Abufagin phosphate derivative and application thereof

Abstract

The invention discloses arenobufagin phosphate ester derivatives or pharmaceutical salts thereof, the structural general formula of the arenobufagin phosphate ester derivatives is as shown in the specification: # imgabs0. Compared with arenobufagin, the toxicity of the compound prepared by the invention is reduced due to the introduction of phosphate ester groups, and the compound has smaller toxic and side effects when being used for preparing antitumor drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a class of bufotoxin phosphate derivatives and applications thereof. Background Art

[0002] Arenobufagin is a toad venom lactone compound extracted from the traditional Chinese medicine toad venom. It has multiple biological activities such as cardiotonic, anti-tumor, and anti-viral hepatitis. Its chemical structure is as follows:

[0003]

[0004] Studies have found that safflower venom induces ferroptosis in human gastric cancer SGC-7901 cells through the Nrf2-HO-1 / SLC7A11 pathway, thereby exerting anti-tumor activity (Wang Wenjun et al., Central South Pharmacy, 2023, 21: 285-290). Studies have shown that safflower venom can inhibit the proliferation of HepG2 cells and induce their apoptosis in a dose-dependent manner, and its mechanism of action may be related to the regulation of the JAK / STAT3 signaling pathway (Kong Lingqi et al., Shandong Medicine, 2022, 62: 6-10). Safflower venom has a significant inhibitory effect on the growth of human colon cancer cell lines, and is dose- and time-dependent. At the same time, safflower venom can promote cell apoptosis and autophagy through the mTOR pathway (Chen Jinbao et al., Hebei Traditional Chinese Medicine, 2020, 42, 1216-1221). Safflower venom can induce cell apoptosis in a caspase-dependent manner, and reduce the vitality of colon cancer cells in a dose- and time-dependent manner. Toad venom inhibits the expression of Drp1 siRNA or MDIVI-1 and induces the accumulation of Bax protein in mitochondria (Xu Wenjuan, doctoral dissertation of Southern Medical University, 2015). Toad venom can reduce the adhesion ability of HepG2 cells and significantly reduce the number of liver cancer cells on the membrane of Transwell chambers in a dose-dependent manner. In addition, toad venom can significantly inhibit the expression of matrix metalloproteinases MMP 2 and MMP 9 (Cao Huihui et al., Chinese Pharmacological Bulletin, 2011, 27, 19-23). ​​However, toad venom also has obvious defects such as cardiotoxicity, which limits its development as a drug (Kan Jun et al., Shandong Medicine, 2019, 59, 98-101). Therefore, it is of great significance to find toad venom derivatives with high efficiency and low toxicity through the study of the structure-activity relationship of toad venom.

[0005] At present, there are relatively few studies on the structural modification of bufotoxin. The patent application with publication number CN102038690A reports the application of bufotoxin in anti-tumor drug preparations. The patent application with publication number WO2013165924A1 reports 3-substituted amine derivatives of bufotoxin and their anti-tumor applications. The patent application with publication number CN110483608A reports the preparation method and use of a class of bufotoxin 3-position carbamate derivatives. The patent application with publication number CN107955059A also reports the preparation method and use of bufotoxin carbamate derivatives. The patent application with publication number CN112062804A reports a class of disubstituted bufotoxin derivatives and their preparation methods, as well as their use in the preparation of anti-tumor, cardiovascular and anti-nervous system drugs.

[0006] Although bufotoxin has excellent anti-tumor activity, its therapeutic index is relatively narrow. Therefore, it is necessary to carry out structural modification on this basis to enhance the activity of the compound and discover new anti-tumor drugs. Summary of the invention

[0007] The purpose of the present invention is to provide a class of bufotoxin phosphate derivatives.

[0008] Another object of the present invention is to provide a use of the bufotoxin phosphate derivative in the preparation of anti-tumor drugs.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In the first aspect of the present invention, a class of bufotoxin phosphate derivatives or pharmaceutically acceptable salts thereof are provided, and the general structural formula is as follows:

[0011]

[0012] in,

[0013] n is an integer selected from 1 to 8 (preferably 1, 2, 3, 4);

[0014] R 1 Selected from C1-C30 alkyl, C3-C10 cycloalkyl;

[0015] R 2 Selected from C1~C30 alkyl, C3~C10 cycloalkyl.

