Abufagin diphosphate derivative and application thereof

Abstract

The invention discloses a arenobufagin diphosphate derivative or a pharmaceutical salt thereof. The structural general formula of the arenobufagin diphosphate derivative is as follows: # imgabs0. The arenobufagin diphosphate derivative disclosed by the invention has relatively good anti-tumor activity and relatively low cardiotoxicity. Compared with arenobufagin, toxicity is reduced due to introduction of the diphosphate group, and toxic and side effects are smaller when the arenobufagin is used for preparing anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to a bisphosphate derivative of bufotoxin and application thereof. Background Art

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

[0003]

[0004] Studies have found that toad venom can activate the Rho A / ROCK1 signaling pathway, promote the translocation of BAX to mitochondria, induce mitochondrial damage, and lead to cell apoptosis; toad venom can also activate the AMPK / Drp1 pathway, inhibit the activity of mitochondrial respiratory chain complex enzymes, reduce mitochondrial ATP production, induce mitochondrial energy metabolism disorders, lead to mitochondrial damage, and ultimately lead to cell apoptosis (Li Zhiqiang, Master's thesis of Army Medical University, 2023). Studies have shown that toad venom promotes autophagy-dependent ferroptosis of HepG2 cells by regulating the p62-Keap1-Nrf2 pathway through induced autophagy (Liu Chun, Master's thesis of Anhui University of Traditional Chinese Medicine, 2023). Toad 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, toad venom can promote cell apoptosis and autophagy through the mTOR pathway (Chen Jinbao et al., Hebei Traditional Chinese Medicine, 2020, 42, 1216-1221). Toad venom can induce apoptosis in a caspase-dependent manner and reduce the viability 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 bufotoxin 3-substituted amine derivatives 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. Studies have found that although the anti-proliferative activity of the disubstituted derivatives of toad venom is significantly reduced in vitro, they still maintain good anti-tumor activity in vivo and their cardiotoxicity is significantly reduced (Dong Lian, Master's thesis, Shanghai University of Technology, 2022).

[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 bisphosphate derivative of bufotoxin.

[0008] Another object of the present invention is to provide a use of the bisphosphate derivative of bufotoxin 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 bisphosphate derivative of bufotoxin or a pharmaceutically acceptable salt thereof is provided, and the general structural formula is as follows:

[0011]

[0012] in,

[0013] R1 is selected from C1-C30 alkyl, C3-C10 cycloalkyl;

[0014] R2 is selected from C1-C30 alkyl, C3-C10 cycloalkyl;

[0015] R3 is selected from C1-C30 alkyl, C3-C10 cycloalkyl;

[0016] R4 is selected from C1-C30 alkyl and C3-C10 cycloalkyl.

[0017] More preferably, in the bisphosphate derivative of bufotoxin,

[0018] 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,

[0019] 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,

[0020] R3 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,

[0021] R4 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,

[0022] Most preferably, the bisphosphate derivative of bufotoxin is selected from one of the following compounds:

[0023]

[0024]

[0025] The pharmaceutically acceptable salt is a salt formed by the bisphosphate derivative of bufotoxin 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.

[0026] The second aspect of the present invention provides a pharmaceutical composition, which uses the bufotoxin bisphosphate derivative or its pharmaceutically acceptable salt as the sole active ingredient.

[0027] The mass percentage of the bisphosphate derivative of bufotoxin is 0.1-99wt%.

[0028] The third aspect of the present invention provides a pharmaceutical preparation, which is prepared from the bisphosphate derivative of bufotoxin or its pharmaceutically acceptable salt and excipients commonly used in pharmacy.

[0029] 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.

[0030] The fourth aspect of the present invention provides a use of the bisphosphate derivative of bufotoxin or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

[0031] 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.

