Betulinic acid derivative and application of betulinic acid derivative in preparation of anti-pressure and anti-aging drugs

Abstract

The invention discloses a betulinic acid derivative and application of the betulinic acid derivative in preparation of anti-stress and anti-aging drugs, and belongs to the field of medicines.The betulinic acid derivative has the structural formula shown in the formula I. # imgabs0 # R1 is H or acyl; x is O or NH; # imgabs 1 # represents a double bond or a single bond; r2 is H, C1-C5 straight-chain alkyl, C3-C6 mono-cycloalkyl, polycycloalkyl or substituted / unsubstituted six-membered heterocyclic radical containing one nitrogen or oxygen atom. The betulinic acid derivative can improve the activity of cell telomerase, prolong the telomere length, practically prolong the life of drosophila melanogaster and play a role in delaying senescence.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a betulinic acid derivative and application thereof in preparing anti-stress and anti-aging drugs. Background Art

[0002] Aging is closely related to many age-related diseases, including Alzheimer's disease, cognitive impairment, decreased immune system function, type II diabetes, autoimmune diseases, heart disease, and various cancers. Therefore, it is of great significance to discover highly effective anti-aging active ingredients or drugs.

[0003] Telomere shortening or telomere DNA damage is considered to be the root cause of aging and many aging-related diseases (Rossiello F et al. Nat Cell Biol. 2022, 24 (2): 135-147). The commonly believed mechanism is that telomeres shorten, normal DNA is damaged, the rate of cell repair and recycling molecules slows down, damage accumulates and causes cells to lose normal function, and eventually the cells no longer divide and die. Telomerase, an enzyme responsible for telomere extension in cells, is a basic nuclear protein reverse transcriptase that can extend telomere DNA to the ends of eukaryotic cell chromosomes, fill in the telomeres lost by DNA replication, extend telomere repair, and increase the number of cell divisions. Telomerase can extend and shorten telomeres, thereby enhancing the proliferation ability of cells in vitro. The activity of telomerase is inhibited in normal human tissues and reactivated in tumors, which may be involved in malignant transformation. Telomerase plays an important role in maintaining telomere stability, genome integrity, long-term cell activity and potential continued proliferation ability.

[0004] Betulinic acid (BA) can be extracted from Syzygium wilfordii leaves, birch bark, and Ziziphus jujuba seeds; it can also be obtained by chemical synthesis using betulinol as a raw material. It is a lupine-type pentacyclic triterpenoid compound. In 1995, betulinic acid was found to selectively inhibit human melanoma (Pisha E et al. Nat Med. 1995, 1(10): 1046-1051). Since then, great progress has been made in the study of the anticancer activity of BA. These results show that betulinic acid has inhibitory activity against many different cancer cells, including Ewing sarcoma, malignant brain tumors, ovarian cancer, prostate cancer, breast cancer, and leukemia HL-60 cells. However, there has been no report on the anti-aging effect of betulinic acid. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a betulinic acid derivative and its use in the preparation of anti-stress and anti-aging drugs. The betulinic acid derivative in the present invention can increase the activity of cell telomerase, extend the length of telomeres, and can effectively extend the lifespan of fruit flies, thereby playing a role in delaying aging.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention:

[0008] A betulinic acid derivative, the structural formula of which is shown in Formula I:

[0009]

[0010] Among them, R 1 is H or acyl; X is O or NH; Represents a double bond or a single bond; R 2 is H, C1-C5 straight-chain alkyl, C3-C6 monocyclic alkyl, polycyclic alkyl, or substituted / unsubstituted six-membered heterocyclic group containing one nitrogen or oxygen atom;

[0011] The substituent of the six-membered heterocyclic group containing one nitrogen or oxygen atom is selected from C1-C5 alkyl.

[0012] Preferably, in the betulic acid derivative, R 1 is H, n-propionyl, n-butyryl, benzoyl, X is O or NH; Represents a double bond or a single bond; R 2 H, CH 3 ,

[0013] Preferably, in the betulic acid derivative, R 1 H, n-butyryl, X is O or NH; Represents a double bond or a single bond; R 2 H, CH 3 ,

[0014] Preferably, in the betulic acid derivative, R 1 is H, X is O or NH, Represents a double bond or a single bond, R 2 For H.

[0015] or R 1 H, n-butyryl, X is O, represents a double bond or a single bond, R 2 For H.

[0016] More preferably, the betulic acid derivative is selected from the following compounds:

[0017]

[0018] The second technical solution of the present invention:

[0019] The present invention also provides a method for preparing the betulic acid derivative, comprising the following steps:

[0020] Betulinic acid (1) undergoes a condensation reaction with an alcohol or an amine compound to obtain a compound of formula II; betulinic acid (1) undergoes an esterification reaction with an acid anhydride compound to obtain a compound of formula III; the compound of formula II and the compound of formula III are successively reacted with selenium dioxide (SeO 2 ) oxidation, Pinnick oxidation reaction, or further hydrogenation reduction to obtain a compound of formula I, the reaction scheme is as follows:

[0021]

[0022] Among them, R 1 is H or acyl; X is O or NH; Represents a double bond or a single bond; R 2 is H, C1-C5 straight-chain alkyl, C3-C6 monocyclic alkyl, polycyclic alkyl, or substituted / unsubstituted six-membered heterocyclic group containing one nitrogen or oxygen atom;

[0023] The substituent of the six-membered heterocyclic group containing one nitrogen or oxygen atom is selected from C1-C5 alkyl.

