Vinblastine derivatives, preparation method therefor and application thereof
10377774 ยท 2019-08-13
Assignee
Inventors
- Wencai Ye (Guangdong, CN)
- Heru Chen (Guangdong, CN)
- Dongmei Zhang (Guangdong, CN)
- Minfeng Chen (Guangdong, CN)
- Nanhui Xu (Guangdong, CN)
Cpc classification
C07D519/04
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides a new kind of vinca alkaloid derivatives, new applications thereof and preparation methods therefor. The vinca alkaloid derivatives comprise hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivative. The hydrazinolyzed vinca alkaloids are the compounds obtained from the reaction of vinca alkaloids or salts thereof with hydrazinolyzed hydrate; and the vinca alkaloid dipeptide derivatives are the compounds obtained from the condensation of hydrazinolyzed vinca alkaloids with N-benzyloxycarbonylglycyl proline. The present invention provides the uses of the vinca alkaloids derivatives or the pharmaceutical compositions thereof in anti-tumor, preventing or treating diabetic retinopathy, rheumatoid arthritis and serving as angiogenesis inhibitors or vascular disrupting agents.
Claims
1. A method for treating a disease in a subject comprising: administering to a subject a composition comprising a vinca alkaloid dipeptide derivative or a physiologically acceptable salt thereof, wherein the vinca alkaloid dipeptide derivative is selected from the group consisting of BX-CCXJ, BX-CCJ, BX-CCRB and BX-CCFN shown as follows ##STR00013## wherein, -PG-Z represents a benzyloxycarbonylglycylprolyl group having the structure as follows ##STR00014## wherein the disease is a cancer selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, testicular cancer, non-small cell lung cancer, stomach cancer, nasopharyngeal cancer, breast cancer, intestinal cancer, liver cancer, leukemia, prostate cancer, cervical cancer, melanoma, ovarian cancer, neuroblastoma, nephroblastoma, rheumatoid arthritis, and diabetic retinopathy.
2. The method of claim 1, wherein the physiologically acceptable salt is selected from the group consisting of hydrochloride, sulfate, acetate, tartrate and citrate.
3. A method for preparing the vinca alkaloid dipeptide derivative of claim 1 or a pharmaceutically acceptable salt thereof, comprising the steps of: (1) reacting a vinca alkaloid or a physiologically acceptable salt thereof with hydrazinolyzed hydrate to obtain a hydrazinolyzed vinca alkaloid; (2) reacting the hydrazinolyzed vinca alkaloid with benzyloxycarbonylglycyl-proline under a condensing agent to obtain a vinca alkaloid dipeptide derivative; wherein the hydrazinolyzed vinca alkaloid is selected from JJ-CCXJ or JJ-CCJ shown as follows ##STR00015##
4. The method according to claim 3, wherein the condensing agent is selected from the group consisting of ethyl chloroformate, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N,N-diisopropyl carbodiimide, benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate and 1-chloro-N,N,2-trimethylacrylamide and any combination of ethyl chloroformate, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N,N-diisopropyl carbodiimide, benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate and 1-chloro-N,N,2-trimethylacrylamide.
5. A method for treating a disease in a subject comprising: administering to a subject a composition comprising a vinca alkaloid derivative, wherein the vinca alkaloid derivative is selected from a hydrazinolyzed vinca alkaloid and physiologically acceptable salts thereof, and a vinca alkaloid dipeptide derivative and physiologically acceptable salts thereof; wherein the hydrazinolyzed vinca alkaloid is JJ-CCXJ or JJ-CCFN shown as follows: ##STR00016## and wherein the vinca alkaloid dipeptide derivative is selected from the group consisting of BX-CCXJ, BX-CCJ, BX-CCRB and BX-CCFN shown as follows ##STR00017## wherein, -PG-Z represents a benzyloxycarbonylglycylprolyl group having the structure as follows ##STR00018## wherein the disease is a cancer selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, testicular cancer, non-small cell lung cancer, stomach cancer, nasopharyngeal cancer, breast cancer, intestinal cancer, liver cancer, leukemia, prostate cancer, cervical cancer, melanoma, ovarian cancer, neuroblastoma, nephroblastoma, rheumatoid arthritis, and diabetic retinopathy.
6. The method of claim 5, the vinca alkaloid dipeptide derivative or a physiologically acceptable salt thereof serves as a substrate for specific hydrolysis by tumor stroma fibroblast activating protease (FAP).
7. The method according to claim 5, wherein the physiologically acceptable salt is selected from hydrochloride, sulfate, acetate, tartrate and citrate.
8. The method according to claim 5, wherein the composition, serves as an angiogenesis inhibitor or a vascular disrupting agent.
9. The method according to claim 5, wherein the physiologically acceptable salt is selected from hydrochloride, sulfate, acetate, tartrate and citrate.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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PARTICULAR EMBODIMENTS
(18) The present invention will be further described in detail below in combination of the examples, but the embodiments of the present invention are not limited thereto.
Example 1. Preparation, Separation and Purification of Vinca Alkaloid Derivatives
(19) 1.1.1. Hydrazinolysis of Vinblastine.