[0032] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0033] The bisphosphate derivative of bufotoxin of the present invention has good anti-tumor activity and low hERG cardiac toxicity. Compared with bufotoxin, the toxicity is reduced due to the introduction of bisphosphate groups, and the toxic and side effects of the derivative when used in the preparation of anti-tumor drugs are smaller. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1

[0036] Preparation of compound 1A

[0037]

[0038] (3S, 5R, 8R, 9S, 10S, 11S, 13R, 14S, 17R)-14-hydroxy-10, 13-dimethyl-12-oxo-17-(2-oxo-2H-pyran-5-yl) hexahydro-1H-cyclopenta[a]phenanthrene-3, 11-diylbis(4-nitrophenyl)biscarbonate (compound 1, 80 mg, 0.11 mmol, prepared by the synthesis method in the patent application with publication number CN112062804A) and 10 mL of dichloromethane were added to a three-necked flask, and triethylamine (53 mg, 0.52 mmol) was added under argon protection. Then a solution of ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate (compound 2, 164 mg, prepared by the synthesis method in the patent application with publication number CN116063370A) in dichloromethane (5 mL) was added dropwise. After the addition was completed, the mixture was reacted at room temperature for 30 minutes, and then DMAP (63 mg, 0.52 mmol) was added and reacted at room temperature for 12 hours. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography to obtain 63 mg of white powder, namely compound 1A, with a yield of 43%. 1 H NMR(501MHz,Chloroform-d)δ7.73(d,J=9.7Hz,1H),7.37(s,1H),6.23(d,J=9.7Hz,1H),5.45–5.22(m,1H),4.97(d,J= 20.6Hz,1H),4.73(d,J=8.0Hz,1H),4.60(d,J=7.9Hz,1H),4.05(q,J=6.9Hz,9H),3.43(d,J=48.2Hz,2H),3.01(s,4H), 2.82–2.73(m,4H),2.32(d,J=12.9Hz,4H),2.21–2.10(m,1H),2.09–2.00(m,2H),1.91(d,J=11.9Hz,6H),1.84–1.71(m ,4H),1.65(p,J=6.9Hz,10H),1.57–1.42(m,5H),1.41–1.15(m,46H),1.11(s,4H),0.96(s,3H),0.87(t,J=6.7Hz,12H); 13C NMR(126MHz,Chloroform-d)δ207.50,162.18,155.41,154.31,150.27,146.91,1 20.87,115.39,84.87,75.45,66.19,62.93,54.34,53.99,53.04,47.93,47.45,3 8.56,37.20,36.69,33.05,32.55,31.76,31.10,30.61,30.56,29.18,29.13,27. 90,26.32,25.76,25.53,23.32,22.62,21.75,16.57,14.09; HRMS(ESI)m / z:calcd for C 70 H 122 N4O 14 P2[M+H] + 1305.84 found 1305.8512.

[0039] Example 2

[0040] Preparation of Compound 1B

[0041]

[0042] Referring to the synthesis method of 1A in Example 1, ((4-aminopiperidin-1-yl)methyl)dipentyl phosphate was used instead of ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate to obtain 80 mg of white solid, namely compound 1B, with a yield of 54%. 1 H NMR(501MHz,Chloroform-d)δ7.73(d,J=9.7Hz,1H),7.35(s,1H),6.21(d,J=9.7Hz,1H),5.29(s,1H),4.93( s,1H),4.78(d,J=8.1Hz,1H),4.65(d,J=7.8Hz,1H),4.07–3.97(m,9H),3.43(s,5H),2.98(s,4H),2.76(dd,J =11.6,5.0Hz,4H),2.35–2.22(m,4H),2.19–2.08(m,1H),2.02(s,2H),1.89(d,J=10.3Hz,6H),1.81–1.58(m ,15H),1.48(dd,J=23.5,12.1Hz,5H),1.39–1.26(m,20H),1.10(s,3H),0.94(s,3H),0.88(d,J=6.6Hz,12H); 13C NMR (126MHz, CDCl3) δ208.26,162.24,155.46,154.35,150.25,147.05,120.99,115. 31,84.76,77.35,77.10,76.85,66.16,62.98,54.32,53.98,53.03,50.53,47.91,47 .50,40.46,40.02,38.55,37.20,36.68,33.04,32.51,32.31,31.10,30.26,30.21,2 7.92,27.64,26.33,25.74,23.31,22.20,21.73,16.58,13.95; HRMS(ESI)m / z:calcd for C 58 H 98 N4O 14 P2[M+H] + 1137.6555 found 1137.6636.