[0024] The pharmaceutically acceptable salt of the compound of formula I is synthesized by general chemical methods. For example, in general, the preparation of the salt can be prepared by reacting a free base or acid with an equal chemical equivalent or excess acid (inorganic acid or organic acid) or base (inorganic base or organic base) in a suitable solvent or solvent composition. For example, the preparation method of the pharmaceutically acceptable potassium salt of the compound of formula I is: add 5g of KOH to 50mL of ethanol solution, heat and stir under ultrasound or 40°C until dissolved, that is, prepare a 10% W / V KOH ethanol solution. Add the compound of formula I (20g) to a 1000mL round-bottom flask and dissolve it in 434mL of ethanol, add KOH-ethanol solution (10% W / V, 24.7mL) dropwise, stir at room temperature for 24h, spin dry, and dry to obtain 18g of white solid (yield: 90%).

[0025] The third technical solution of the present invention:

[0026] The present invention also provides the use of the betulic acid derivative, its pharmaceutically acceptable salt, tautomer, stereoisomer, nitrogen oxide, hydrate or prodrug in the preparation of telomerase activity regulating drugs.

[0027] Preferably, the betulic acid derivative, its pharmaceutically acceptable salt, tautomer, stereoisomer, nitrogen oxide, hydrate or prodrug has the function of positively regulating telomerase activity, maintaining telomere length and maintaining the RNA expression level of telomerase Tert.

[0028] Preferably, the drug is in the form of tablets, granules, powders, capsules, emulsions or injections, with the betulic acid derivative or a pharmaceutically acceptable salt thereof described in the present invention as an effective ingredient.

[0029] The fourth technical solution of the present invention:

[0030] The present invention also provides the use of the betulic acid derivatives, pharmaceutically acceptable salts, tautomers, stereoisomers, nitrogen oxides, hydrates or prodrugs thereof in the preparation of drugs for preventing or treating stress resistance and aging-related diseases.

[0031] Preferably, the stress resistance and aging-related diseases include Alzheimer's disease, cognitive impairment or decreased immune system function.

[0032] Preferably, the dosage form of the drug is tablets, granules, powders, capsules, emulsions or injections.

[0033] Compared with the prior art, the present invention has the following advantages and technical effects:

[0034] The betulinic acid derivatives of the present invention can increase the activity of cell telomerase, extend the length of telomeres, and effectively extend the life span of fruit flies, thereby playing a role in delaying aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0036] Figure 1The results are the effects of betulinic acid (compound 1), betulinic acid derivative 2 and compounds I-1 to I-12 on telomerase activity of 293T cells, wherein a is the control group and compound 1, b is the control group and betulinic acid derivative 2, c is the control group and I-1, d is the control group and I-2, e is the control group and I-3, f is the control group and I-4, g is the control group and I-5, h is the control group and I-6, i is the control group and I-7, j is the control group and I-8, k is the control group and I-9, l is the control group and I-10, m is the control group and I-11, and n is the control group and I-12;

[0037] Figure 2 The results of the effects of compound I-9 on cell telomerase activity at different concentrations, where "ns", "*", and "**" represent no difference, significant difference (p<0.05), and significant difference (p<0.01), respectively;

[0038] Figure 3 The results of detecting the survival period and crawling ability of fruit flies by oral administration (taking compound I-9 as an example), wherein a is a time course diagram of oral administration of I-9 to fruit flies, b is the survival period of male fruit flies when I-9 = 50 μM is administered, c is the survival period of female fruit flies when I-9 = 50 μM, d is the crawling ability of male fruit flies when I-9 = 50 μM, e is the crawling ability of female fruit flies when I-9 = 50 μM, f is the survival period of male fruit flies when I-9 = 20 μM, g is the survival period of male fruit flies when I-9 = 20 μM; h is the crawling ability of male fruit flies when I-9 = 20 μM; i is the crawling ability of female fruit flies when I-9 = 20 μM.

[0039] Figure 4 The results of the rotating rod fatigue test of compound I-9;

[0040] Wherein: "Control Group" is Vehicle, "I-1" is Compound I-1, "I-9" is Compound I-9, and the same goes for the others. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0047] All raw materials used in the embodiments of the present invention are commercially available.

[0048] The technical solution of the present invention is further illustrated by the following embodiments.

