Betulinic acid hydroximic acid derivative and application thereof

Abstract

The invention discloses a betulinic acid hydroxamic acid derivative or a pharmaceutical salt thereof. The structure of the betulinic acid hydroxamic acid derivative is selected from one of the following structures: # imgabs0 #. The invention also provides an application of the betulinic acid hydroxamic acid derivative or the pharmaceutical salt thereof in preparation of a CD73 small-molecule inhibitor. The invention provides a novel betulinic acid hydroximic acid derivative which has good CD73 inhibitory activity, and part of compounds show activity superior to that of betulinic acid, so that the betulinic acid hydroximic acid derivative can be used as a CD73 inhibitor antitumor drug.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a betulinic acid hydroxamic acid derivative and application thereof. Background Art

[0002] CD73, also known as extracellular-5'-nucleotidase (ecto-5'-nucleotidase, eNT), belongs to the metallophosphatase superfamily. It is a multifunctional glycoprotein encoded by the NT5E gene. It mainly exerts its biological function by catalyzing the hydrolysis of AMP into ADO. CD73 is widely distributed on the surface of various types of cells in human tissues, including lymphocytes, endothelial cells, and epithelial cells. The results of clinical tumor sample analysis showed that CD73 is highly expressed in a variety of solid tumors, such as lung cancer, prostate cancer, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, kidney cancer, head and neck cancer, etc. It is a potential biomarker that is closely related to tumor staging, pathological type and prognosis. Therefore, the development of drugs targeting CD73 has received widespread attention from many researchers. At present, multiple CD73 monoclonal antibodies and small molecule inhibitor drugs have entered clinical trial research. Small molecule inhibitors have the characteristics of being orally ingestible, relatively small in molecular weight, and able to penetrate cell membranes, making the research on CD73 small molecule inhibitors increasingly attract the attention of drug researchers. Several candidate drugs such as AB680, ORIC-533, LY3475070, ATG-037, BPI-472372, and ABSK051 have entered clinical trials for the treatment of patients with hematological tumors, solid tumors, etc. New structures of CD73 small molecule inhibitors are also constantly being reported, but so far, no drugs have been approved for marketing. Therefore, there is still a need to continuously develop new structures and highly active CD73 small molecule inhibitors. Summary of the invention

[0003] The purpose of the present invention is to provide a betulinic acid hydroxamic acid derivative.

[0004] Another object of the present invention is to provide a use of the betulic acid hydroxamic acid derivative in the preparation of CD73 small molecule inhibitors.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect of the present invention, there is provided a betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof, wherein the structure is selected from one of the following structures:

[0007]

[0008] in:

[0009] X is selected from -(CH2) m1 NH-,

[0010] Y is selected from -(CH2) m1 NH-,

[0011] m1 is selected from 0 to 20 (preferably 0, 1, 2, 3, 4, 5, 6, 7, 8);

[0012] n is selected from 0 to 20 (preferably 0, 1, 2, 3, 4, 5);

[0013] m2 is selected from 0 to 20 (preferably 0, 1, 2, 3, 4, 5, 6, 7, 8).

[0014] Preferably, in the betulic acid hydroxamic acid derivative,

[0015] and

[0016]

[0017] Y is selected from -NH-, -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2CH2CH2CH2NH-, -CH2CH2CH2CH2CH2CH2NH-, -CH2CH2CH2CH2CH2CH2CH2NH-,

[0018]

[0019] Most preferably, the structure of the betulic acid hydroxamic acid derivative is selected from one of the following structures:

[0020]

[0021]

[0022] The pharmaceutically acceptable salt refers to a salt formed by the reaction of a betulinic acid hydroxamic acid derivative with a pharmaceutically acceptable inorganic acid or organic acid; wherein the inorganic acid is at least one of hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid or sulfuric acid; and the organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalenedisulfonic acid, asiatic acid, carbenoxolone acid, glycyrrhetinic acid, oleanolic acid, hawthorn acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acids.

[0023] The second aspect of the present invention provides a use of the betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof in the preparation of a CD73 small molecule inhibitor.

[0024] The third aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof as the sole active ingredient.

[0025] The mass percentage of the betulic acid hydroxamic acid derivative or its pharmaceutically acceptable salt is 0.1-99wt%.

[0026] The fourth aspect of the present invention provides a pharmaceutical preparation, which is prepared from the betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof and excipients commonly used in pharmacy.