[0016] More preferably, in the bufotoxin phosphate derivative,

[0017] n is selected from 1, 2, 3, 4;

[0018] R 1is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,

[0019] R 2 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,

[0020] Most preferably, the bufotoxin phosphate derivative is selected from one of the following compounds:

[0021]

[0022]

[0023] The pharmaceutically acceptable salt is a salt formed by the bufotoxin phosphate derivative and a pharmaceutically acceptable inorganic acid or organic acid; the inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid; the organic acid is selected from 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, and amino acids.

[0024] The second aspect of the present invention provides a pharmaceutical composition, which uses the bufotoxin phosphate derivative or a pharmaceutically acceptable salt thereof as the sole active ingredient.

[0025] The mass percentage of the bufotoxin phosphate derivative is 0.1-99wt%.

[0026] The third aspect of the present invention provides a pharmaceutical preparation, which is prepared from the bufotoxin phosphate derivative or its pharmaceutically acceptable salt and common pharmaceutical excipients.

[0027] The dosage form of the pharmaceutical preparation is selected from injections (including small-volume injections, medium-volume injections, large-volume injections, and powder injections), injection emulsions, tablets, pills, capsules, ointments, creams, patches, liniments, powders, sprays, implants, drops, suppositories, and ointments.

[0028] The fourth aspect of the present invention provides a use of the bufotoxin phosphate derivative or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0029] The tumor is selected from cancers occurring 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, as well as thyroid cancer, leukemia, Hodgkin's disease, lymphoma, myeloma, etc.; preferably lung cancer and colon cancer.

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

[0031] Compared with bufotoxin, the compound prepared by the present invention has lower cardiac toxicity after the introduction of phosphate groups, and has smaller toxic and side effects when used for preparing 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] Preparation of compound 1a

[0035]

[0036] (3S, 5R, 8R, 9S, 10S, 11S, 13R, 14S, 17R)-11, 14-dihydro-10, 13-dimethyl-12-oxo-17-(2-oxo-2H-pyran-5-yl) hexahydro-1H-cyclopenta[a]phenanthrene-3-yl(4-nitrophenyl) carbonate (100 mg, 0.17 mmol, prepared according to the patent application with publication number CN110483608A) was added to a 50 mL two-necked flask, and 15 mL of dry dichloromethane, triethylamine (69 mg, 0.68 mmol) and ((4-aminopiperidin-1-yl)methyl) diisopropyl phosphate (98 mg, 0.34 mmol, prepared according to the patent application with publication number CN116063370A) were added under argon protection, and the reaction was carried out at room temperature for 30 minutes. Then, DMAP (82 mg, 0.68 mmol) was added and stirred at room temperature overnight. The solvent was evaporated and the mixture was purified by column chromatography to obtain 80 mg of a white solid, namely compound 1a, with a yield of 66%. 1H NMR(501MHz,Chloroform-d)δ7.79–7.74(m,1H),7.39(d,J=2.6Hz,1H),6.24(d,J=9.7Hz,1H),4.93(s,1H),4.7 5–4.62(m,3H),4.31(d,J=11.1Hz,1H),4.08(t,J=8.3Hz,1H),3.83(s,1H),3.50–3.40(m,1H),2.98(d,J=12.9H z,2H),2.70(d,J=11.5Hz,2H),2.39(d,J=14.1Hz,1H),2.30(t,J=11.7Hz,2H),2.11–1.97(m,2H),1.96–1.82(m ,4H),1.82–1.56(m,8H),1.51–1.39(m,3H),1.37–1.21(m,19H),1.16(s,3H),0.90(s,3H).HRMS(ESI)m / z:calcd for C 37 H 57 N 2 O 10 P[M+H] + 721.3751 found 721.3814.