[0049] Example 1

[0050] Preparation of 3β-hydroxylupin-20(29)-ene-28,30-dioic acid (2) The synthetic route is as follows:

[0051]

[0052] The specific method is as follows:

[0053] In a 100mL round-bottom flask, betulinic acid (compound 1) (3g, 6.6mmol) was dissolved in 1,4-dioxane (20mL), selenium dioxide (1.83g, 16.5mmol) was added, heated to 100℃, and stirred for 3 hours. Filtered, concentrated under reduced pressure, and dried to obtain a solid crude product. In a 100mL round-bottom flask, the above crude product (3g, 6.4mmol) was added, dissolved with a mixed solution of tert-butyl alcohol / 2-methyl-2-butene (1:1, V / V, 30mL in total), and then an aqueous solution (15mL) of sodium chlorite (2.89g, 32mmol) and potassium dihydrogen phosphate (4.29g, 32mmol) was added, and the reaction mixture was stirred at room temperature to complete the reaction. The starting material disappeared as determined by TLC. The pH was adjusted to acidic with 1M HCl aqueous solution (10 mL). The solid crude product was obtained by filtration and purified by silica gel column chromatography (eluent volume ratio: dichloromethane / methanol = 60 / 1) to give white solid compound 2 (600 mg, yield 19%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.36-12.03(s,2H),5.95-5.90(s,1H),5.59-5.53(s,1H),4.25-4.19(s,1H),3.22-3.17(d,J=9. 0Hz,1H),2.96-2.90(s,1H),2.16-2.07(t,J=14.3Hz,2H),1.95-1.89(d,J=15.9Hz,1H),1.77-1.71( t,J=10.5Hz,2H),1.58-1.50(s,2H),1.42-1.35(d,J=12.3Hz,5H),1.28-1.20(d,J=25.0Hz,6H),1.1 3-1.02(t,J=13.4Hz,3H),0.91-0.82(d,J=15.1Hz,10H),0.76-0.69(s,4H),0.62-0.57(s,4H); HRMS calcd for C 30 H 47 O 5 [M+H] + m / z 487.69300, found 487.33700.

[0054] Example 2

[0055] Preparation of 3β-hydroxylupin-20(29)-ene-30-acid-28-carboxymethyl ester (I-1), the synthetic route is as follows:

[0056]

[0057] In a 100 mL round-bottom flask, betulinic acid (compound 1) (3 g, 6.6 mmol) was dissolved in N, N-dimethylformamide (20 mL), iodomethane (1.1 g, 7.9 mmol) and potassium carbonate (1.8 g, 13.2 mmol) were added, and the reaction was stirred at room temperature for 6 h. The raw material disappeared after TLC determination, and the mixture was concentrated by vacuum distillation. The residue was treated with aqueous solution (10 mL) and ethyl acetate (20 mL), and the organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain white solid compound 3 (2.8 g, yield 90%).

[0058] Referring to the preparation method of compound 2 in Example 1, only compound 1 was replaced by compound 3 to obtain white solid compound I-1 (500 mg, yield 18%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.36-12.03(s,1H),5.95-5.90(s,1H),5.59-5.53(s,1H),4.25-4.19(s,1H),3.69-3.52(s,3H),3.22 -3.17(d,J=9.0Hz,1H),2.96-2.90(s,1H),2.16-2.07(t,J=14.3Hz,2H),1.95-1.89(d,J=15.9Hz,1H),1. 77-1.71(t,J=10.5Hz,2H),1.58-1.50(s,2H),1.42-1.35(d,J=12.3Hz,5H),1.28-1.20(d,J=25.0Hz,6H) ,1.13-1.02(t,J=13.4Hz,3H),0.91-0.82(d,J=15.1Hz,10H),0.76-0.69(s,4H),0.62-0.57(s,4H); HRMS calcd for C 31 H 49 O 5 [M+H] + m / z 501.72000, found 501.69000.

[0059] Example 3

[0060] Preparation of 3β-hydroxylupin-20(29)-ene-30-acid-28-carboxycyclohexyl ester (I-2), the synthetic route is as follows:

[0061]

[0062] In a 100 mL round-bottom flask, betulinic acid (compound 1) (3 g, 6.6 mmol) and cyclohexanol (1 g, 7.92 mmol) were dissolved in DCM (dichloromethane), and DMAP (4-dimethylaminopyridine) (319 mg, 2.64 mmol) was added. The reaction was carried out at 40 ° C for 5 min, and DCC (N, N'-dicyclohexylcarbodiimide) (2.04 g, 9.9 mmol) was added and stirred until the reaction was completed. The TLC spot plate monitored the disappearance of the raw material, and the vacuum distillation was concentrated. The residue was treated with aqueous solution (10 mL) and ethyl acetate (20 mL), and the organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white crude product, which was purified by silica gel column chromatography (eluent volume ratio: petroleum ether / ethyl acetate = 3 / 1) to obtain a white solid compound 4 (2.8 g, yield 79%).