[0027] The dosage form of the pharmaceutical preparation is selected from at least one of injection, injection emulsion, tablet, pill, capsule, ointment, cream, patch, liniment, powder, spray, implant, drop, suppository, ointment or nano preparation, wherein: the injection is at least one of small volume injection, medium volume injection or large volume injection, and the nano preparation is liposome.

[0028] The fifth aspect of the present invention provides a use of the betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0029] The anti-tumor drug is used to treat at least one malignant tumor growing in the esophagus, stomach, intestine, rectum, oral cavity, pharynx, larynx, lung, colon, breast, uterus, endometrium, ovary, prostate, testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain and central nervous system of human or animal, and at least one of thyroid cancer, leukemia, Hodgkin's disease, lymphoma and myeloma.

[0030] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0031] The natural product betulinic acid has certain CD73 inhibitory activity (Yanming Zhang, et al. Bioorganic & Medicinal Chemistry, 2022, 59, 116672). In order to further improve the CD73 inhibitory activity of betulinic acid, the present invention provides a new betulinic acid hydroxamic acid derivative with good CD73 inhibitory activity. Some compounds show activity better than betulinic acid, and thus can be used as CD73 inhibitor anti-tumor drugs. DETAILED DESCRIPTION

[0032] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0033] Example 1

[0034] A method for preparing a betulinic acid hydroxamic acid derivative comprises the following steps:

[0035]

[0036] Place betulinic acid (4.00 g, 8.8 mmol) in a 250 mL two-necked flask, add DMF (60 mL) and stir to dissolve. Then add anhydrous potassium carbonate (3.03 g, 21.9 mmol) and benzyl bromide (1.79 g, 10.5 mmol), heat to 65 ° C and react for 3.5 h. After the reaction is complete, cool to room temperature, add 200 mL of water, and extract with ethyl acetate. The organic phase is washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure. The residue is purified by column chromatography to obtain 4.30 g of white solid, namely intermediate 1, with a yield of 89.9%. 1H NMR(400MHz,Chloroform-d)δ7.42–7.29(m,5H),5.12(q,J=12.3Hz,2H),4.72(s,1H),4.59(s,1H),3.17(d d,J=11.2,5.0Hz,1H),3.02(td,J=11.0,4.6Hz,1H),2.28(dt,J=11.6,3.0Hz,1H),2.23–2.13(m,1H),1.95 –1.83(m,2H),1.67(s,5H),1.62–1.54(m,3H),1.54–1.43(m,2H),1.43–1.22(m,11H),1.13–1.06(m,1H),0 .95(d,J=5.4Hz,6H),0.87(dd,J=12.9,4.3Hz,1H),0.80(s,3H),0.75(d,J=4.0Hz,6H),0.69–0.63(m,1H).

[0037] Place intermediate 1 (2.37 g, 4.3 mmol) in a 250 mL single-necked flask, add DCM (20 mL) and stir to dissolve. Then add DAMP (0.58 g, 4.8 mmol) and 4-nitrophenyl chloroformate (1.75 g, 8.7 mmol), and react at room temperature for 12 h. After the reaction, add 100 mL of saturated brine and extract with dichloromethane. The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure. The residue is purified by column chromatography to obtain 2.09 g of white solid, namely intermediate 2, with a yield of 67.7%. 1H NMR(400MHz,Chloroform-d)δ8.27(d,J=9.1Hz,2H),7.43–7.30(m,7H),5.12(q,J=12.3Hz,2H),4.72(s,1H),4.60(s,1H),4.43(d d,J=10.7,5.5Hz,1H),3.02(td,J=10.9,4.6Hz,1H),2.28(dt,J=11.8,3.0Hz,1H),2.19(td,J=12.2,3.4Hz,1H),1.95–1.86(m,2H ),1.80(dd,J=9.2,4.7Hz,2H),1.77–1.71(m,2H),1.68(s,3H),1.56(s,3H),1.50(s,1H),1.43–1.37(m,4H),1.36–1.30(m,4H),1 .28–1.22(m,3H),1.10(d,J=12.9Hz,1H),1.02(d,J=4.9Hz,1H),0.97(s,3H),0.94(s,3H),0.89(s,3H),0.85(s,3H),0.77(s,4H).