[0037] Example 2

[0038] Preparation of compound 1b

[0039]

[0040] Referring to the synthesis method of compound 1a in Example 1, diethyl ((4-aminopiperidin-1-yl)methyl)phosphate was substituted for diisopropyl ((4-aminopiperidin-1-yl)methyl)phosphate to obtain 34 mg of white solid, namely compound 1b, with a yield of 29%. 1H NMR(501MHz,Chloroform-d)δ7.76–7.70(m,1H),7.41(d,J=2.6Hz,1H),6.30(d,J=9.7Hz,1H),5.32(s,1H),4.98(s,1H), 4.57(s,1H),4.38–4.31(m,1H),4.21–4.09(m,4H),3.84(d,J=3.6Hz,1H),3.51(s,2H),3.15(q,J=7.5Hz,1H),3.04(s,2H) ,2.81(d,J=11.5Hz,2H),2.44(d,J=13.5Hz,1H),2.37(t,J=11.2Hz,2H),2.13–2.00(m,2H),1.95(d,J=12.2Hz,2H),1.87– 1.71(m,5H),1.67(s,10H),1.51(d,J=11.6Hz,3H),1.36(q,J=7.1Hz,9H),1.20(s,3H),0.94(s,3H).HRMS(ESI)m / z:calcd for C 35 H 53 N 2 O 10 P[M+H] + 693.3438 found 693.3511.

[0041] Example 3

[0042] Preparation of compound 1c

[0043]

[0044] Referring to the synthesis method of compound 1a in Example 1, diethyl ((4-aminopiperidin-1-yl)ethyl)phosphate was substituted for diisopropyl ((4-aminopiperidin-1-yl)methyl)phosphate to obtain 25 mg of white solid, namely compound 1c, with a yield of 21%. 1H NMR(600MHz,Chloroform-d)δ7.73(dd,J=9.8,2.6Hz,1H),7.41(dd,J=2.6,1.1Hz,1H),6.30(dd,J=9.8,1.1Hz,1H), 4.98(s,1H),4.74(s,1H),4.34(dd,J=11.0,3.1Hz,1H),4.17–4.09(m,5H),3.84(d,J=3.5Hz,1H),3.64(s,1H),3.18( s,1H),3.14(q,J=7.3Hz,2H),2.92(s,1H),2.43(s,2H),2.16(s,2H),2.11–1.92(m,7H),1.89–1.82(m,2H),1.82–1. 72(m,6H),1.70–1.65(m,2H),1.54–1.48(m,1H),1.42–1.32(m,12H),1.20(s,3H),0.94(s,3H).HRMS(ESI)m / z:calcd for C 36 H 55 N 2 O 10 P[M+H] + 707.3594 found 707.3656.

[0045] Example 4

[0046] Preparation of compound 1d

[0047]

[0048] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)diheptyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 96 mg of white solid, namely compound 1d, with a yield of 68%. 1H NMR(501MHz,Chloroform-d)δ7.79–7.72(m,1H),7.40(s,1H),6.27(d,J=9.8Hz,1H),4.95(s,1H),4.61(d,J=8 .0Hz,1H),4.36–4.29(m,1H),4.13–3.99(m,5H),3.84(d,J=3.7Hz,1H),3.49(s,1H),3.02(s,2H),2.80(d,J=11 .5Hz,2H),2.69(s,1H),2.45–2.28(m,3H),2.11–2.00(m,2H),1.91(t,J=14.8Hz,4H),1.82–1.69(m,5H),1.69– 1.57(m,7H),1.49(d,J=13.5Hz,3H),1.40–1.20(m,22H),1.17(s,3H),0.94–0.83(m,9H).HRMS(ESI)m / z:calcd forC 45 H 73 N 2 O 10 P[M+H] + 833.5003 found 833.5088.

[0049] Example 5

[0050] Preparation of compound 1e

[0051]

[0052] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 67 mg of white solid, namely compound 1e, with a yield of 46%. 1H NMR(501MHz,Chloroform-d)δ7.77–7.70(m,1H),7.41(d,J=2.6Hz,1H),6.30(d,J=9.7Hz,1H),4.98( s,1H),4.57(s,1H),4.38–4.31(m,1H),4.18–4.00(m,6H),3.83(d,J=3.6Hz,1H),3.52(s,1H),3.04(s ,2H),2.87–2.76(m,2H),2.49–2.26(m,3H),2.13–2.02(m,3H),1.98(d,J=24.8Hz,4H),1.88–1.72(m ,6H),1.72–1.59(m,7H),1.51(d,J=12.0Hz,3H),1.43–1.22(m,24H),1.20(s,3H),0.96–0.84(m,9H). 1 HRMS(ESI)m / z:calcd for C 47 H 77 N 2 O 10 P[M+H] + 861.5316 found 861.5378.