[0063] Referring to the preparation method of compound 2 in Example 1, only compound 1 was replaced by compound 4 to obtain white solid compound I-2 (560 mg, yield 19%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.36-12.03(s,1H),5.95-5.90(s,1H),5.59-5.53(s,1H),4.25-4.19(s,1H),4.15-4.09(d,J=15.9Hz,1H),3.22-3.17 (d,J=9.0Hz,1H),2.96-2.90(s,1H),2.16-2.07(t,J=14.3Hz,2H),1.95-1.89(d,J=15.9Hz,1H),1.85-1.79(d,J=11.9Hz,2 H),1.77-1.71(t,J=10.5Hz,2H),1.58-1.53(s,2H),1.52-1.45(d,J=12.3Hz,8H),1.42-1.35(d,J=12.3Hz,5H),1.28-1.20 HRMS calcd for C 36 H 57 O 5 [M+H] + m / z 569.83900,found 559.69700.

[0064] Example 4

[0065] Preparation of 3β-hydroxylupin-20(29)-ene-30-acid-28-carboxytetrahydro-4-pyranyl ester (I-3), the structural formula of which is The specific method is as follows:

[0066] Referring to the preparation method of compound I-2 in Example 3, only cyclohexanol was replaced by tetrahydro-4-pyranol to obtain white solid compound I-3 (610 mg, yield 21%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.36-12.03(s,1H),5.95-5.90(s,1H),5.59-5.53(s,1H),4.25-4.19(m,2H),3.76-3.61(d,J=8.0Hz,4H),3.22- 3.17(d,J=9.0Hz,1H),2.96-2.90(s,1H),2.16-2.07(t,J=14.3Hz,2H),1.95-1.89(d,J=15.9Hz,3H),1.85-1.79(d,J =11.9Hz,2H),1.77-1.71(t,J=10.5Hz,2H),1.58-1.53(s,2H),1.42-1.35(d,J=12.3Hz,5H),1.28-1.20(d,J=25.0H z, 6H), 1.13-1.02 (t, J=13.4Hz, 3H), 0.91-0.82 (d, J=15.1Hz, 10H), 0.76-0.69 (s, 4H), 0.62-0.57 (s, 4H); HRMScalcd for C 35 H 55 O 6 [M+H] + m / z 571.81100, found 571.47200.

[0067] Example 5

[0068] Preparation of 3β-hydroxylupin-20(29)-ene-30-acid-28-carboxyl (1-methyl-4-piperidinyl) ester (I-4), the structural formula of which is The specific method is as follows:

[0069] Referring to the preparation method of compound I-2 in Example 3, only cyclohexanol was replaced by 1-methyl-4-piperidinol to obtain white solid compound I-4 (600 mg, yield 23%). 1 H NMR (400 MHz, DMSO-d 6)δ12.36-12.03(s,1H),5.95-5.90(s,1H),5.59-5.53(s,1H),5.05-4.98(m,1H),4.25-4.19(s,1H),3.22-3.17(d,J=9.0Hz, 1H),2.96-2.90(s,1H),2.56-2.41(d,J=11.30Hz,4H),2.23-2.19(s,3H),2.16-2.07(t,J=14.3Hz,2H),1.95-1.89(d,J=15.9 Hz,1H),1.85-1.79(d,J=11.9Hz,2H),1.77-1.71(t,J=10.5Hz,4H),1.58-1.53(s,2H),1.42-1.35(d,J=12.3Hz,5H),1.28-1. 20(d,J=25.0Hz,6H),1.13-1.02(t,J=13.4Hz,3H),0.91-0.82(d,J=15.1Hz,10H),0.76-0.69(s,4H),0.62-0.57(s,4H); HRMS calcd for C 36 H 58 NO 5 [M+H] + m / z 584.85400,found584.63100.

[0070] Example 6

[0071] Preparation of N-cyclohexyl-3β-hydroxylupin-20(29)-ene-30-acid-28-amide (I-5), the structural formula of which is The specific method is as follows:

[0072] Referring to the preparation method of compound I-2 in Example 3, only cyclohexanol was replaced by cyclohexylamine to obtain white solid compound I-5 (595 mg, yield 21%). 1 H NMR (400 MHz, DMSO-d 6)δ12.36-12.03(s,1H),8.15-8.03(s,1H),5.95-5.90(s,1H),5.59-5.53(s,1H),4.25-4.19(s,1H),3.52-3.47(d,J=8.4Hz, 1H),3.22-3.17(d,J=9.0Hz,1H),2.96-2.90(s,1H),2.16-2.07(t,J=14.3Hz,2H),1.95-1.89(d,J=15.9Hz,1H),1.77-1.71( t,J=10.5Hz,2H),1.70-1.61(m,J=11.6Hz,2H),1.58-1.50(s,2H),1.48-1.44(m,4H),1.42-1.35(d,J=12.3Hz,5H),1.28-1. 20(d,J=25.0Hz,8H),1.13-1.02(t,J=13.4Hz,5H),0.91-0.82(d,J=15.1Hz,10H),0.76-0.69(s,4H),0.62-0.57(s,4H); HRMS calcd for C 36 H 58 NO 4 [M+H] + m / z 567.85500, found 567.31700.