[0038] Place intermediate 2 (1.00 g, 1.4 mmol) in a 250 mL single-necked flask, add 20 mL of dichloromethane and stir to dissolve. Then add triethylamine (1.00 g, 9.8 mmol) and 3-aminopropionic acid methyl ester hydrochloride (0.98 g, 7.0 mmol), and react at room temperature for 12 h. After the reaction is complete, add 100 mL of saturated brine and extract with dichloromethane. The organic phase is washed with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure. The residue is purified by column chromatography to obtain 0.90 g of white solid, namely intermediate 3, with a yield of 94.8%.

[0039] Place intermediate 3 (200 mg, 0.3 mmol) in a 100 mL single-necked flask, add 5 mL of methanol and 5 mL of dichloromethane, stir and dissolve. Then add excess 50% hydroxylamine aqueous solution (2 mL) and sodium hydroxide (23.7 mg, 0.6 mmol), and react at room temperature for 12 hours. After the reaction is complete, dichloromethane and methanol are evaporated under reduced pressure, and the residue is extracted with ethyl acetate. The organic phase is washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure. The residue is purified by column chromatography to obtain 0.22 g of light yellow solid, namely intermediate 4, with a yield of 99.5%.

[0040] Intermediate 4 (217 mg, 0.3 mmol) was placed in a 100 mL single-necked flask, and THF (25 mL) was added and stirred to dissolve. Then 10% Pd / C (481 mg, 0.03 mmol) was added and hydrogen was catalyzed for hydrogenolysis. After the reaction was completed, it was filtered with diatomaceous earth, and the filtrate was evaporated under reduced pressure to remove the solvent. The residue was purified by column chromatography to obtain a white solid, namely compound Ia, with a yield of 19.2%. 1 H NMR (400MHz, DMSO-d6) δ12.08(s,1H),10.41(s,1H),8.73(s,1H),6.96(t,J=5.6Hz,1H),4.70(d,J= 2.6Hz,1H),4.61–4.54(m,1H),4.19(t,J=8.1Hz,1H),3.24–3.08(m,2H),2.95(td,J=10.4,4.8Hz,1H ),2.23(t,J=12.0Hz,1H),2.12(s,3H),1.88–1.74(m,2H),1.65(s,5H),1.57–1.49(m,3H),1.39(s, 11H),1.25–1.07(m,3H),0.95(s,5H),0.88(s,3H),0.78(d,J=12.9Hz,9H).HRMS(ESI,positive)m / z calcd for C 34 H 54 N2O6[M+Na] + :609.3879; found609.3879.

[0041] Example 2

[0042]

[0043] The preparation route and specific operation process of compound Ib are basically the same as the preparation route and specific operation process of compound Ia in Example 1, except that 4-aminobutyric acid methyl ester hydrochloride is used instead of 3-aminopropionic acid methyl ester hydrochloride in Example 1 to obtain a white solid, namely compound Ib, with a yield of 17.6%. 1H NMR (400MHz, DMSO-d6) δ11.94(s,1H),10.36(s,1H),8.69(s,1H),6.98(s,1H),4.69(d,J=3.0Hz,1H),4.5 7(s,1H),4.19(t,J=8.2Hz,1H),2.94(s,3H),2.17(dd,J=38.0,10.7Hz,3H),1.93(t,J=7.7Hz,2H),1.80(d ,J=7.5Hz,2H),1.65(s,3H),1.61–1.55(m,4H),1.52(s,2H),1.44(s,2H),1.39(s,2H),1.34(s,3H),1.24 (s,6H),0.94(d,J=7.8Hz,4H),0.88(s,3H),0.79(d,J=10.4Hz,9H),0.69(s,1H).HRMS(ESI,negative)m / z calcd for C 35 H 56 N2O6[MH] - :599.4060; found599.4060.