(20) To a 35-mL thick wall pressure pipe, 182 mg (0.2 mmol) vinblastine sulphate, following with 8 mL methanol and 0.9 mL 80 wt % hydrazinolyzed hydrate (23 mmol), were added. The mixture was stirred for 5 min by a sonic oscillator and was degassed through N.sub.2 bubbling. The container was then covered with plug and protected from light with tinfoil coverage. Then the mixture was stirred over an oil-bath at 60 C. for 24 h. Water was added to terminate the reaction. Multiple extractions with dichloromethane were carried on. After combining all the organic layers, the extract was washed with water and saturated brine, respectively, and dried over anhydrous Na.sub.2SO.sub.4. Removal of excess solvent was carried on. The crude residues were then purified by RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) with eluant of MeOH:H.sub.2O:Et.sub.3N=70:30:0.005 (in V/V/V), resulted in a slight yellow solid 116 mg with a yield of 76.1%. .sup.1H NMR (300 MHz, CDCl.sub.3) : 8.19 (s, 1H), 8.03 (s, 1H), 7.51 (d, J=9.0 Hz, 1H), 7.077.22 (m, 3H), 6.54 (s, 1H), 6.09 (s, 1H), 5.745.88 (m, 2H), 4.13 (m, 2H), 3.844.00 (m, 3H), 3.78 (s, 3H), 3.60 (s, 3H), 3.453.56 (m, 2H), 3.34 (d, J=6.0 Hz, 1H), 3.29 (d, J=6.0 Hz, 1H), 3.123.27 (m, 3H), 2.812.93 (m, 3H), 2.78 (s, 3H), 2.61 (s, 1H), 2.392.54 (m, 3H), 2.262.38 (m, 2H), 1.942.08 (m, 2H), 1.641.80 (m, 4H), 1.431.58 (m, 3H), 1.321.45 (m, 4H), 0.810.96 (m, 6H); .sup.13C NMR (75 MHz, CDCl.sub.3) : 175.3, 173.4, 158.0, 152.5, 135.0, 131.4, 130.4, 129.3, 123.9, 123.7, 122.5, 122.3, 120.0, 118.9, 118.4, 116.7, 110.5, 93.4, 84.1, 80.5, 73.7, 69.3, 66.4, 64.0, 55.8, 55.7, 53.3, 52.4, 50.4, 50.2, 49.7, 47.7, 45.1, 42.2, 41.0, 40.8, 38.3, 34.5, 32.8, 29.7, 22.6, 8.6, 6.9; ESI-MS (m/z): 769.9[M+H].sup.+. All the data support that the provided compound is hydrazinolyzed vinblastine (JJ-CCJ) with the exact structure as shown below.
(21) ##STR00005##
1.1.2. Hydrazinolysis of Vinorelbine.
(22) To a 35-mL thick wall pressure pipe, 185.6 mg (0.2 mmol) vinorelbine tartrate, following 8 mL methanol and 9 mL 80 wt % hydrazinolyzed hydrate (0.23 mol), were added. The mixture was stirred for 5 min by a sonic oscillator and was degassed through N.sub.2 bubbling. The container was then covered with plug and protected from light with tinfoil coverage. Then the mixture was stirred over an oil-bath at 52 C. for 60 h. Water was added to terminate the reaction. Multiple extractions with dichloromethane were carried on. After combining all the organic layers, the extract was washed with water and saturated brine, respectively, and dried over anhydrous Na.sub.2SO.sub.4. Removal of excess solvent was carried on. The crude residues were then purified by HPLC with eluant of MeOH:H.sub.2O:Et.sub.3N=70:30:0.005 (in V/V/V), resulted in a yellow solid 89.8 mg with a yield of 61%. .sup.1H NMR (300 MHz, CDCl.sub.3) : 8.61 (s, 1H), 8.22 (br s, 1H), 8.02 (d, J=6.0 Hz, 1H), 7.167.25 (m, 2H), 6.27 (s, 1H), 6.09 (s, 1H), 5.795.92 (m, 2H), 5.71 (d, J=9.0 Hz, 1H), 4.92 (d, J=15.0 Hz, 1H), 4.52 (d, J=15.0 Hz, 1H), 4.19 (d, J=15.0 Hz, 1H), 4.02 (s, 1H), 3.84 (s, 3H), 3.70 (s, 3H), 3.65 (s, 1H), 3.403.55 (m, 3H), 3.30 (dd, J=3.0, 15.0 Hz, 1H), 3.103.23 (m, 2H), 2.93 (d, J=15.0 Hz, 1H), 2.85 (s, 1H), 2.80 (s, 1H), 2.75 (d, J=3.0 Hz, 1H), 2.602.73 (m, 3H), 2.362.59 (m, 3H), 2.002.14 (m, 4H), 1.881.99 (m, 2H), 1.651.84 (m, 4H), 1.221.34 (m, 4H), 1.10 (t, J=9.0, 3H), 0.84 (t, J=9.0 Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) : 174.2, 158.1, 152.9, 134.7, 134.4, 131.3, 130.0, 128.2, 124.7, 124.0, 123.4, 123.0, 122.4, 121.4, 119.2, 117.3, 110.6, 104.8, 93.0, 83.7, 73.9, 65.0, 55.7, 54.4, 53.4, 53.3, 53.0, 50.1, 48.9, 47.2, 44.7, 43.5, 42.1, 37.7, 34.5, 31.8, 29.7, 27.6, 27.3, 11.9, 8.5; ESI-MS (m/z): 737.5[M+H].sup.+. All the data support that the provided compound is hydrazinolyzed vinorelbine (JJ-CCRB) with the exact structure as shown below.