[0073] Example 7

[0074] Preparation of N-(tetrahydropyran-4-yl)-3β-hydroxylupin-20(29)-ene-30-acid-28-amide (I-6), the structural formula of which is The specific method is as follows:

[0075] Referring to the preparation method of compound I-2 in Example 3, only cyclohexanol was replaced by 4-aminotetrahydropyran to obtain white solid compound I-6 (630 mg, yield 24%). 1 H NMR (400 MHz, DMSO-d 6)δ12.36-12.03(s,1H),8.15-8.03(s,1H),5.95-5.90(s,1H),5.59-5.53(s,1H),4.25-4.19(s,1H),3.72-3.57(m,J=8. 4Hz, 5H), 3.22-3.17 (d, J = 9.0Hz, 1H), 2.96-2.90 (s, 1H), 2.16-2.07 (t, J = 14.3Hz, 2H), 1.95-1.89 (d, J = 15.9Hz, 2H), 1. 77-1.71(t,J=10.5Hz,2H),1.70-1.61(m,J=11.6Hz,3H),1.58-1.50(s,2H),1.42-1.35(d,J=12.3Hz,5H),1.28-1.20(d HRMS calcd for C 35 H 56 NO 5 [M+H] + m / z 570.82700, found 570.76900.

[0076] Example 8

[0077] Preparation of N-(1-methylpiperidin-4-yl)-3β-hydroxylupin-20(29)-ene-30-acid-28-amide (I-7), the structural formula of which is The specific method is as follows:

[0078] Referring to the preparation method of compound I-2 in Example 3, only cyclohexanol was replaced by 4-amino-1-methylpiperidine to obtain white solid compound I-7 (630 mg, yield 22%). 1 H NMR (400 MHz, DMSO-d 6)δ12.36-12.03(s,1H),8.15-8.03(s,1H),5.95-5.90(s,1H),5.59-5.53(s,1H),4.25-4.19(s,1H),3.75-3.58(m,1H),3.22-3.1 7(d,J=9.0Hz,1H),2.96-2.90(s,1H),2.56-2.41(d,J=11.30Hz,4H),2.23-2.19(s,3H),2.16-2.07(t,J=14.3Hz,2H),1.95-1.89( d,J=15.9Hz,1H),1.85-1.79(d,J=11.9Hz,2H),1.77-1.71(t,J=10.5Hz,4H),1.58-1.53(s,2H),1.42-1.35(d,J=12.3Hz,5H),1.2 HRMS calcd for C 36 H 59 N 2 O 4 [M+H] + m / z583.87000, found 583.79100.

[0079] Example 9

[0080] Preparation of 3β-butyryloxylupin-20(29)-ene-28,30-dioic acid (I-8), the synthetic route is as follows:

[0081]

[0082] The specific preparation method is as follows:

[0083] In a 100 mL round-bottom flask, betulinic acid (1) (3 g, 6.6 mmol) was dissolved in pyridine (10 mL), and butyric anhydride (1.1 g, 7.26 mmol) and 4-dimethylaminopyridine (319 mg, 2.64 mmol) were added. The reaction mixture was stirred at room temperature for 24 h to complete the reaction. TLC showed that the starting material disappeared, and the mixture was concentrated by vacuum distillation. The residue was treated with aqueous solution (10 mL) and ethyl acetate (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white crude product, which was purified by silica gel column chromatography (eluent volume ratio: petroleum ether / ethyl acetate = 90 / 1) to obtain white solid compound 5 (3.0 g, yield 86%).

[0084] Referring to the preparation method of compound 2 in Example 1, only compound 1 was replaced by compound 5 to obtain white solid compound I-8 (720 mg, yield 23%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.10-12.03(s,2H),5.67-5.63(s,1H),5.53-5.50(s,1H),4.37-4.31(dd,J=11.4,4.9H z,1H),2.94-2.86(td,J=10.7,4.8Hz,1H),2.24-2.16(m,J=10.1,5.0Hz,3H),2.09-2.03(d, J=9.3Hz,1H),1.81-1.72(m,2H),1.53-1.45(m,5H),1.39-1.24(dd,J=20.9,11.2Hz,11H), 1.14-1.03(m,2H),0.93-0.89(s,4H),0.85-0.81(m,7H),0.77-0.71(d,J=5.8Hz,11H); HRMS calcd for C 34 H 53 O 6 [M+H] + m / z557.78400, found 557.49300.