[0044] Example 3

[0045]

[0046] The preparation route and specific operation process of compound Ic are basically the same as the preparation route and specific operation process of compound Ia in Example 1, except that 5-aminovaleric acid methyl ester hydrochloride is used instead of 3-aminopropionic acid methyl ester hydrochloride in Example 1 to obtain a white solid, namely compound Ic, with a yield of 36.5%. 1H NMR (400MHz, DMSO-d6) δ12.05(s,1H),10.33(s,1H),8.66(s,1H),6.94(t,J=5.7Hz,1H),4.70(d,J=2.6Hz,1H),4.57(t,J=2.0Hz,1H),4.23–4.13(m, 1H),3.01–2.85(m,3H),2.29–2.17(m,1H),2.12(d,J=8.6Hz,1H),1.93(t, J=7.2Hz,2H),1.80(q,J=6.3,5.5Hz,2H),1.65(s,3H),1.63–1.57(m,2H), 1.53(t,J=11.3Hz,3H),1.46(dd,J=9.0,6.0Hz,3H),1.41(d,J=4.2Hz,1H) ,1.36(dd,J=14.5,8.6Hz,8H),1.30(q,J=2.3Hz,1H),1.27–1.23(m,1H),1 .17(d,J=12.4Hz,1H),1.10(dd,J=11.1,3.8Hz,1H),1.01(d,J=4.3Hz,1H),0.95(s,5H),0.88(s,3H),0.79(d,J=9.8Hz,9H).HRMS(ESI,negative)m / z calcd for C 36 H 58 N2O6[MH] - :613.4217; found613.4211.

[0047] Example 4

[0048]

[0049] The preparation route and specific operation process of compound Id are basically the same as the preparation route and specific operation process of compound Ia in Example 1, except that 2-(piperazine-1-yl)acetic acid methyl ester hydrochloride is used instead of 3-aminopropionic acid methyl ester hydrochloride in Example 1 to obtain a light yellow solid, namely compound Id, with a yield of 37.6%. 1H NMR (400MHz, DMSO-d6) δ10.46(s,1H),8.79(s,1H),4.70(d,J=2.6Hz,1H),4.57(t,J=2.1Hz,1H),4.25–4.18(m,1H),3.41( d,J=23.8Hz,4H),2.93(d,J=24.3Hz,3H),2.40(s,4H),2.30–2.19(m,1H),2.12(d,J=7.6Hz,1H),1.80(q,J=7.1,5.8Hz,2H ),1.65(s,3H),1.64–1.52(m,5H),1.51–1.44(m,2H),1.40–1.30(m,8H),1.25(d,J=5.0Hz,3H),1.17(dd,J=12.9,4.3Hz,1 H),1.13–1.08(m,1H),1.03(dd,J=18.5,5.6Hz,1H),0.95(s,3H),0.88(s,3H),0.82–0.79(m,9H); HRMS(ESI,negative)m / z calcd for C 37 H 59 N3O6[MH] - :640.4326; found640.4321.

[0050] Example 5

[0051]

[0052] The preparation route and specific operation process of compound Ie are basically the same as the preparation route and specific operation process of compound Ia in Example 1, except that 4-(aminomethyl)benzoic acid methyl ester is used instead of 3-aminopropionic acid methyl ester hydrochloride in Example 1 to obtain a light yellow solid, namely compound Ie, with a yield of 47.5%. 1H NMR (400MHz, DMSO-d6) δ11.96(s,1H),11.21(s,1H),7.71(d,J=7.9Hz,2H),7.63(s,1H),7.29(d,J=8.1Hz,2H),4.69(d,J=2 .6Hz,1H),4.56(s,1H),4.21(d,J=6.1Hz,3H),3.01–2.90(m,1H),2.22(s,1H),2.12(d,J=8.8Hz,1H),1.80(q,J=6.7,5.7Hz, 2H),1.65(s,4H),1.56–1.51(m,2H),1.50–1.41(m,3H),1.35(dd,J=15.5,7.0Hz,6H),1.30(s,2H),1.24(s,3H),1.16(d,J=1 0.6Hz,1H),1.12–1.08(m,1H),0.95(s,3H),0.88(s,3H),0.84–0.77(m,9H),0.63(d,J=8.6Hz,1H).HRMS(ESI,negative)m / z calcd for C 39 H 56 N2O6[MH] - :647.4060; found647.4066.