(23) ##STR00006##
1.1.3. Hydrazinolysis of Vincristine.
(24) To a 100-mL thick wall pressure pipe, 461 mg (0.5 mmol) vincristine sulphate, following 20 mL methanol and 20 mL 80 wt % hydrazinolyzed hydrate (0.51 mol), were added. The mixture was stirred for 5 min by a sonic oscillator and was degassed through N.sub.2 bubbling. The container was then cover with plug and protected from light with tinfoil coverage. Then the mixture was stirred over an oil-bath at 60 C. for 24 h. Water was added to terminate the reaction. Multiple extractions with dichloromethane (DCM) were carried on. After combining all the organic layers, the extract was washed with water and saturated brine, respectively, and dried over anhydrous Na.sub.2SO.sub.4. Removal of excess solvent was carried on. The crude residues were then purified by RP-HPLC with eluant of Acetonitrile:H.sub.2O:Et.sub.3N=55:45:0.005 (in V/V/V), resulted in a slight yellow solid 324 mg with a yield of 86%. .sup.1H NMR (300 MHz, CDCl.sub.3) : 9.77 (s, 1H), 8.06 (s, 1H), 7.51 (d, J=9.0 Hz, 1H), 7.057.19 (m, 3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.765.94 (m, 3H), 5.65 (d, J=9.0 Hz, 1H), 5.48 (s, 1H), 4.00 (s, 1H), 3.853.96 (m, 1H), 3.83 (s, 1H), 3.72 (s, 3H), 3.58 (s, 3H), 3.253.47 (m, 4H), 3.043.24 (m, 3H), 2.722.88 (m, 3H), 2.352.52 (m, 3H), 2.27 (d, J=15.0 Hz, 1H), 2.022.15 (m, 2H), 1.98 (s, 3H), 1.791.93 (m, 1H), 1.481.65 (m, 2H), 1.361.48 (m, 2H), 0.81.00 (m, 8H); ESI-MS (m/z): 755.6 [M+H].sup.+. All the data support that the provided compound is hydrazinolyzed vincristine (JJ-CCXJ) with the exact structure as shown below.
(25) ##STR00007##
1.1.4. Hydrazinolysis of Vinflunine.
(26) To a 35-mL thick wall pressure pipe, 163.2 mg (0.2 mmol) vinflunine, following by added 8 mL methanol and 9 mL 80 wt % hydrazinolyzed hydrate. The mixture was stirred for 5 min by a sonic oscillator and was degassed through N.sub.2 bubbling. The container was then covered with plug and protected from light with tinfoil coverage. Then the mixture was stirred over an oil-bath at 52 C. for 60 h. Water was added to terminate the reaction. Multiple extractions with dichloromethane (DCM) were carried on. After combining all the organic layers, the extract was washed with water and saturated brine, respectively, and dried over anhydrous Na.sub.2SO.sub.4. Removal of excess solvent was carried on. The crude residues were then purified by RP-HPLC with eluant of Acetonitrile:H.sub.2O:Et.sub.3N=55:45:0.005, resulted in a slight yellow solid 80.5 mg with a yield of 52%. .sup.1H NMR (300 MHz, CDCl.sub.3) : 9.53 (br s, 1H), 8.48 (s, 1H), 8.28 (s, 1H), 7.72 (d, J=6.0 Hz, 1H), 7.18 (m, 3H), 6.32 (s, 1H), 6.08 (s, 1H), 5.555.90 (m, 3H), 4.484.64 (m, 2H), 4.06 (s, 1H), 3.80 (s, 3H), 3.69 (s, 3H), 3.373.45 (m, 3H), 3.233.37 (m, 2H), 3.133.22 (m, 1H), 2.893.12 (m, 2H), 2.79 (s, 3H), 2.66 (d, J=6.0 Hz, 1H), 2.61 (s, 1H), 2.51 (s, 1H), 2.292.47 (m, 2H), 1.912.05 (m, 3H), 1.791.91 (d, J=12.0 Hz, 1H), 1.531.78 (m, 6H), 1.221.36 (m, 2H), 1.091.23 (m, 1H), 0.81 (t, J=9.0 Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) : 174.7, 173.5, 157.8, 152.5, 134.6, 133.3, 130.2, 128.5, 124.0, 122.8, 122.7, 122.4, 120.0, 119.0, 118.3, 110.6, 109.2, 92.9, 83.8, 80.4, 73.7, 65.5, 55.6, 55.3, 53.4, 53.1, 52.7, 50.2, 49.3, 47.0, 46.3, 44.7, 42.0, 37.9, 33.7, 32.0, 29.9, 28.5, 22.5, 21.5, 8.4; ESI-MS (m/z): 775.4 [M+H].sup.+. All the data support that the provided compound is hydrazinolyzed vinflunine (JJ-CCFN) with the exact structure as shown below.