[0085] Example 10

[0086] Preparation of 3β-(succinic acid monoacyloxy) lupin-20(29)-ene-28,30-dioic acid (I-9), the structural formula of which is The specific method is as follows: referring to the preparation method of compound I-8 in Example 9, only butyric anhydride is replaced by succinic anhydride to obtain white solid compound I-9 (700 mg, yield 21%). 1 H NMR (400 MHz, DMSO-d 6)δ12.26-11.96(s,3H),5.66-5.63(s,1H),5.54-5.50(s,1H),4.37-4.31(dd,J=11.6,4.7Hz ,1H),2.93-2.86(m,1H),2.49-2.45(d,J=1.9Hz,4H),2.21-2.14(m,1H),2.11-2.04(d,J=9.0 Hz,1H),1.79-1.72(m,2H),1.58-1.53(m,2H),1.51-1.40(m,4H),1.37-1.24(m,10H),1.14-1 .03(m,2H),0.93-0.89(s,4H),0.84-0.82(s,3H),0.78-0.75(s,4H),0.75-0.70(s,6H); HRMS calcd for C 34 H 51 O 8 [M+H] + m / z 587.76600, found 587.78600.

[0087] Embodiment 11

[0088] Preparation of 3β-(succinic acid monoacyloxy) lupin-20(29)-ene-28,30-dioic acid (I-10), the structural formula of which is The specific method is as follows: Referring to the preparation method of compound I-8 in Example 9, only butyric anhydride is replaced by phthalic anhydride to obtain white solid compound I-10 (590 mg, yield 20%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.10-12.03(s,3H),8.17-8.03(m,2H),7.67-7.63(m,2H),5.67-5.63(s,1H),5.53- 5.50(s,1H),4.37-4.31(dd,J=11.4,4.9Hz,1H),2.24-2.16(m,J=10.1,5.0Hz,3H),2.0 9-2.03(d,J=9.3Hz,1H),1.81-1.72(m,2H),1.53-1.45(m,3H),1.39-1.24(dd,J=20.9, 11.2Hz,11H),1.14-1.03(m,2H),0.85-0.81(m,7H),0.77-0.71(d,J=5.8Hz,11H); HRMS calcd for C 38 H 51 O 8 [M+H]+ m / z 635.81000, found 635.57300.

[0089] Example 12

[0090] Preparation of 3β-hydroxylupin-30-acid-28-carboxyl (1-methyl-4-piperidinyl) ester (I-11), the synthetic route is as follows:

[0091]

[0092] The specific preparation method is as follows:

[0093] In a 100 mL round-bottom flask, compound I-5 (3.9 g, 6.6 mmol) was dissolved in tetrahydrofuran (20 mL), 5% Pd-C (319 mg, 2.6 mmol) was added, hydrogen was passed, and the reaction was stirred at room temperature. The raw material disappeared after TLC determination, and the product was filtered and concentrated by vacuum distillation to obtain a solid crude product, which was purified by silica gel column chromatography (eluent volume ratio: petroleum ether / ethyl acetate = 90 / 1) to obtain a white solid compound I-11 (3.2 g, yield 83%). 1 H NMR (400 MHz, DMSO-d 6 )δ10.36-10.03(s,1H),5.05-4.98(m,1H),4.25-4.19(s,1H),3.22-3.17(d,J=9.0Hz,1H),2.96-2.90(s,1H),2.56-2.41(d, J=11.30Hz,4H),2.29-2.24(m,1H),2.23-2.19(s,3H),2.16-2.07(t,J=14.3Hz,2H),1.95-1.89(d,J=15.9Hz,1H),1.85-1.79 (d,J=11.9Hz,2H),1.77-1.71(t,J=10.5Hz,4H),1.58-1.53(s,2H),1.42-1.35(d,J=12.3Hz,5H),1.28-1.20(d,J=25.0Hz,6H ),1.13-1.02(t,J=13.4Hz,3H),1.03-0.93(m,3H),0.91-0.82(d,J=15.1Hz,10H),0.76-0.69(s,4H),0.62-0.57(s,4H); HRMS calcd for C 36 H 60 NO 5 [M+H] + m / z586.87000, found 586.75000.

[0094] Embodiment 13

[0095] Preparation of 3β-(succinic acid monoacyloxy) lupin-28,30-diacid (I-12), the structural formula of which is The specific method is as follows: Referring to the preparation method of compound I-11 in Example 12, only compound I-4 is replaced by compound I-9 to obtain white solid compound I-12 (1.3 mg, yield 84%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.26-11.96(s,2H),10.11(s,1H),4.37-4.31(dd,J=11.6,4.7Hz,1H),2.93-2.86( m,1H),2.49-2.45(d,J=1.9Hz,4H),2.21-2.14(m,2H),2.11-2.04(d,J=9.0Hz,1H),1.7 9-1.72(m,5H),1.58-1.53(m,2H),1.51-1.40(m,4H),1.37-1.24(m,10H),1.14-1.03(m ,2H),0.93-0.89(s,4H),0.84-0.82(s,3H),0.78-0.75(s,4H),0.75-0.70(s,6H); HRMS calcd for C 34 H 53 O 8 [M+H] + m / z 589.78200, found 589.76500.