[0053] Example 6

[0054]

[0055] The preparation route and specific operation process of compound If are basically the same as those of compound Ia in Example 1, except that 4-aminobenzoic acid methyl ester is used instead of 3-aminopropionic acid methyl ester hydrochloride in Example 1 to obtain a light yellow solid, namely compound If, with a yield of 96.6%. 1H NMR (400MHz, DMSO-d6) δ12.06(s,1H),11.09(s,1H),9.75(s,1H),8.92(s,1H),7.69(d,J=8.4Hz,2H),7.53(d,J=8.4Hz,2H),4.70(d,J=2.6Hz ,1H),4.57(s,1H),4.36(d,J=8.9Hz,1H),3.03–2.88(m,1H),2.32–2.1 9(m,1H),2.12(d,J=10.0Hz,1H),1.81(d,J=7.6Hz,2H),1.65(d,J=8.6H z,6H),1.55(d,J=11.3Hz,1H),1.46(d,J=11.7Hz,2H),1.39(d,J=7.6Hz,4H),1.33(d,J=7.1Hz,3H),1.24(s,3H),1.19(d,J=12.9Hz,1H),1.1 1(d,J=9.8Hz,1H),1.02(d,J=1.5Hz,1H),0.96(s,3H),0.89(s,3H),0.87(s,6H),0.84(s,3H),0.74(d,J=6.6Hz,1H).HRMS(ESI,negative)m / z calcd for C 38 H 54 N2O6[MH] - :633.3904; found 633.3909.

[0056] Example 7

[0057]

[0058] 1 g (2.20 mmol) of betulinic acid was placed in a 250 mL single-necked flask, and 100 mL of MeOH / THF (MeOH, THF volume ratio is 1:1) was added and stirred to dissolve, and then 10% Pd / C (3.30 g, 2.20 mmol) was added, and the reaction was completed by hydrogen catalysis. Pd / C was removed by filtration under reduced pressure on diatomaceous earth, and the solvent was evaporated from the filtrate under reduced pressure to obtain 0.971 g of white solid, namely intermediate 5, with a yield of 96.7%.

[0059] The synthesis method was similar to the preparation method of compound Ia in Example 1, except that intermediate 5 was used instead of betulinic acid to obtain a light yellow solid, compound IIa, with a yield of 36.0%. 1H NMR (400MHz, DMSO-d6) δ11.98(s,1H),10.40(s,1H),8.72(s,1H),6.95(t,J=5.6Hz,1H),4.20(t,J=8.1Hz,1 H),3.16(dt,J=12.8,6.9Hz,2H),2.24(d,J=11.4Hz,1H),2.13(t,J=7.5Hz,4H),1.73(dd,J=12.5,6.5Hz,2H ),1.67–1.50(m,5H),1.45–1.32(m,8H),1.25(d,J=5.7Hz,5H),1.18(t,J=11.8Hz,2H),1.11–1.06(m,1H),0 .93(s,3H),0.88(s,3H),0.82(t,J=5.2Hz,9H),0.78(s,3H),0.74(d,J=6.6Hz,3H).HRMS(ESI,negative)m / z calcd for C 34 H 56 N2O6[MH] - :587.4060; found 587.4046.

[0060] Example 8

[0061]

[0062] The preparation route and specific operation process of compound IIb are basically the same as those of compound IIa in Example 7, except that 4-aminobutyric acid methyl ester hydrochloride is used instead of 3-aminopropionic acid methyl ester hydrochloride in Example 7 to obtain a light yellow solid, namely compound IIb, with a yield of 40.7%. 1H NMR(400MHz, DMSO-d6)δ11.98(s,1H),10.35(d,J=1.7Hz,1H),8.68(s,1H),7.05–6.93(m,1H),4.20(t, J=8.1Hz,1H),2.95(s,2H),2.27–2.10(m,3H),2.05–1.89(m,2H),1.73(dd,J=12.8,6.4Hz,2H),1.66–1. 52(m,6H),1.49–1.28(m,12H),1.25(d,J=9.2Hz,6H),1.18(t,J=10.3Hz,2H),1.13–1.05(m,2H),0.93( s,3H),0.88(s,3H),0.82(d,J=7.4Hz,9H),0.79(s,3H),0.74(d,J=6.7Hz,3H).HRMS(ESI,negative)m / z calcd for C 35 H 58 N2O6[MH] - :601.4217; found601.4218.