(27) ##STR00008##
1.2.1. Preparation of Z-GP-NHNH-Vinblastine (BX-CCJ).
(28) 36.7 mg (0.12 mmol) of N-carbobenzoxyglycyl proline were dissolved in 5 mL of acetonitrile and sealed with a rubber plug. The solution was put over an ice-bath with stirring for 5 min. Then, 0.031 mL (0.2 mmol) of DIC was added. The reaction was kept on with stirring over ice-bath for 20 min Afterward, 76.8 mg (0.1 mmol) of JJ-CCJ in 1 mL DCM was added slowly. The reaction mixture was then warmed up to room temperature and lasted for 24 h. The reaction was quenched with the addition of water. Multiple extractions with dichloromethane were carried on. After combining all the organic layers, the extract was washed with water and saturated brine, respectively, and dried over anhydrous Na.sub.2SO.sub.4. Removal of excess solvent was carried on. The crude residues were then purified by RP-HPLC with eluant of MeOH:H.sub.2O:Et.sub.3N=70:30:0.005 (in V/V/V), resulted in a slight yellow solid 86.6 mg with a yield of 82.2%. .sup.1H NMR (300 MHz, CDCl.sub.3) : 8.04 (br, 1H), 7.53 (d, J=9.0 Hz 1H), 7.277.38 (m, 5H), 7.057.21 (m, 3H), 6.54 (s, 1H), 6.08 (s, 1H), 5.615.84 (m, 2H), 5.005.20 (m, 2H), 4.63 (d, J=6.0 Hz, 1H), 3.81409 (m, 4H), 3.76 (s, 3H), 3.60 (s, 3H), 3.56 (s, 2H), 3.253.49 (m, 4H), 3.083.24 (m, 3H), 3.04 (dd, J=6.0, 15.0 Hz, 1H), 2.86 (s, 2H), 2.81 (s, 3H), 2.60 (s, 1H), 2.372.54 (m, 2H), 2.242.37 (m, 2H), 1.902.10 (m, 4H), 1.581.79 (m, 2H), 1.401.53 (m, 2H), 1.141.39 (m, 8H), 0.811.00 (m, 8H); .sup.13C NMR (75 MHz, CDCl.sub.3) : 175.1, 171.2, 169.6, 168.8, 158.0, 156.5, 152.8, 136.5, 134.9, 131.4, 130.3, 129.3, 128.4, 128.0, 124.0, 123.5, 122.4, 122.3, 119.8, 118.9, 118.3, 116.4, 110.5, 93.5, 83.4, 80.8, 73.7, 69.4, 66.8, 66.2, 63.6, 58.8, 55.8, 55.7, 53.4, 53.2, 52.4, 50.3, 49.6, 47.3, 46.3, 45.3, 44.8, 43.4, 42.3, 40.8, 38.6, 34.5, 32.7, 29.6, 29.4, 28.2, 27.4, 24.8, 14.1, 8.7, 8.6, 6.8; ESI-MS (m/z): 1057.9 [M+H].sup.+. All the data support that the provided compound is Z-GP-NHNH-Vinblastine (BX-CCJ) with the exact structure as shown below.
(29) ##STR00009##
1.2.2. Preparation of Z-GP-NHNH-Vinorelbine (BX-CCRB).
(30) 33.7 mg (0.11 mmol) of N-carbobenzoxyglycyl proline (Z-GP-OH) were dissolved in 5 mL of acetonitrile and sealed with a rubber plug. The solution was put over an ice-bath with stirring for 5 min. Then, 0.031 mL (0.2 mmol) of DIC was added. The reaction was kept on with stirring over ice-bath for 20 min, thus reaction mixture A was obtained. Afterward, 73.6 mg (0.1 mmol) of JJ-CCRB was dissolved in 1 mL DCM, dropped into the reaction mixture A, then warmed up to room temperature and lasted for 24 h. The reaction was quenched with the addition of water. Multiple extractions with DCM were carried on. After combining all the organic layers, the extract was washed with water and saturated brine, respectively, and dried over anhydrous Na.sub.2SO.sub.4. Removal of excess solvent was carried on. The crude residues were then purified by HPLC with eluant of MeOH:H.sub.2O:Et.sub.3N=70:30:0.005 (in V/V/V), resulted in a white solid 80.2 mg with a yield of 77%. .sup.1H NMR (300 MHz, CDCl.sub.3) : 8.69 (s, 1H), 8.43 (br, 1H), 7.84 (s, 1H), 7.147.40 (m, 9H), 6.41 (s, 1H), 6.32 (s, 1H), 6.08 (s, 1H), 5.79 (s, 2H), 5.60 (d, J=9.0 Hz, 1H), 5.005.18 (m, 2H), 4.94 (d, J=15.0 Hz 1H), 4.61 (s, 1H), 4.46 (d, J=12.0 Hz, 1H), 3.884.11 (m, 4H), 3.81 (s, 3H), 3.69 (s, 3H), 3.483.65 (m, 4H), 3.36 (m, 4H), 2.983.27 (m, 8H), 2.87 (m, 1H), 2.83 (s, 3H), 2.67 (m, 2H), 2.52 (t, J=12.0 Hz, 2H), 2.142.30 (m, 1H), 1.832.14 (m, 6H), 1.661.81 (m, 1H), 1.50166 (m, 1H), 1.30 (t, J=9.0 Hz, 3H), 1.09 (t, J=9.0 Hz, 3H), 0.79 (m, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) : 174.4, 171.1, 170.0, 168.5, 167.9, 157.9, 156.5, 153.0, 136.4, 134.6, 134.2, 131.7, 130.2, 128.2, 128.1, 127.7, 127.6, 124.3, 123.3, 123.1, 122.6, 122.4, 120.6, 118.5, 117.3, 110.5, 105.6, 92.8, 82.8, 80.5, 73.8, 66.4, 64.5, 58.7, 55.6, 54.3, 52.9, 52.7, 52.1, 49.9, 48.9, 46.1, 46.0, 45.1, 44.2, 43.2, 42.8, 42.2, 38.0, 34.4, 31.5, 28.6, 27.4, 27.1, 24.4, 11.7, 8.3, 8.1; ESI-MS (m/z): 1025.6[M+H].sup.+. All the data support that the provided compound is Z-GP-NHNH-Vinorelbine (BX-CCRB) with the exact structure as shown below.
(31) ##STR00010##
1.2.3. Preparation of Z-GP-NHNH-Vincristine (BX-CCXJ).
(32) 137.7 mg (0.45 mmol) of N-carbobenzoxyglycyl proline (Z-GP-OH) were dissolved in 7.5 mL of acetonitrile. The solution was put over an ice-bath with stirring for 5 min. Then, 0.09 mL (0.6 mmol) of DIC was added. The reaction was kept on with stirring over ice-bath for 20 min, thus reaction mixture A was obtained. Afterward, 339.0 mg (0.45 mmol) of JJ-CCXJ was dissolved in 4.5 mL DCM, dropped into the reaction mixture A, then warmed up to room temperature and lasted for 24 h. The reaction was quenched with addition of water. Multiple extractions with DCM were carried on. After combining all the organic layers, the extract was washed with water and saturated brine, respectively, and dried over anhydrous Na.sub.2SO.sub.4. Removal of excess solvent was carried on. The crude residues were then purified by HPLC with eluant of MeOH:H.sub.2O:Et.sub.3N=80:20:0.005 (in V/V/V), resulted in a slight yellow solid 286.1 mg with a yield of 61%, which was named as compound B.