[0053] Example 6

[0054] Preparation of compound 1f

[0055]

[0056] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)dipentyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 87 mg of white solid, namely compound 1f, with a yield of 66%. 1H NMR(501MHz,Chloroform-d)δ7.80–7.71(m,1H),7.41(d,J=2.7Hz,1H),6.28(d,J=9.7Hz,1H),5.31(s, 4H),4.96(s,1H),4.61(d,J=8.0Hz,1H),4.37–4.30(m,1H),4.16–4.00(m,5H),3.85(d,J=3.7Hz,1H),3 .55–3.46(m,1H),3.02(s,2H),2.81(d,J=11.6Hz,2H),2.50–2.29(m,4H),2.13–2.01(m,3H),1.99–1.5 7(m,16H),1.56–1.44(m,3H),1.42–1.27(m,12H),1.18(s,3H),0.98–0.82(m,9H).HRMS(ESI)m / z:calcd for C 41 H 65 N 2 O 10 P[M+H] + 777.4377 found 777.4448

[0057] Example 7

[0058] Preparation of compound 1g

[0059]

[0060] Referring to the synthesis method of compound 1a in Example 1, diisopropyl ((4-aminopiperidin-1-yl)methyl)phosphate was replaced with dihexyl ((4-aminopiperidin-1-yl)methyl)phosphate to obtain 105 mg of white solid, namely compound 1g, with a yield of 77%. 1H NMR(501MHz,Chloroform-d)δ7.74(dd,J=9.7,2.6Hz,1H),7.41(d,J=2.7Hz,1H),6.29(d,J=9.7Hz,1H),4.97(s, 1H),4.59(d,J=8.0Hz,1H),4.34(dd,J=11.2,3.3Hz,1H),4.16–4.01(m,5H),3.84(d,J=3.6Hz,1H),3.52(s,1H), 3.05(s,2H),2.83(d,J=11.5Hz,2H),2.43(d,J=15.8Hz,2H),2.18(s,1H),2.12–2.02(m,3H),2.00–1.89(m,3H), 1.89–1.59(m,11H),1.59–1.45(m,4H),1.42–1.22(m,17H),1.19(s,3H),0.95–0.84(m,9H).HRMS(ESI)m / z:calcd for C 43 H 69 N 2 O 10 P[M+H] + 805.469 found 805.4767.

[0061] Example 8

[0062] Preparation of compound 1h

[0063]

[0064] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)dipropyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 114 mg of white solid, namely compound 1h, with a yield of 93%. 1H NMR(501MHz,Chloroform-d)δ7.80–7.73(m,1H),7.38(d,J=2.6Hz,1H),6.23(d,J=9.7Hz,1H),5.28(s,1H),4.91 (s,1H),4.69(d,J=7.9Hz,1H),4.35–4.24(m,1H),4.12–3.91(m,5H),3.84(d,J=3.7Hz,1H),3.38(d,J=45.3Hz,4 H),2.97(t,J=12.9Hz,2H),2.77(d,J=11.5Hz,2H),2.48–2.22(m,4H),2.11–1.97(m,2H),1.89(d,J=12.5Hz,4H) ,1.81–1.53(m,12H),1.52–1.39(m,3H),1.37–1.18(m,5H),1.14(s,3H),0.97–0.83(m,9H).HRMS(ESI)m / z:calcd for C 37 H 57 N 2 O 10 P[M+H] + 721.3751 found 721.3818.