[0096] Embodiment 14

[0097] Preparation of potassium 3β-hydroxylupin-20(29)-ene-28-oate (6), the synthetic route is as follows:

[0098]

[0099] The specific method is as follows:

[0100] Add 5g of KOH to 50mL of ethanol solution, stir under ultrasound or heating at 40°C until dissolved, and prepare a 10% W / V KOH ethanol solution. Add betulinic acid (compound 1, 20g) to 434mL of ethanol in a 1000mL round-bottom flask, add KOH-ethanol solution (10% W / V, 24.7mL) dropwise, stir at room temperature for 24h, spin dry, and dry to obtain 18g of white solid (yield: 90%).

[0101] Embodiment 15

[0102] Preparation of potassium 3β-hydroxylupin-20(29)-ene-28,30-dioate (7), the synthetic route is as follows:

[0103]

[0104] The specific method is as follows:

[0105] Add 10g of KOH to 50mL of ethanol solution, stir under ultrasound or heating at 40°C until dissolved, and prepare a 20% W / V KOH ethanol solution. Add betulinic acid (compound 1, 20g) to 434mL of ethanol in a 1000mL round-bottom flask, add KOH-ethanol solution (20% W / V, 24.7mL) dropwise, stir at room temperature for 24h, spin dry, and dry to obtain 22g of white solid (yield: 90%).

[0106] Application Example 1 Effects of betulinic acid (compound 1), betulinic acid derivative 2, and I-1 to I-12 on telomerase activity in 293T cells

[0107] Experimental materials and main instruments:

[0108] Microplate reader (brand: Bio-Tek model: SYNERGY H1), centrifuge (Shanghai Anting Feige centrifuge TGL-16G), telomerase activity assay kit (Merck Millipore, USA), ultrapure water prepared by ultrapure water system, and other reagents were of analytical grade.

[0109] Experimental methods:

[0110] (1) Experimental grouping and drug administration: 293T cells were used and seeded in six-well plates. The top three wells on each plate were set as the control group, and the bottom three wells were set as the drug administration group. Drugs (BA series compounds (i.e., compound 1, betulinic acid derivative 2, and I-1 to I-12), one at a time) were dissolved in DMSO (dimethyl sulfoxide). The final drug concentration of the drug administration group was 20 μM. The control group was given the same amount of DMSO as the drug administration group, and the drug administration time was 2 hours.

[0111] (2) Experimental operation steps:

[0112] 1) Sample preparation:

[0113] Wash the attached 293T cells, collect the cells in a 15mL centrifuge tube, and centrifuge at 4°C and 1000 rpm for 5 minutes. After centrifugation, discard the supernatant, add 200μL chaps lysis solution (provided with the kit) to resuspend the cells, blow and mix, transfer the liquid to a new 1.5mL enzyme-free EP tube, and place it on ice for 30 minutes. After the lysis is completed, use a high-speed centrifuge to centrifuge at 4°C and 12000 rpm for 20 minutes. The resulting supernatant is the sample, ready for PCR loading.

[0114] 2) PCR amplification:

[0115] Set up sample wells (12 wells, n=6), Yang ginseng wells, Yin ginseng wells, and Taq enzyme-free wells. For sample wells and Yang ginseng wells, add 10μL 5x TRAPEXE@XL reaction Mix, 0.4μL Taq Polymerase (5 units / μL), 37.6μL PCRGrade water, and 2μL sample or Yang ginseng (Yang ginseng in the kit, i.e. TSR8) to each well; for Yin ginseng wells, directly add 50μL chaps lysis lysis buffer; for Taq enzyme-free wells, add 10μL 5x TRAPEXE@XL reaction Mix, 37.6μL PCRGrade water, and 2.4μL chaps lysis lysis buffer, and the total system is 50μL. Centrifuge after sample loading and run PCR on the machine.

[0116] 3) PCR program settings

[0117]

[0118] 4) Fluorescence microplate reader reading

[0119] Add 150 μL of fluorescence buffer (1 mL of Tris HCl (tris(hydroxymethyl)aminomethane hydrochloride, 0.05 mol / L), 0.88 g of NaCl and 0.04 g of MgCl per 100 mL of pure water) to each well of a black 96-well plate. 2 6H 2 O), then add 50 μL of the sample after PCR, and place it in an enzyme-labeled fluorometer to read the readings at a wavelength of 485-535 nm using Fluorescence intensity and at a wavelength of 590-620 nm using TR Fluorescence intensity.

[0120] 5) Data calculation

[0121] Sample wells, Yang Shen Yin Shen, and Taq enzyme-free wells all have two readings, FL and TR. For each sample well, calculate ΔFL (sample FL reading minus Yin Shen FL reading) and ΔR (sample TR reading minus Taq enzyme-free TR reading), and calculate LOG (ΔFL / ΔR) to get the relative telomerase activity reading.