[0043] Example 3

[0044] Preparation of Compound 1C

[0045]

[0046] Referring to the synthesis method of 1A in Example 1, ((4-aminopiperidin-1-yl)ethyl)dihexyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate to obtain 79 mg of white solid, namely compound 1C, with a yield of 51%. 1H NMR(501MHz,Chloroform-d)δ7.73(d,J=9.8Hz,1H),7.37(s,1H),6.23(d,J=9.7Hz,1H),5.44–5.24(m,1H),4.97(d,J=20.6H z,1H),4.72(d,J=8.1Hz,1H),4.60(d,J=8.4Hz,1H),4.05(p,J=7.3Hz,9H),3.54–3.35(m,2H),3.07–2.92(m,4H),2.77(dd,J =11.5,4.7Hz,5H),2.31(q,J=11.5,11.0Hz,4H),2.14(d,J=11.9Hz,1H),2.10–1.99(m,3H),1.91(d,J=12.5Hz,6H),1.83–1. 70(m,4H),1.65(p,J=6.9Hz,10H),1.57–1.42(m,6H),1.41–1.19(m,30H),1.12(s,3H),0.96(s,3H),0.89(t,J=6.7Hz,12H); 13 C NMR (126MHz, CDCl3) δ207.51,162.14,155.41,154.30,150.27,146.87,120.85,115.40, 84.87,77.32,77.07,76.81,75.42,66.16,62.92,54.40,54.01,53.11,47.95,47.52,40 .72,40.13,37.22,36.70,33.06,32.59,32.37,31.33,31.11,30.57,30.52,27.89,27.6 6,26.33,25.76,25.19,23.32,22.53,22.22,21.76,16.56,13.98; HRMS(ESI)m / z:calcd for C 62 H 106 N4O 14 P2[M+H] + 1193.7181 found 1193.7252.

[0047] Example 4

[0048] Preparation of Compound 1D

[0049]

[0050] Referring to the synthesis method of Example 1A, ((4-aminopiperidin-1-yl)methyl)diheptyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate to obtain 107 mg of white solid, namely compound 1D, with a yield of 66%. 1 H NMR(501MHz,Chloroform-d)δ7.73(d,J=9.7Hz,1H),7.35(s,1H),6.20(d,J=9.8Hz,1H),5.27(d,J=11.2Hz,1H),4.95(d,J =19.5Hz,1H),4.74(d,J=8.0Hz,1H),4.67–4.55(m,1H),4.10–3.94(m,9H),3.45(s,2H),3.20(s,1H),2.97(s,4H),2.75(dd ,J=11.5,3.6Hz,4H),2.28(d,J=10.1Hz,4H),2.15(t,J=12.2Hz,1H),2.08–1.97(m,2H),1.89(d,J=13.5Hz,6H),1.82–1.6 7(m,5H),1.63(t,J=7.1Hz,10H),1.55–1.40(m,6H),1.39–1.18(m,37H),1.10(s,3H),0.94(s,3H),0.86(t,J=6.7Hz,12H); 13 C NMR (126MHz, CDCl3) δ207.61,162.18,155.42,154.33,150.25,147.03,120.98,115.2 9,84.75,77.36,77.10,76.85,75.45,66.15,62.98,54.38,54.00,53.09,47.95,47.52 ,40.50,40.03,37.19,36.68,33.05,32.55,32.34,31.69,31.10,30.59,30.54,28.80 ,27.93,26.35,25.76,25.47,23.31,22.53,21.73,16.58,14.03; HRMS(ESI)m / z:calcd for C 66 H 114 N4O 14 P2[M+H] + 1249.7807 found 1249.7880.

