethoxy) ethoxy ] acetyl } -dammarane-12 β, 25-diol;
compound 5(42mg,0.088mmol) and 2- [ 2- (2-Fmoc-aminoethoxy) ethoxy ] ethoxy]Acetic acid (48mg,0.124mmol) was dissolved in anhydrous DMF (0.8mL), and after stirring and dissolving under argon protection, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 51mg,0.158mmol) and triethylamine (50. mu.L) were added at 0 ℃ to react at room temperature for 10min, and the reaction was terminated. By using BAfter extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (52mg,0.062mmol, 70%).1H NMR(400MHz,CDCl3)δ7.76(d,J＝7.3Hz,2H),7.60(d,J＝7.3Hz,2H),7.40(td,J＝7.5Hz,2H),7.31(td,J＝7.3,0.9Hz,2H),6.56(d,J＝10.1Hz,1H),5.22(s,1H),4.40(d,J＝6.9Hz,2H),4.22(t,J＝6.9Hz,1H),4.03(d,J＝10.1Hz,1H),4.00(d,J＝10.1Hz,1H),3.84(dd,J＝8.7,6.9Hz,1H),3.72-3.56(m,6H),3.51(td,J＝10.5,4.6Hz,1H),3.42-3.38(m,2H),2.18(td,J＝9.9,2.9Hz,1H),2.11-1.82(m,5H),1.71-1.39(m,10H),1.28(s,3H),1.26(s,3H),1.09(s,3H),0.96(s,3H),0.88(s,3H),0.87(s,3H),0.83(s,3H),0.78(s,3H)。
Example 25
(20S,24S) -epoxy-3 β -N- { 2- [ 2- (2-Fmoc-aminoethoxy) ethoxy ] acetyl } -dammarane-12 β, 25-diol;
compound 8(30mg,0.063mmol) and 2- [ 2- (2-Fmoc-aminoethoxy) ethoxy ] ethanol]Acetic acid (34mg,0.088mmol) was dissolved in anhydrous DMF (0.6mL) under stirring under argon, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate, 36mg,0.113mmol) and triethylamine (20. mu.L) were added at 0 ℃ and the reaction was terminated after 10min at room temperature. After extraction with ethyl acetate and washing with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated. Column chromatography gave the title compound (34mg,0.040mmol, 64%).1H NMR(400MHz,CDCl3)δ7.77(d,J＝7.3Hz,2H),7.60(d,J＝7.3Hz,2H),7.40(t,J＝7.3Hz,2H),7.31(t,J＝7.3Hz,2H),6.55(d,J＝10.1Hz,1H),5.73(s,1H),5.20(s,1H),4.40(d,J＝6.9Hz,2H),4.22(t,J＝6.6Hz,1H),4.01(s,2H),3.87(dd,J＝10.7,5.3Hz,1H),3.73-3.49(m,8H),3.44-3.38(m,2H),2.25(td,J＝10.3,4.3Hz,1H),2.10-1.65(m,10H),1.27(s,3H),1.23(s,3H),1.10(s,3H),0.99(s,3H),0.90(s,3H),0.88(s,3H),0.87(s,3H),0.79(s,3H)。
Pharmacological tests prove that the ocotillol type sapogenin derivative has tumor drug resistance reversal activity, is used for preparing MDR reversal agents, is combined with common antitumor drugs, and exerts good antitumor activity.
The following are the pharmacological tests and data for some of the compounds of the invention.