[0063] Example 9

[0064]

[0065] The preparation route and specific operation process of compound IIc are basically the same as the preparation route and specific operation process of compound IIa in Example 7, except that 5-aminovaleric acid methyl ester hydrochloride is used instead of 3-aminopropionic acid methyl ester hydrochloride in Example 7 to obtain a light yellow solid, namely compound IIc, with a yield of 80.9%. 1H NMR (400MHz, DMSO-d6) δ11.98(s,1H),10.40(s,1H),8.68(s,1H),6.96(t,J=5.5Hz,1H),4.19(t,J=8.1Hz,1H ),2.93(d,J=7.0Hz,2H),2.27–2.09(m,3H),1.94(t,J=7.3Hz,2H),1.78–1.69(m,2H),1.62–1.50(m,4H),1.4 7–1.41(m,5H),1.39–1.33(m,5H),1.30(d,J=3.1Hz,2H),1.24(d,J=5.4Hz,6H),1.16(d,J=11.4Hz,2H),1.10 –1.05(m,1H),0.93(s,3H),0.88(s,3H),0.84–0.77(m,12H),0.73(d,J=6.6Hz,3H).HRMS(ESI,negative)m / z calcd for C 36 H 60 N2O6[MH] - :615.4373; found 615.4371.

[0066] Example 10

[0067]

[0068] The preparation route and specific operation process of compound IId are basically the same as those of compound IIa in Example 7, except that 2-(piperazine-1-yl)acetic acid methyl hydrochloride is used instead of 3-aminopropionic acid methyl hydrochloride in Example 7 to obtain a light yellow solid, namely compound IId, with a yield of 63.6%. 1H NMR (400MHz, DMSO-d6) δ11.97(s,1H),10.48(s,1H),8.80(s,1H),4.23(t,J=8.1Hz,1H),3.42(s, 4H),2.91(s,2H),2.51(p,J=1.8Hz,9H),2.25(s,1H),2.21–2.07(m,2H),1.80–1.69(m,2H),1.65– 1.54(m,4H),1.47–1.30(m,10H),1.25(q,J=5.2,4.4Hz,5H),1.16(d,J=11.5Hz,1H),1.10(s,1H), 0.93(s,3H),0.88(s,3H),0.81(d,J=7.3Hz,12H),0.74(d,J=6.7Hz,3H).HRMS(ESI,negative)m / z calcd for C 37 H 61 N3O6[MH] - :642.4482; found642.4482.

[0069] Embodiment 11

[0070] A method for preparing a pharmaceutical salt of a betulic acid hydroxamic acid derivative:

[0071] The compounds Ia-If and IIa-IId prepared in the present invention are reacted with pharmaceutically acceptable inorganic acids or organic acids to obtain the corresponding pharmaceutical salts of betulinic acid hydroxamic acid derivatives.

[0072] In the preparation process of the above-mentioned pharmaceutical salt of betulic acid hydroxamic acid derivative: the inorganic acid is at least one of hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid or sulfuric acid; the organic acid is at least one of formic acid, acetic acid, propionic acid, succinic acid, 1,5-naphthalene disulfonic acid, asiatic acid, carbenoxolone acid, glycyrrhetinic acid, oleanolic acid, hawthorn acid, ursolic acid, corosolic acid, betulic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid or amino acid.

[0073] Example 12

[0074] A pharmaceutical composition prepared from betulinic acid hydroxamic acid derivatives:

[0075] A therapeutically effective amount of the compounds Ia-If and IIa-IId prepared in the present invention is mixed or dissolved with one or more pharmaceutically acceptable carriers, excipients and auxiliary materials to obtain a corresponding pharmaceutical composition.

[0076] Embodiment 13

[0077] A pharmaceutical preparation prepared from a betulinic acid hydroxamic acid derivative:

[0078] Take compounds Ia~If, IIa~IId prepared by the present invention, and perform corresponding operations according to the preparation methods of existing pharmaceutical preparations to prepare self-injections, injection emulsions, tablets, pills, capsules, ointments, creams, patches, liniments, powders, sprays, implants, drops, suppositories, ointments or nano preparations.

[0079] Embodiment 14

[0080] CD73 inhibitory activity of the betulinic acid hydroxamic acid derivatives prepared in Examples 1 to 10 of the present invention:

[0081] The recombinant human CD73 protein used in the experiment was purchased from Beijing Biopsies Biotechnology Co., Ltd. and stored in a -78°C refrigerator by the Department of Medicinal Chemistry of the Naval Medical University of the Chinese People's Liberation Army. The free inorganic phosphate released by AMP hydrolysis was quantified using the green malachite phosphate detection kit to evaluate the CD73 inhibitory activity of the target compound.