[0051] Example 5

[0052] Preparation of Compound 1E

[0053]

[0054] Referring to the synthesis method of 1A in Example 1, ((4-aminopiperidin-1-yl)methyl)dicyclopentyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate to obtain 95 mg of white solid, namely compound 1E, with a yield of 65%. 1 H NMR(501MHz,Chloroform-d)δ7.75(d,J=10.2Hz,1H),7.35(d,J=9.5Hz,1H),6.22(d,J=10.2Hz,1H),5.33(d,J=57.2Hz,1H),4.80(d,J=118.1Hz,8H),4.02 (s,1H),3.54–3.22(m,5H),3.07–2.87(m,4H),2.70(d,J=10.9Hz,4H),2.49–2 .09(m,8H),2.08–1.19(m,60H),1.10(d,J=9.5Hz,3H),0.94(d,J=9.2Hz,3H); 13 C NMR (126MHz, CDCl3) δ207.66,162.24,155.47,154.13,150.23,147.06,121.02 ,115.29,84.77,79.29,77.34,77.09,76.84,75.27,63.00,55.03,53.86,50.53 ,48.00,47.53,40.27,40.05,38.73,37.19,36.69,34.21,32.55,32.36,31.28 ,27.91,26.46,25.75,23.08,23.04,21.74,17.12,16.61; HRMS(ESI)m / z:calcd for C 58 H 90 N4O 14 P2[M+H] + 1129.5929found1129.6017.

[0055] Example 6

[0056] Preparation of Compound 1F

[0057]

[0058] Referring to the synthesis method 1A of Example 1, ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate was replaced with ((4-aminopiperidin-1-yl)methyl)dipropyl phosphate to obtain 72 mg of white solid, namely compound 1F, with a yield of 54%. 1H NMR(600MHz,Chloroform-d)δ7.72(d,J=9.7,2.5Hz,1H),7.33(d,J=3.3Hz,1H),6.18(d,J=9.6Hz,1H),5.40–5. 19(m,1H),4.92(d,J=25.2Hz,1H),4.78(d,J=7.8Hz,1H),4.74–4.62(m,1H),4.02–3.91(m,9H),3.53–3.28(m,3 H),2.96(s,4H),2.78–2.69(m,4H),2.27(q,J=12.2,11.6Hz,4H),2.14(t,J=10.9Hz,1H),2.05–1.94(m,2H),1. 94–1.81(m,6H),1.80–1.58(m,15H),1.54–1.38(m,5H),1.37–1.23(m,5H),1.07(s,3H),0.90(t,J=7.4Hz,15H); 13 C NMR (151MHz, CDCl3) δ207.66,162.18,155.42,154.34,150.20,147.09,121.04,115.21,84.67,77.36,77.3 3,77.14,77.12,76.93,76.91,75.42,70.47,70.20,67.63,67.58,67.53,63.01,60.34,54.19,53.98,53.1 1,48.34,47.91,47.50,45.78,40.47,39.92,38.56,37.17,36.67,33.03,32.47,32.29,31.09,29.61,27.9 2,26.35,25.74,23.89,23.86,23.30,21.71,20.98,16.60,14.14,10.04,8.54,-0.05; HRMS(ESI)m / z:calcd forC 50 H 82 N4O 14 P2[M+H] + 1025.5303 found 1025.5374.

[0059] Example 7

[0060] Preparation of Compound 1G

[0061]

[0062] Referring to the synthesis method of 1A in Example 1, ((4-aminopiperidin-1-yl)methyl)dibutyl phosphate was used instead of ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate to obtain 81 mg of white solid, namely compound 1G, with a yield of 58%. 1 H NMR(600MHz,Chloroform-d)δ7.72(d,J=9.8Hz,1H),7.34(s,1H),6.19(d,J=9.8Hz,1H),5.30–5.22( m,1H),4.91(s,1H),4.75(s,1H),4.63(s,1H),4.07–3.96(m,9H),3.44(s,2H),2.96(s,4H),2.77–2. 70(m,4H),2.28(d,J=11.3Hz,4H),2.15(t,J=12.4Hz,1H),2.06–1.95(m,2H),1.95–1.83(m,6H),1.8 3–1.66(m,5H),1.65–1.55(m,10H),1.54–1.17(m,19H),1.09(s,3H),0.91(dd,J=16.7,9.0Hz,15H); 13 C NMR (151MHz, CDCl3) δ207.65,162.17,155.42,154.33,150.22,147.05,121.01,115.24,84.70,7 7.31,77.10,76.89,75.42,70.20,65.85,65.80,65.75,63.00,54.71,54.22,53.99,53.14,47.9 2,47.51,45.75,40.48,39.96,38.57,37.18,36.67,36.58,33.04,32.56,32.52,32.31,31.09,2 8.57,27.92,26.96,26.35,25.75,23.30,21.72,18.69,16.59,13.58,8.53; HRMS(ESI)m / z:calcd for C 54 H 90 N4O 14 P2[M+H] + 1081.5929 found 1081.6000.