Instruments and devices:
super clean bench
High-pressure steam sterilizer
Carbon dioxide incubator
Enzyme-linked immunosorbent assay (ELISA) instrument
Analytical balance
Refrigerator with a door
Liquid-transfering gun
Cell culture dish
96-well plate
Filter
Cell lines and reagents:
dimethyl sulfoxide (DMSO)
Tetramethyl azo blue (MTT)
Complete culture medium
Oral epidermoid carcinoma cell KBV
Pancreatin digestive juice (0.25% trypsin + 0.02% EDTA)
The ocotillol type sapogenin and the derivatives thereof are dissolved in DMSO to prepare raw medicine liquid
The experimental method comprises the following steps:
firstly, the method comprises the following steps: drug screening:
the MTT method, also known as MTT colorimetric method, is a method for detecting cell survival and growth. The principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit in cells, but the phenomenon does not occur in dead cells. Formazan crystals can be dissolved in DMSO, and absorbance is measured at a wavelength of 570nm by an enzyme linked immunosorbent assay (ELISA) detector, and the absorbance value is directly proportional to the number of living cells in a certain cell number range, thereby indirectly reflecting the number of the living cells.
1. Cell plating: KBV drug-resistant strain cells were presented friendlily to the photo teacher's subject group at the institute of Chinese medical academy of sciences. Taking adherent KBV cells which grow in logarithmic phase and are in good state, and digesting the adherent KBV cells into single cell suspension by pancreatin. After counting, the cells are diluted to 3-4 multiplied by 104one/mL and 100. mu.L/well in 96-well plates at 37 ℃ with 5% CO2CulturingAnd (5) standing and culturing in a box.
2. Cell administration: after 24h of cell plating to the wall, 10 μ M of each compound was added and cultured in combination with 100nM paclitaxel and corresponding solvent control, with 3 parallel wells per group. And (4) after the medicine is added, placing the 96-well plate in an incubator, and performing static culture for 72 hours.
3. MTT detection: after culturing the cells for 72 hours by administering the corresponding drug, 20. mu.L of MTT solution at a concentration of 5mg/mL was added to each well, and the culture medium containing MTT was discarded after incubation at 37 ℃ for 2 hours. After 150. mu.L of DMSO was added to each well to dissolve formazan, absorbance at 570nm was measured for each well after shaking and mixing, and inhibition of the compound was calculated using tumor cell group treated with DMSO as a control group and verapamil as a positive control group.
Inhibition (%) - (control group mean OD value-administration group mean OD value)/control group mean value%
II, secondly: MTT method for detecting reverse activity of compound in tumor drug-resistant cell strain KBV cell of antitumor drug paclitaxel
KBV cells in logarithmic growth phase were trypsinized and then seeded into 96-well plates at 3000-. After 24h of cell plating, 5 μ M and 10 μ M of test compound are added respectively to be combined with paclitaxel with different concentrations and corresponding solvent control for culture. And (3) discarding the supernatant after 72h, adding 20 mu L MTT into each well, continuing culturing for 2h, discarding the supernatant, adding 150 mu L DMSO, placing the supernatant on a micro-oscillator, uniformly oscillating, measuring absorbance (OD) at 570nM, taking the tumor cells treated by the solvent control as a control group, calculating the inhibition rate of the compound on the tumor cells, and calculating to obtain IC50 and the reversal multiple of the compound.
IC50 when the reversal factor is single antineoplastic medicine/IC 50 when the reversal agent is used together
Thirdly, the method comprises the following steps: example 3 drug resistance reversing Activity in nude mice
Collecting KBV cells under aseptic conditions, counting with a cell counter, inoculating 5X 10 cells subcutaneously on the right side of each nude mouse6And (4) KBV cells. The tumor mass is 100-300 mm long3Thereafter, the control group, paclitaxel (30mg/kg) group, example 3(10mg/kg) group, and paclitaxel and example 3 combination treatment group were randomly divided. Chemical combinationThe preparation is administered once a day by intragastric administration, and paclitaxel is administered by intraperitoneal injection twice a week. Animal body weights and tumor volumes were recorded.
Tumor volume was calculated: volume (V) ═ a × b2And/2 (a: long diameter, b: short diameter), and a tumor growth curve is prepared. After two weeks, the animals were euthanized and stripped of tumors, and the tumor growth inhibition (%) was calculated by weighing the tumors.