[0065] Example 9

[0066] Preparation of compound 1i

[0067]

[0068] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)dibutyl phosphate was used instead of ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 42 mg of white solid, namely compound 1i, with a yield of 33%. 1H NMR(501MHz,Chloroform-d)δ7.75(d,J=9.8Hz,1H),7.41(s,1H),6.29(d,J=9.7Hz,1H),4.96(s,1H ),4.61(s,1H),4.37–4.30(m,1H),4.15–4.02(m,5H),3.84(d,J=3.7Hz,1H),3.52(s,1H),3.05(s,2H ),2.84(d,J=11.4Hz,2H),2.42(d,J=14.5Hz,2H),2.27(s,1H),2.12–2.01(m,3H),1.95(d,J=13.3Hz ,3H),1.89–1.46(m,16H),1.46–1.23(m,10H),1.19(s,3H),0.98–0.86(m,9H).HRMS(ESI)m / z:calcd for C 39 H 61 N 2 O 10 P[M+H] + 749.4064 found 749.4140.

[0069] Example 10

[0070] Preparation of compound 1j

[0071]

[0072] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)dicyclobutyl phosphate was used instead of ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 82 mg of white solid, namely compound 1j, with a yield of 67%. 1HNMR(600MHz,Chloroform-d)δ7.74(dd,J=9.8,2.6Hz,1H),7.41–7.38(m,1H),6.30–6.25(m,1H),4.95(s,1H),4.81–4.73(m,2H ),4.60(d,J=7.8Hz,1H),4.32(dd,J=11.2,3.3Hz,1H),4.15–4.07(m,2H),3.83(d,J=3.6Hz,1H),3.48(s,1H),3.00(s,2H),2.73( d,J=11.3Hz,2H),2.48–2.38(m,2H),2.38–2.27(m,6H),2.21–2.12(m,4H),2.10–2.02(m,4H),1.97–1.89(m,3H),1.89–1.82(m, 1H),1.82–1.70(m,7H),1.69–1.60(m,2H),1.56–1.45(m,5H),1.39–1.30(m,4H),1.18(s,3H),0.92(s,3H).HRMS(ESI)m / z:calcd for C 39 H 57 N 2 O 10 P[M+H] + 745.3751 found 745.3839.

[0073] Embodiment 11

[0074] Preparation of compound 1k

[0075]

[0076] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)dicyclopentyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 76 mg of white solid, namely compound 1k, with a yield of 58%. 1HNMR(600MHz,Chloroform-d)δ7.74(dd,J=9.8,2.6Hz,1H),7.40(m,1H),6.27(d,J=9.66Hz,1H),5.31(s,1H),4.9 8–4.92(m,3H),4.59(s,1H),4.32(dd,J=11.1,3.6Hz,1H),4.15–4.07(m,1H),3.83(d,J=3.6Hz,1H),3.49(s,1H), 3.01(s,2H),2.77–2.69(m,2H),2.52–2.29(m,4H),2.10–2.02(m,2H),1.93(s,3H),1.89–1.70(m,19H),1.69–1.5 5(m,7H),1.54–1.44(m,3H),1.40–1.29(m,5H),1.29–1.24(m,2H),1.18(s,3H),0.92(s,3H).HRMS(ESI)m / z:calcd for C 41 H 61 N 2 O 10 P[M+H] + 773.4064 found 773.4156.

[0077] Example 12

[0078] Preparation of compound 11

[0079]

[0080] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)dicyclohexyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 83 mg of white solid, namely compound 11, with a yield of 61%. 1HNMR(600MHz,Chloroform-d)δ7.74(dd,J=9.8,2.6Hz,1H),7.40(d,J=1.98Hz,1H),6.27(d,J=9.72,1H),4.95(s,1H),4.61( d,J=8.1Hz,1H),4.47–4.39(m,2H),4.32(d,J=11.1Hz,1H),4.14–4.07(m,1H),3.83(s,1H),3.48(s,2H),3.02(d,J=13.5Hz, 2H),2.77(d,J=11.5Hz,2H),2.45–2.34(m,3H),2.10–2.02(m,3H),1.97–1.88(m,7H),1.86(d,J=11.7Hz,1H),1.82–1.69(m, 10H),1.64(s,2H),1.56–1.44(m,9H),1.38–1.29(m,8H),1.28–1.22(m,3H),1.18(s,3H),0.92(s,3H).HRMS(ESI)m / z:calcd for C 43 H 65 N 2 O 10 P[M+H] + 801.4377 found 801.4442.