[0082] Take 20uL of CD73 protein and add it to the reaction buffer (20mM Tris, 150mM NaCl, pH=8.0) containing a series of concentration gradient compounds (the starting concentration of the compound is 100uM, 2-fold or 3-fold dilution) and place it at room temperature for 1min. Add 20uM substrate AMP 20uL and incubate at 37°C for 2h. Add green malachite reagent to quench the reaction, and read the absorbance value at 620nm with BiotekSynergy H2 to quantify the free inorganic phosphate produced. The raw data were standardized according to the independent phosphate range and fitted using GraphPad Prism to determine the IC of the inhibitory compound. 50 The test results are shown in Table 1:

[0083] Table 1 CD73 inhibitory activity of betulinic acid hydroxamic acid derivatives

[0084] Test compound <![CDATA[IC 50 (μM)]]> Ia 3.70 Ib 25.67 Ic 15.69 If 11.03 IIa 8.00 IIb 3.90 IIc 15.90 Betulinic acid 4.11

[0085] The experimental data in Table 1 show that some betulinic acid hydroxamic acid derivatives have good CD73 inhibitory activity, IC 50The values ​​were 3.70uM-25.67uM, among which compounds Ia and IIb were more active than betulinic acid.

[0086] Although Table 1 only lists the in vitro CD73 inhibitory activity data of some betulinic acid hydroxamic acid derivatives, according to relevant similar experiments, it can be concluded that the betulinic acid hydroxamic acid derivatives or their pharmaceutically acceptable salts prepared by the present invention have CD73 inhibitory activity similar to that of the above-mentioned betulinic acid hydroxamic acid derivatives. Accordingly, the betulinic acid hydroxamic acid derivatives or their pharmaceutically acceptable salts prepared by the present invention can be used to prepare CD73 inhibitor anti-tumor drugs.

[0087] In summary, we can see that:

[0088] First, the betulic acid hydroxamic acid derivatives provided by the present invention are obtained by structurally modifying the existing betulic acid and introducing different hydroxamic acid groups to obtain new betulic acid hydroxamic acid series derivatives or their pharmaceutical salts. They have excellent anti-tumor activity and can be used to prepare a variety of anti-tumor drugs, cardiovascular and cerebrovascular disease treatment drugs, and nervous system disease treatment drugs.

[0089] Secondly, the betulinic acid hydroxamic acid derivatives provided by the present invention have a variety of optional structural forms or their pharmaceutically acceptable salts, and therefore can meet the needs of preparing a variety of pharmaceutical compositions, pharmaceutical preparations, etc., and provide a basis and ideas for preparing new pharmaceutical preparations with better efficacy and lower toxic side effects, and have broad application prospects.

[0090] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A betulinic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof, characterized in that: The structure is selected from one of the following structures: in: X is selected from -(CH2) m1 NH-, Y is selected from -(CH2) m1 NH-, m1 is selected from 0 to 20; n is selected from 0 to 20; m2 is selected from 0 to 20.

2. The betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the betulic acid hydroxamic acid derivative, and Y is selected from -NH-, -CH2NH-, -CH2CH2NH-, -CH2CH2CH2NH-, -CH2CH2CH2CH2NH-, -CH2CH2CH2CH2CH2NH-, -CH2CH2CH2CH2CH2CH2NH-, -CH2CH2CH2CH2CH2CH2CH2NH-, 3. The betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The structure of the betulic acid hydroxamic acid derivative is selected from one of the following structures:

4. Use of the betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a CD73 small molecule inhibitor.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3 as the sole active ingredient.

6. A pharmaceutical preparation, characterized in that The pharmaceutical preparation is prepared from the betulic acid hydroxamic acid derivative or its pharmaceutically acceptable salt according to any one of claims 1 to 3 and excipients commonly used in pharmacy.

7. The pharmaceutical preparation according to claim 6, characterized in that The dosage form of the pharmaceutical preparation is selected from at least one of injection, injection emulsion, tablet, pill, capsule, ointment, cream, patch, liniment, powder, spray, implant, drop, suppository, ointment or nano preparation.

8. Use of the betulic acid hydroxamic acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The anti-tumor drug is used to treat at least one malignant tumor growing in the esophagus, stomach, intestine, rectum, oral cavity, pharynx, larynx, lung, colon, breast, uterus, endometrium, ovary, prostate, testis, bladder, kidney, liver, pancreas, bone, connective tissue, skin, eye, brain or central nervous system of human or animal, and at least one of thyroid cancer, leukemia, Hodgkin's disease, lymphoma and myeloma.

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