(33) Afterward, 208 mg (0.2 mmol) of compound B was dissolved in 3 mL of DCM and added appropriate amount of acetic formic anhydride solution, which was prepared by mixing 1.1 mol acetic anhydride and 0.5 mL formic acid solution, fully stirred with a proportion of 11:5. The mixture was stirred at room temperature for 2 h. Then, excess reagent was removed by evaporation. The crude residues were then purified by RP-HPLC with eluant of MeOH:H.sub.2O:Et.sub.3N=65:35:0.005, resulted in a slight yellow solid 70.3 mg with a yield of 32.9%. .sup.1H NMR (300 MHz, CD.sub.3OD) : 8.50 (m, 1H), 7.43 (d, J=6.0 Hz, 2H), 7.207.57 (m, 11H), 7.15 (d, J=6.0 Hz, 1H), 7.08 (t, J=6.0 Hz, 2H), 7.00 (t, J=6.0 Hz, 2H), 6.54 (s, 1H), 6.27 (s, 1H), 5.80 (m, 2H), 5.565.72 (m, 2H), 5.005.14 (m, 4H), 4.42461 (m, 2H), 3.90410 (m, 9H), 3.773.89 (m, 3H), 3.723.75 (m, 1H), 3.73 (s, 3H), 3.613.68 (m, 4H), 3.60 (s, 3H), 3.493.57 (m, 3H), 3.44 (d, J=6.0 Hz, 1H), 3.353.41 (m, 1H), 3.293.33 (m, 4H), 3.163.29 (m, 6H), 3.003.15 (m, 3H), 2.702.87 (m, 5H), 2.63 (d, J=12.0 Hz, 1H), 2.392.52 (m, 4H), 2.212.38 (m, 4H), 2.052.21 (m, 7H), 1.922.05 (m, 4H), 1.802.02 (m, 2H), 1.62174 (m, 1H), 1.44161 (m, 4H), 1.20144 (m, 13H), 0.66100 (m, 17H); .sup.13C NMR (75 MHz, CD.sub.3OD) : 177.3, 174.0, 173.9, 173.3, 170.3, 170.2, 159.3, 158.9, 158.5, 151.4, 138.1, 136.9, 136.5, 132.1, 131.8, 131.1, 130.4, 130.2, 129.4, 129.0, 128.9, 128.8, 126.4, 125.4, 125.2, 124.0, 123.3, 120.6, 119.9, 119.1, 118.2, 117.6, 112.0, 111.3, 102.4, 94.3, 82.8, 81.7, 75.7, 75.0, 69.5, 69.3, 69.0, 67.7, 64.0, 60.4, 60.3, 60.1, 57.5, 56.9, 56.6, 56.3, 54.4, 54.0, 53.0, 52.9, 51.9, 51.8, 47.6, 44.7, 44.1, 43.5, 41.1, 40.9, 35.8, 33.6, 33.3, 33.0, 30.7, 30.5, 30.4, 27.7, 25.8, 23.7, 14.4, 9.0, 7.3; ESI-MS (m/z): 1071.5 [M+H].sup.+. All the data support that the provided compound is Z-GP-NHNH-Vincristine (BX-CCXJ) with the exact structure as shown below.
(34) ##STR00011##
1.2.4. Preparation of Z-GP-NHNH-Vinflunine (BX-CCFN).
(35) 30.6 mg (0.1 mmol) of N-carbobenzoxyglycyl proline (Z-GP-OH) were dissolved in 5 mL of acetonitrile and sealed with a rubber plug. The solution was put over an ice-bath with stirring for 5 min. Then, 0.031 mL (0.2 mmol) of DIC was added. The reaction was kept on with stirring over ice-bath for 20 min Afterward, 77.4 mg (0.1 mmol) of JJ-CCFN in 1 mL DCM was added slowly. The reaction mixture was then warmed up to room temperature and lasted for 24 h. The reaction was quenched with the addition of water. Multiple extractions with DCM were carried on. After combining all the organic layers, the extract was washed with water and saturated brine, respectively, and dried over anhydrous Na.sub.2SO.sub.4. Removal of excess solvent was carried on. The crude residues were then purified by HPLC with eluant of MeOH:H.sub.2O:Et.sub.3N=75:25:0.005 (in V/V/V), resulted in a slight yellow solid 77.2 mg with a yield of 74%. .sup.1H NMR (300 MHz, CDCl.sub.3) : 8.54 (s, 1H), 7.73 (d, J=6.0 Hz, 1H), 7.267.38 (m, 5H), 7.18 (m, 3H), 6.31 (s, 1H), 6.07 (s, 1H), 5.79 (dd, J=3.0, 9.0 Hz, 1H), 5.64 (d, J=12.0 Hz, 1H), 5.015.16 (m, 2H), 4.444.75 (m, 2H), 3.854.13 (m, 2H), 3.79 (s, 3H), 3.68 (s, 3H), 3.513.65 (m, 2H), 3.313.50 (m, 3H), 3.26 (dd, J=3.0, 15 Hz, 1H), 3.073.19 (m, 1H), 2.903.07 (m, 3H), 2.82 (s, 3H), 2.78 (s, 1H), 2.64 (dd, J=6.0, 15.0 Hz, 1H), 2.55 (s, 1H), 2.332.49 (m, 2H), 1.651.73 (m, 2H), 1.591.68 (m, 3H), 1.311.43 (m, 1H), 1.201.31 (m, 5H), 1.061.20 (m, 2H), 0.820.92 (m, 1H), 0.80 (t, J=9.0 Hz, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) : 174.6, 173.3, 171.2, 170.0, 168.6, 157.8, 156.5, 155.7, 153.0, 136.4, 134.6, 133.5, 130.3, 128.5, 128.4, 127.9, 127.8, 124.3, 122.9, 122.5, 122.4, 120.1, 118.8, 118.3, 93.1, 83.1, 80.7, 73.8, 66.7, 65.3, 58.7, 55.6, 55.3, 53.1, 52.7, 50.2, 49.5, 49.4, 49.2, 46.4, 46.3, 45.8, 45.0, 44.5, 43.3, 42.3, 38.2, 33.6, 32.0, 29.6, 28.6, 28.1, 24.7, 22.5, 8.6, 8.3; ESI-MS (m/z): 1063.4 [M+H].sup.+. All the data support that the provided compound is Z-GP-NHNH-Vinflunine (BX-CCFN) with the exact structure as shown below.