[0081] Embodiment 13

[0082] Preparation of compound 1m

[0083]

[0084] Referring to the synthesis method of compound 1a in Example 1, ((4-aminopiperidin-1-yl)methyl)dicycloheptyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)diisopropyl phosphate to obtain 70 mg of white solid, namely compound 1m, with a yield of 50%. 1HNMR(600MHz,Chloroform-d)δ7.75(dd,J=9.8,2.6Hz,1H),7.40(d,J=2.04Hz,1H),6.26(d,J=9.72,1H),5.30(s,2H),4.9 5(s,1H),4.64–4.57(m,3H),4.32(dd,J=11.1,3.2Hz,1H),4.13–4.07(m,1H),3.83(d,J=3.6Hz,1H),3.48(s,1H),3.01(s, 2H),2.72(d,J=11.4Hz,2H),2.67(s,1H),2.41(d,J=13.8Hz,1H),2.33(s,2H),2.10–2.02(m,2H),2.00–1.89(m,7H),1.89 –1.83(m,1H),1.82–1.70(m,10H),1.70–1.59(m,6H),1.58–1.29(m,20H),1.17(s,3H),0.92(s,3H).HRMS(ESI)m / z:calcd for C 45 H 69 N 2 O 10 P[M+H] + 829.469 found829.4766.

[0085] Embodiment 14

[0086] In vitro antitumor activity test of the compounds of the present invention

[0087] The compounds of the present invention were subjected to a tumor cell proliferation inhibition test using the conventional CCK-8 method (Lv Qiujun, chief editor, "New Drug Pharmacology Research Methods", 2007: 242-243).

[0088] The cell lines used were A549 (human lung adenocarcinoma cells) and HCT116 (human colon cancer cells), which were cryopreserved and passaged by the Pharmacology Laboratory of Shanghai Institute of Pharmaceutical Industry. The culture medium was DMEM+10% FBS+double antibodies.

[0089] In vitro activity test: cells in the logarithmic growth phase were taken and 7×10 3 The cells were seeded into 96-well plates at a density of 100 μl / well and incubated at 37°C in 5% CO 2Incubate for 24 h in an environment to maintain normal physiological pH. After cell adhesion, different concentrations of test compounds were added to three sets of wells, and 0.1% DMSO was added as a control. After 72 h, 10 μL of a cell counting kit (CCK-8) was added to each well, and the plate was incubated at 37 ° C for another 0.5 h-1 h. The absorbance (OD) was read at 450 nm on a Biotek Synergy H2. The concentration that causes 50% cell growth inhibition (IC 50 ).

[0090] The test results are shown in Table 1, wherein the sample refers to the bufotoxin phosphate derivative prepared in the corresponding example.

[0091] Table 1 In vitro antitumor activity of compounds

[0092]

[0093] The above experimental results show that the compounds of the present invention have excellent anti-tumor activity and show excellent activity against lung cancer, colon cancer, etc. Most of the compounds are better than or equivalent to the positive drug doxorubicin. In addition, the activity of compounds 1a, 1b and 1c against two tumor cell lines reaches several nM. Therefore, the compounds of the present invention and their salts can be used to prepare anti-tumor drugs.

[0094] Embodiment 15

[0095] In vivo antitumor activity test of compound 1a of the present invention on colon cancer HCT116

[0096] Animals: BALB / C nude mice (SPF grade), male, 18-20 g.

[0097] A well-growing colon cancer HCT116 tumor was taken and cut into uniform small pieces of about 3 mm under sterile conditions. One piece was inoculated subcutaneously in the right armpit of each mouse using a trocar. On the 13th day after inoculation, the average tumor volume was about 130-140 mm 3 , regrouped according to tumor size, eliminated animals with tumors that were too large or too small, and the average tumor volume of each group was basically the same. The following regimen was used to start drug administration, with a volume of 0.2 mL / 20 g body weight. From the 9th day of inoculation, the long diameter a (mm) of the tumor and the perpendicular short diameter b (mm) of the tumor were measured twice a week with a digital electronic caliper. The tumor volume was calculated as follows: TV = ab 2 / 2, the relative tumor volume calculation formula is: RTV = Vt / Vo, Vo is the tumor volume measured at the time of cage division (i.e. d1), and Vt is the tumor volume at each measurement. The animals were killed 30 days after inoculation (d18), weighed, and the tumor was dissected and weighed. The results were determined according to the following formula:

[0098]

[0099] Table 2 In vivo antitumor activity of compound 1a in colon cancer HCT116

[0100]

[0101] Compared with the Control group: **P<0.01.