[0122] Experimental results:

[0123] like Figure 1 As shown in the results, betulinic acid (compound 1) and betulinic acid derivative 2 can effectively increase the telomerase activity of 293T cells, betulinic acid derivatives I-9 and I-10 can more significantly increase the telomerase activity of 293T cells, and the remaining compounds have a tendency to increase the telomerase activity. The results of the effect of compound I-9 on cell telomerase activity at different concentrations are shown in the figure below. Figure 2 As shown (Vehicle is the control group, "ns", "*", and "**" are no difference, significant difference (p<0.05), and significant difference (p<0.01), respectively), compound I-9 can be effective at concentrations of 20μM, 10μM, 1μM, and 0.1μM.

[0124] Application Example 2 Effect of Compound I-9 on Natural Aging of Drosophila

[0125] The life span and crawling ability of fruit flies were tested by oral administration. The experimental method is as follows:

[0126] like Figure 3 The control group and the treatment group of fruit flies were set up as shown: the control group had a normal diet, and the treatment group added compound I-9 to the diet, with concentrations of 50 μM and 20 μM, respectively, and the administration time was long-term administration until the end of the life of the fruit flies. The number of fruit flies was tested every week, and the crawling ability of the fruit flies was tested after one and a half months.

[0127] Experimental results:

[0128] The effects of the compounds obtained in Examples 1 to 12 on the telomerase activity of 293T cells are as follows: Figure 3 As shown, compound I-9 can significantly increase telomerase activity.

[0129] Application Example 3 Rotarod test

[0130] Compound I-9 was tested in a rotating rod fatigue test on mice. The experimental method was as follows: two days before the formal test, the mice were subjected to adaptive training, and the training intensity was set to 1+0 revolutions per minute (10r / min), and each training lasted for 10 minutes. A 20-minute rest period was left between each training session so that the mice could recover from fatigue. During the formal experiment, the mice were placed on the rotating rod, and the rotation speed was gradually increased from 4 revolutions per minute (4r / min) and fixed to 40 revolutions per minute (40r / min), and the duration was 5 minutes. The time the mice remained on the rotating rod without falling was recorded. If the mouse can stay on the rotating rod for more than 300 seconds, the recording time is 300 seconds.

[0131] Experimental results: Figure 4 As shown, compound I-9 was found to have a slight anti-fatigue effect.

[0132] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A betulinic acid derivative, characterized in that: The structural formula is shown in Formula I: Among them, R 1 is H or acyl; X is O or NH; Represents a double bond or a single bond; R 2 It is H, C1-C5 straight-chain alkyl, C3-C6 monocyclic alkyl, polycyclic alkyl or substituted / unsubstituted six-membered heterocyclic group containing one nitrogen or oxygen atom.

2. The betulinic acid derivative according to claim 1, characterized in that R 1 is H, n-propionyl, n-butyryl, benzoyl, X is O or NH; Represents a double bond or a single bond; R 2 H, CH3, 3. The betulic acid derivative according to claim 2, characterized in that R 1 H, n-butyryl, X is O or NH; Represents a double bond or a single bond; R 2 H, CH3, 4. The betulic acid derivative according to claim 3, characterized in that R 1 is H, X is O or NH, Represents a double bond or a single bond, R 2 For H. or R 1 H, n-butyryl, X is O, Represents a double bond or a single bond, R 2 For H.

5. The betulic acid derivative according to claim 1, characterized in that Selected from the following compounds:

6. A method for preparing the betulic acid derivative according to any one of claims 1 to 5, characterized in that: The following steps are involved: Betulinic acid (1) undergoes a condensation reaction with an alcohol or an amine compound to obtain a compound of formula II; betulinic acid (1) undergoes an esterification reaction with an acid anhydride compound to obtain a compound of formula III; the compound of formula II and the compound of formula III are sequentially subjected to selenium dioxide oxidation, Pinnick oxidation reaction, or further hydrogenation reduction to obtain a compound of formula I. The reaction scheme is as follows: Among them, R 1 is H or acyl; X is O or NH; Represents a double bond or a single bond; R 2 It is H, C1-C5 straight-chain alkyl, C3-C6 monocyclic alkyl, polycyclic alkyl or substituted / unsubstituted six-membered heterocyclic group containing one nitrogen or oxygen atom.

7. Use of the betulic acid derivative according to any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof in the preparation of a drug for regulating telomerase activity.

8. The use according to claim 7, characterized in that: The betulinic acid derivative, its pharmaceutically acceptable salt, tautomer, stereoisomer, nitrogen oxide, hydrate or prodrug has the function of positively regulating telomerase activity, maintaining telomere length and maintaining the RNA expression amount of telomerase Tert.

9. Use of the betulic acid derivative according to any one of claims 1 to 5, or its pharmaceutically acceptable salt, tautomer, stereoisomer, nitrogen oxide, hydrate or prodrug in the preparation of a drug for preventing or treating stress-related and aging-related diseases.

10. The use according to claim 9, characterized in that: The stress resistance and aging-related diseases include Alzheimer's disease, cognitive impairment, and decreased immune system function.

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