The experimental results are as follows:
according to results obtained on tumor-resistant oral epidermal cancer cell lines (KBV cells), the prepared ocotillol type sapogenin derivatives have better drug resistance reversal activity, which is obviously superior to ocotillol type sapogenins 1 and 2, as shown in Table 1. And examples 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25 showed better enhancement of cytotoxicity of the antitumor drug paclitaxel to tumor cells at concentrations of 5 and 10 μ M compared to the positive control drug verapamil.
In addition, the product of the invention has good gastrointestinal stability and better drug applicability, which is a characteristic that the compounds disclosed in the prior art do not have. For example, a study of half-maximal inhibition of KBV cells using representative example 3 in combination with paclitaxel was selected and shown in Table 2, resulting in paclitaxel having IC50 of 48.41 + -3.16 and 28.29 + -2.90 nM at 5 and 10 μ M concentrations, respectively. The fold reversal reached 28 and 48 fold respectively. Esterified derivatives of natural products are modified and often are digested by esterases in the small intestine after oral administration, thereby becoming inactive. The drug designed by our patent, found in a nude mouse xenograft tumor model (see fig. 1), oral administration of the representative drug example 3 in a KBV xenograft tumor model, significantly enhanced the in vivo antitumor activity of paclitaxel, showing significant resistance-reversing activity, which indicates that our drug has better gastrointestinal stability, enabling it to reach tumor sites, producing resistance-reversing activity.
TABLE 1 Effect of ocotillol-type sapogenins and their derivatives on paclitaxel cytotoxicity in KBV cells
Figure BDA0001797659160000131
Figure BDA0001797659160000141
Table 2 example 3 determination of drug resistance reversal activity
Figure BDA0001797659160000142
The above description is only a preferred embodiment of the application and is illustrative of the principles of the technology employed.
It will be appreciated by a person skilled in the art that the scope of the invention as referred to in the present application is not limited to the embodiments with a specific combination of the above-mentioned features, but also covers other embodiments with any combination of the above-mentioned features or their equivalents without departing from the inventive concept. For example, the above features may be interchanged with other features disclosed in this application, but not limited to those having similar functions.

Claims (3)

1. An ocotillol-type sapogenin derivative, characterised in that it is selected from the following compounds:
(20S,24R) -epoxy-3 β -N- (4-Boc-aminobutyryl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (6-Boc-aminocaproyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (8-Boc-aminooctanoyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (8-aminocapryl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -benzoylaminodammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β - (4-Boc-aminobenzamidoyl) dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β - (2-hydroxybenzamido) dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- (2-Boc-aminoacetyl) -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- (4-Boc-aminobutyryl) -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- (6-Boc-aminocaproyl) -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- (8-Boc-aminocapryl) -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β - (2-hydroxybenzamido) dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (N' -Boc-prolyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -prolylamido dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (N' -Boc-lysyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (N' -Boc-aspartyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- (N' -Boc-tyrosyl) -dammarane-12 β, 25-diol;
(20S,24R) -epoxy-3 β -N- { 2- [ 2- (2-Fmoc-aminoethoxy) ethoxy ] acetyl } -dammarane-12 β, 25-diol;
(20S,24S) -epoxy-3 β -N- { 2- [ 2- (2-Fmoc-aminoethoxy) ethoxy ] acetyl } -dammarane-12 β, 25-diol.
2. Use of ocotillol-type sapogenin derivatives according to claim 1, and pharmaceutically acceptable salts thereof, for the preparation of a tumour resistance-reversing agent for use with paclitaxel.
3. The use of ocotillol-type sapogenin derivatives and pharmaceutically acceptable salts thereof according to claim 2, for the preparation of reversal agents of tumour resistance,
the ocotillol type sapogenin derivative and the medically acceptable salt thereof with effective dose are used together with paclitaxel which is a clinical antitumor drug.
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