(36) ##STR00012##
1.3.1. Preparation of BX-CCJ Sulphate.
(37) 106.0 mg (0.1 mmol) of BX-CCJ were dissolved in 12 mL of 0.01 mmol/L sulphuric acid in 1:1 methanol/dichloromethane solution. The solution was stirred at 0 C. for 3 h. Then removal of excess reagents was carried on at room temperature by vacuum evaporation. The resulted solid was washed with cold ether for triple times and removed diethyl ether by centrifugation. The solid compounds was composited and then re-dissolved in water. After lyophilization, 111.4 mg of BX-CCJ sulphate were collected with a yield of 96.2%.
(38) 1.3.2. Preparation of BX-CCRB Tartrate.
(39) 102.5 mg (0.1 mmol) of BX-CCRB were dissolved in 15 mL of 0.01 mmol/L tartaric acid in 1:1 methanol/dichloromethane solution. The solution was stirred 0 C. for 3 h. Then removal of excess reagents was carried on at room temperature by vacuum evaporation. The resulted solid was washed with cold ether for triple times and removed diethyl ether by centrifugation. The solid compounds was composited and then re-dissolved in water. After lyophilization, 115.1 mg of BX-CCRB tartrate were collected with a yield of 98%.
Example 2. In Vitro Cell Growth Inhibitory Activities of Vinca Alkaloid Derivatives
(40) Experimental method: Cell lines (human non-small-cell lung cancer cell line A549, human colon cancer cell line LOVO, human nasopharyngeal carcinoma cell line CNE-2, human liver cancer cell line HepG2, human cervical carcinoma cell line Hela, human breast cancer cell lines MCF-7 and MDA-MB-231, human gastric carcinoma cell line NCI-N87, human prostatic cancer cell lines PC-3 and DU145, human leukemia cell line K562, human melanoma cell line A375, human neuroblastoma cell line SH-SY5H, human promyelocytic leukemia cell line HL-60, 5-FU-resistant human hepatocellular carcinoma cell line BEL-7402/5-Fu, doxorubicin-resistant hepatocellular carcinoma cell line HepG2/ADM, doxorubicin-resistant human breast cancer cell lines MCF-7/ADR) at logarithmic phase were resuspended in RPMI 1640 medium (containing 10% fetal bovine serum, 100 U/mL penicillin-streptomycin). Then, 100 L of cells (cell concentration was 510.sup.5/mL) were seeded into 96 wells plate. After 24 h incubation at 37 C. with 5% CO.sub.2, tested compounds were added (the control group without compound) for an additional 72 h incubation. After that, 30 L of MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (5 mg/mL) was added into each well and incubated for another 4 h at 37 C. and then the formazan crystals were solubilized with 100 L DMSO. Finally, absorbance of each well was determined at 570 nm by a microplate reader (Thermo).
(41) Cell growth inhibition rates were measured using the following formula:
Inhibition rates (%)=(1OD value of drug group/OD value of control group)100%
(42) The standard curve was drew with compound concentrations as the abscissa and cell growth inhibition rates as the ordinate. The concentration required to inhibit cell growth by 50% (IC.sub.50) was calculated from survival curves.
(43) Results: From table 2-1 and 2-2, vinca alkaloids, hydrazinolyzed vinca alkaloids and of vinca alkaloid dipeptide derivatives all had a broad spectrum of anticancer activity. At FAP negative expressed tumor cell lines, cell growth inhibitory activities were similar among vinca alkaloids and hydrazinolyzed vinca alkaloids and superior to their vinca alkaloid dipeptide derivatives. Besides, the cell growth inhibitory activities were similar among hydrazinolyzed vinca alkaloids and of vinca alkaloid dipeptide derivatives at FAP positive expressed LOVO cells. These results showed that the vinca alkaloid dipeptide derivatives could be hydrolied by FAP enzyme, and corresponding hydrazinolyzed vinca alkaloids were produced.