[0102] The above experimental results show that the compound 1a of the present invention has excellent anti-tumor activity at a dose of 1 mg / Kg when intravenously administered, with a tumor inhibition rate of 63.48%, which is better than the clinical drug 5-fluorouracil. Therefore, the compound of the present invention and its salts can be used to prepare anti-tumor drugs.

[0103] Example 16

[0104] Test of the effect of the compounds of the present invention on hERG potassium channels

[0105] CHO-hERG cells were cultured at 175 cm 2 In the culture flask, when the cell density grows to 60-80%, remove the culture medium, wash once with 7mL PBS (Phosphate Buffered Saline), and then add 3mL Detachin for digestion. After digestion is complete, add 7mL culture medium for neutralization, then centrifuge, aspirate the supernatant, and add 5mL culture medium for resuspending to ensure that the cell density is 2-5×10 6 / mL.

[0106] The single cell high impedance sealing and whole cell pattern formation process are all completed automatically by the Qpatch instrument. After obtaining the whole cell recording mode, the cell is clamped at -80 mV. Before giving a 5-second +40 mV depolarizing stimulus, a 50-millisecond -50 mV pre-voltage is given, and then repolarized to -50 mV for 5 seconds, and then returned to -80 mV. This voltage stimulus is applied every 15 seconds. After recording for 2 minutes, the extracellular solution is given for 5 minutes, and then the drug administration process begins. The compound is given for 2.5 minutes for each test concentration, and the positive control compound 0.3 μM cisapride is given. At least 3 cells (n≥3) are tested for each concentration.

[0107] On the day of the test, the compound was prepared into a 15mM DMSO stock solution, and then diluted 500 times with extracellular fluid to obtain the final concentration to be tested. The DMSO content in the final test concentration does not exceed 0.2%, and this concentration of DMSO has no effect on the hERG potassium channel. The compound preparation is completed by the Bravo instrument throughout the dilution process.

[0108] Table 3 hERG potassium channel activity test of compounds

[0109]

[0110] The above experimental results show that the inhibition rate of compound 1a at a concentration of 30 uM is 15.5±4.49%, indicating that the compound of the present invention has lower hERG potassium channel inhibitory activity and lower cardiotoxicity.

[0111] 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 phosphate derivative of bufotoxin or a pharmaceutically acceptable salt thereof, characterized in that: The general structural formula is shown below: in, n is an integer selected from 1 to 8; R1 is selected from C1-C30 alkyl, C3-C10 cycloalkyl; R2 is selected from C1-C30 alkyl and C3-C10 cycloalkyl.

2. The bufotoxin phosphate derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the bufotoxin phosphate derivative, n is selected from 1, 2, 3, 4; R1 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, R2 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 3. The bufotoxin phosphate derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The bufotoxin phosphate derivative is selected from one of the following compounds:

4. A pharmaceutical composition, characterized in that The only active ingredient is the bufotoxin phosphate derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3.

5. A pharmaceutical preparation, characterized in that The invention is prepared from the bufotoxin phosphate derivative or its pharmaceutically acceptable salt as described in any one of claims 1 to 3 and common pharmaceutical auxiliary materials.

6. The pharmaceutical preparation according to claim 5, characterized in that The dosage form of the pharmaceutical preparation is selected from injection, injection emulsion, tablet, pill, capsule, ointment, cream, patch, liniment, powder, spray, implant, drop, suppository, ointment.

7. Use of the bufotoxin phosphate derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

8. Use of the bufotoxin phosphate derivative or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug according to claim 7, characterized in that: The tumor is selected from cancers of 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, central nervous system, as well as thyroid cancer, leukemia, Hodgkin's disease, lymphoma, myeloma.

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