[0063] Example 8

[0064] Preparation of compound 1H

[0065]

[0066] Referring to the synthesis method 1A of Example 1, ((4-aminopiperidin-1-yl)methyl)dicyclohexyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate to obtain 84 mg of white solid, namely compound 1H, with a yield of 55%. 1 H NMR(600MHz,Chloroform-d)δ7.72(dd,J=9.8,2.5Hz,1H),7.34(s,1H),6.20(d,J=9.6Hz,1H),5.28(s,2H),4 .92(s,1H),4.75(d,J=7.9Hz,1H),4.63(d,J=7.9Hz,1H),4.44–4.34(m,4H),4.01(t,J=8.6Hz,1H),3.44(s,2 H),3.34(s,1H),2.97(s,4H),2.75–2.65(m,4H),2.29(d,J=11.3Hz,4H),2.15(t,J=11.4Hz,1H),2.06–1.95( m,2H),1.86(s,14H),1.82–1.56(m,15H),1.55–1.37(m,17H),1.37–1.14(m,17H),1.09(s,3H),0.93(s,3H); 13 C NMR (151MHz, CDCl3) δ207.65,162.17,155.42,154.33,150.22,147.03,121.00,115.26,84.70,77.33,77.3 1,77.12,77.10,76.91,76.88,75.41,75.36,75.31,70.19,63.00,55.35,55.32,54.26,54.23,54.04,53.96 ,53.84,53.43,48.41,48.00,47.57,40.48,39.97,38.57,37.18,36.68,33.81,33.68,33.65,33.04,32.64 ,32.55,32.48,32.40,31.10,27.93,26.38,25.76,25.14,23.55,23.31,21.73,16.59; HRMS(ESI)m / z:calcd forC 62 H 98 N4O 14 P2[M+H] + 1185.6555 found 1185.6633.

[0067] Example 9

[0068] Preparation of Compound 1I

[0069]

[0070] Referring to the synthesis method 1A of Example 1, ((4-aminopiperidin-1-yl)methyl)dicycloheptyl phosphate was substituted for ((4-aminopiperidin-1-yl)methyl)dioctyl phosphate to obtain 52 mg of white solid, namely compound 1I, with a yield of 32%. 1 H NMR(600MHz,Chloroform-d)δ7.74(dd,J=9.8,2.6Hz,1H),7.38(s,1H),6.24(d,J=9.8Hz,1H),5.31 (s,1H),4.96(s,1H),4.74(d,J=8.1Hz,1H),4.66–4.56(m,5H),4.08–4.02(m,1H),3.53–3.35(m,2H ),3.08–2.96(m,4H),2.82(s,1H),2.76–2.67(m,4H),2.32(s,4H),2.16(t,J=10.5Hz,1H),2.10–1. 87(m,16H),1.86–1.71(m,13H),1.71–1.60(m,10H),1.60–1.30(m,34H),1.13(s,3H),0.97(s,3H); 13 C NMR (151MHz, CDCl3) δ207.52,162.13,155.42,154.31,150.26,146.85,120.84,115.3 9,84.87,77.91,77.26,77.05,76.83,75.43,70.19,62.92,55.28,54.19,54.01,48.02 ,47.57,40.50,40.13,38.57,37.22,36.69,36.02,35.83,35.80,33.07,32.62,32.44 ,31.11,28.00,27.98,26.34,25.77,23.32,22.29,21.76,16.56; HRMS(ESI)m / z:calcd for C 66 H 106 N4O 14 P2[M+H] + 1241.7181 found 1241.7252.