(44) TABLE-US-00002 TABLE 2-1 Detection of inhibitory effects of vinca alkaloid derivatives on growth of multiple tumor cell lines by MTT assay IC.sub.50 (nM) Tumor cell Vinblastine JJ-CCJ BX-CCJ Vinorelbine JJ-CCRB BX-CCRB A549 2.31 0.13 6.43 2.42 62.11 3.87 14.54 1.07 73.06 2.36 114.81 5.23 LOVO 1.20 0.24 32.92 6.39 44.79 8.15 7.86 0.16 120.54 10.33 123.07 11.45 CNE-2 1.03 0.07 7.14 0.16 52.67 3.01 9.03 0.35 100.38 5.90 210.32 5.78 HepG2 3.07 0.12 12.44 0.72 46.84 1.17 8.34 0.53 89.31 0.71 167.22 4.37 Hela 4.34 0.36 14.50 1.81 56.77 3.08 11.38 1.04 61.32 3.12 181.53 2.36 MCF-7 3.67 1.34 23.93 3.07 107.88 4.23 49.47 2.01 121.59 5.75 237.76 8.37 MDA-MB-231 0.81 0.35 5.99 3.29 64.22 4.57 39.69 5.79 100.89 7.65 180.69 8.10 NCI-N87 2.23 0.43 17.37 2.20 103.49 8.87 35.78 6.57 89.02 3.34 244.07 9.33 PC-3 1.56 0.39 6.27 0.61 71.10 1.97 23.67 4.31 65.89 3.05 269.76 2.03 DU-145 0.78 0.31 5.33 0.60 57.30 2.07 19.90 0.57 60.44 7.01 218.74 6.29 K562 0.72 0.20 3.67 0.79 11.69 3.04 12.25 0.54 49.74 3.39 108.77 4.66 A375 3.24 0.39 4.56 0.57 49.54 4.53 5.64 0.95 20.37 2.85 120.35 14.51 SH-SY5H 6.13 0.51 6.24 1.31 59.83 6.42 4.78 0.84 32.94 3.27 165.74 15.62 HL-60 5.21 0.84 8.65 1.28 85.49 9.57 8.68 1.35 46.82 5.41 208.41 18.43 BEL-7402/5-Fu 25.77 3.01 80.34 7.53 179.44 8.87 36.09 4.78 143.13 8.57 325.44 11.74 HepG2/ADM 10.07 1.12 43.90 3.77 146.43 5.47 24.40 3.82 182.06 4.38 437.09 14.43
(45) TABLE-US-00003 TABLE 2-2 Detection of inhibitory effects of vinca alkaloid derivatives on growth of multiple tumor cell lines by MTT assay IC.sub.50 (nM) Tumor cell Vinflunine JJ-CCFN BX-CCFN Vincristine JJ-CCXJ BX-CCXJ A549 137.11 4.10 206.34 6.79 334.59 7.31 1.32 0.03 4.34 0.42 47.71 2.64 LOVO 145.78 7.21 312.84 7.64 319.78 8.51 0.74 0.09 24.30 1.09 24.90 1.78 CNE-2 67.43 3.42 198.35 7.71 309.41 5.78 1.51 0.76 8.37 2.70 40.15 2.09 HepG2 32.07 2.17 103.21 6.37 299.10 7.64 0.97 0.12 12.27 1.96 33.43 2.01 Hela 56.66 4.47 132.02 6.67 430.2 10.11 2.33 0.77 23.56 2.97 55.34 1.90 MCF-7 77.03 2.74 303.89 9.44 605.71 11.47 2.74 0.36 26.88 4.38 73.21 3.66 MDA-MB-231 61.29 7.44 332.62 12.47 614.44 9.29 1.03 0.22 7.37 1.18 57.32 3.36 NCI-N87 57.49 3.95 132.60 6.70 430.34 10.39 0.87 0.26 10.73 1.78 43.17 3.53 PC-3 31.14 5.34 98.34 3.19 317.45 31.27 2.93 0.37 27.07 2.18 88.91 6.84 DU-145 22.31 1.67 90.75 5.45 289.43 28.77 1.17 0.31 18.44 3.67 76.90 4.55 K562 19.27 2.48 55.87 7.59 229.31 6.60 1.07 0.33 12.08 1.67 44.90 3.28 A375 39.84 5.29 132.28 9.51 528.41 34.79 0.95 0.16 15.62 2.38 54.82 8.41 SH-SY5H 52.37 6.81 159.46 20.38 459.36 13.48 1.34 0.21 18.69 2.54 61.72 6.14 HL-60 41.75 6.85 197.54 19.52 725.67 98.21 1.81 0.17 32.47 3.51 72.85 4.59 BEL-7402/5-Fu 45.66 3.98 166.48 5.77 389.64 15.70 22.69 3.37 106.44 5.87 326.45 13.32 HepG2/ADM 67.14 4.39 276.89 9.67 603.71 17.88 7.78 2.01 44.90 3.67 134.88 5.54 MCF-7/ADR 154.39 3.95 713.46 25.57 907.93 20.18 9.67 2.20 63.23 7.74 168.80 5.67
Example 3. In Vitro Cytotoxicity of Vinca Alkaloid Derivatives on Normal Cell Lines
(46) Experimental method: In vitro cytotoxicities of vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives on human normal liver cells LO2 and human umbilical vein endothelial cells HUVEC were detected according to the method of Example 2.
(47) Results: The cytotoxicity of hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives on LO2 and HUVEC were much smaller than of the corresponding vinca alkaloids (Table 3). The results showed that cytotoxicity of the targeted compounds were declined dramatically.