[0071] Example 10

[0072] In vitro antitumor activity test of the compounds prepared in Examples 1 to 9 of the present invention:

[0073] The tumor cell proliferation inhibition test method adopts the conventional CCK-8 method (such as "New Drug Pharmacology Research Methods" edited by Lv Qiujun, 2007: 242-243).

[0074] 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.

[0075] In vitro activity test: cells in the logarithmic growth phase were taken and 7×10 3 / well were seeded into 96-well plates and incubated at 37°C in a 5% CO2 environment for 24h to maintain normal physiological pH. After cell adhesion, different concentrations of test compounds (starting concentration of 100uM, 2-fold or 3-fold dilution) were added to triplicate wells, and 0.1% DMSO was added as a control. After 72h, 10μL of a cell counting kit (CCK-8) was added to each well, and the plate was incubated at 37°C for another 0.5h-1h. The absorbance (OD) was read at 450nm on a Biotek Synergy H2. The concentration that causes 50% cell growth inhibition (IC 50 ).

[0076] The test results are shown in Table 1, wherein the sample refers to the bisphosphate derivative of bufotoxin prepared in the corresponding example.

[0077] Table 1 In vitro antitumor activity of compounds

[0078]

[0079] The above experimental results show that the compounds of the present invention have certain in vitro antitumor activity, and their activity against colon cancer is higher than that against lung cancer. The IC values ​​of most compounds against colon cancer are 50 Therefore, the compounds of the present invention and their salts can be used to prepare anti-tumor drugs.

[0080] Embodiment 11

[0081] In vivo antitumor activity test of compound 1F of the present invention on colon cancer HCT116

[0082] Animals: BALB / C nude mice (SPF grade), male, 18-20 g.

[0083] 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:

[0084]

[0085] Table 2 In vivo antitumor activity of compound 1F in colon cancer HCT116

[0086]

[0087] Compared with the Control group: **P<0.01.

[0088] The above experimental results show that the compound 1F of the present invention has excellent anti-tumor activity at a dose of 10 mg / kg when intravenously administered, with a tumor inhibition rate of 58.77%, which is comparable to the clinical drug 5-fluorouracil. Therefore, the compound of the present invention and its salts can be used for intravenous administration.

[0089] Example 12

[0090] Test of the effect of the compounds of the present invention on hERG potassium channels

[0091] 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.

[0092] 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.

[0093] 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. Preparation of the positive control compound cisapride: Take 10L of 150uM cisapride DMSO stock solution, add it to 4990L extracellular fluid, and dilute 500 times to obtain the final concentration of 300nM 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.

[0094] Table 3 hERG potassium channel activity test of compounds

[0095]

[0096] The above experimental results show that the inhibition rates of compound 1C and compound 1F at a concentration of 30 uM are 21.8±0.25% and 35.7±2.44%, respectively, indicating that the compounds of the present invention have lower hERG potassium channel inhibitory activity and lower cardiotoxicity.

[0097] 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 bisphosphate derivative of bufotoxin or a pharmaceutically acceptable salt thereof, characterized in that: The general structural formula is shown below: in, R1 is selected from C1-C30 alkyl, C3-C10 cycloalkyl; R2 is selected from C1-C30 alkyl, C3-C10 cycloalkyl; R3 is selected from C1-C30 alkyl, C3-C10 cycloalkyl; R4 is selected from C1-C30 alkyl and C3-C10 cycloalkyl.

2. The bisphosphate derivative of bufotoxin or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the bisphosphate derivative of bufotoxin, 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, R3 is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, R4 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 bisphosphate derivative of bufotoxin or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The bisphosphate derivative of bufotoxin is selected from one of the following compounds:

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

5. A pharmaceutical preparation, characterized in that The invention is prepared from the bisphosphate derivative of bufotoxin described in any one of claims 1 to 3 or its pharmaceutically acceptable salt 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 bisphosphate derivative of bufotoxin or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

8. The use 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 or central nervous system, as well as thyroid cancer, leukemia, Hodgkin's disease, lymphoma, myeloma.

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