(48) TABLE-US-00004 TABLE 3 Cytotoxicity of vinca alkaloid derivatives on normal cell lines tested by MTT assay IC.sub.50 (nM) Compound HUVEC LO2 Vinblastine 0.76 0.13 0.34 0.07 JJ-CCJ 36.07 1.00 20.10 0.97 BX-CCJ 128.88 1.34 96.27 1.11 Vinorelbine 1.13 0.56 0.97 0.08 JJ-CCRB 44.02 2.27 31.46 2.29 BX-CCRB 166.64 8.87 183.12 5.51 Vinflunine 1.53 0.33 0.79 0.30 JJ-CCFN 64.43 5.04 28.87 2.76 BX-CCFN 269.90 7.70 301.31 14.30 Vincristine 0.42 0.09 0.21 0.05 JJ-CCXJ 48.64 2.13 22.59 2.08 BX-CCXJ 175.70 5.51 100.70 9.02
Example 4. Experiment on Recombinant Humanized FAP (rhFAP)-Specific Enzymolysis of Vinca Alkaloid Dipeptide Derivatives
(49) Experimental method: HPLC chromatographic conditions were as follows: high-performance liquid chromatograph (HPLC): Agilent 1200; chromatographic column: Cosmosil C.sub.18 reverse phase chromatographic column (4.6250 mm.sup.2, 5 m); mobile phase[0 min, 55% methanol and 45% water (containing 2 mM ammonium formate); 10 min, 65% methanol and 35% water (containing 2 mM ammonium formate); 15 min, 75% methanol and 25% water (containing 2 mM ammonium formate); 30 min, 85% methanol and 15% water (containing 2 mM ammonium formate); 40 min, 85% methanol and 15% water (containing 2 mM ammonium formate)]; flow rate: 1 mL/min; determine wavelength: 254 nm; sample size: 2 L. The standard curve of vinca alkaloid dipeptide derivatives was established as follows: vinca alkaloid dipeptide derivatives were dissolved in the buffer of the enzymatic hydrolysis reaction (50 mM Tris-HCl, 1.0 M NaCl, pH7.4) with five different concentrations (6.25, 12.5, 25, 50, 100 M). The standard curve was drew with peak area as the ordinate (Y) and compound concentrations (M) as the abscissa (X). The experiment was repeated three times. 50 M vinca alkaloid dipeptide derivatives was incubated with rhFAP (5 g/ml) buffer of the enzymatic hydrolysis reaction at 37 C. water bath. Then the supernatants were collected and detected at 0, 0.5, 1, 2, 4, 8, 12 and 24 h, enzymatic hydrolysate was measured by HPLC and the enzymolysis rate was calculated (A: BX-CCJ; B: BX-CCRB; C: BX-CCFN; D: BX-CCXJ).
(50) Results: Results showed that vinca alkaloid dipeptide derivatives were hydrolysed by recombinant humanized FAP enzyme in a time-dependent manner (
Example 5. Experiment on Tumor Tissues FAP (rhFAP)-Specific Enzymolysis of Vinca Alkaloid Dipeptide Derivatives
(51) Experimental method: 21 days after the xenografts implantation, the mice were killed by dislocation method after anesthesiaed by CO.sub.2. Then tumors were stripped, washed with saline and cleaned off periadventitial fat, followed by soaking up the surface water with filter. Besides, about 2 g tumor tissues were cut into small tissue pieces, transferred to glass homogenate, then 10 mL enzyme buffer was added. The homogenate was collected and filtered by 200-mesh sieve, and then 10 mL homogenate was mixed with 10 L 50 mM vinca alkaloid dipeptide derivatives with the final concentration 50 M, reacted in 37 C. water bath. 2 mL homogenate was collected at 0, 2, 8, 12 and 24 h, and transferred to 15 mL contrifuge tube, then 5 mL extracting agent (acetonitrile/dichloroethane=1:4) was added, following by vortexed for 1 min and centrifuged with 2500 rpm for 5 min. The lower organic phase to blow dry under nitrogen, and the residue was solubilized with 200 L methanol, then filtered by 0.22 m filter membrane. Finally, enzymatic hydrolysate was measured by HPLC and enzymolysis rate was calculated (A: BX-CCJ; B: BX-CCRB; C: BX-CCFN; D: BX-CCXJ).
(52) Results: Results showed that vinca alkaloid dipeptide derivatives were hydrolysed by tumor tissues FAP enzyme in tumor homogenates in a time-dependent manner (
Example 6. Acute Toxicity Test of Vinca Alkaloid Dipeptide Derivatives
(53) Experimental method: Kunming mice were purchased from Guangdong Medical Experimental Animal Center, weighing 18-22 g, then randomly divided into several groups with 10 mice in each group. Different concentrations of VLB and vinca alkaloid dipeptide derivatives were injected intraperitoneally. Finally, the median lethal dose (LD.sub.50) was calculated according to the surviving animals.
(54) LD.sub.50 was measured using the following formula:
(55) LD.sub.50 (mg/kg)=The median lethal dose/body weight
(56) Results: Results showed that the LD.sub.50 of BX-CCJ, BX-CCRB, BX-CCFN and BX-CCXJ were 10 mg/kg, 12 mg/kg, 15 mg/kg and 10 mg/kg respectively. But, 4 mg/kg of VLB caused half of the mice dead.
Example 7. In Vivo Assay of Vinca Alkaloid Derivatives on Tumor Xenograft Models
(57) Experimental method: The MDA-MB-231 cells at logarithmic phase were digested and washed twice with PBS. Then, 100 L cells (110.sup.7) were inoculated subcutaneously on the backs of female BALB/nu/nu mice. After tumors grew to 70100 mm.sup.3, the mice were divided randomly into saline group (control group), vinca alkaloids groups (VLB, CCRB, CCFN, CCXJ) and vinca alkaloid derivatives groups (JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ and BX-CCXJ), with six mice per group. 1 mg/kg drugs were injected intraperitoneally every two days, meanwhile body weight and tumor sizes were measured. Experiment was ended after 8 times of drug administration, then tumor and organs were stripped. Tumor volumes were measured using the following formula: V=(ab.sup.2), whereas a refers to the longest diameter and b is the shortest diameter.
(58) Tumor xenograft models of HepG2, LOVO, K562 and HL-60 cell lines were performed as the method described above.
(59) Results: 1 mg/kg of vinca alkaloids and vinca alkaloid derivatives all dramatically inhibited tumor xenograft models of MDA-MB-231, HepG2, LOVO, K562 and HL-60 cell lines (Table 4-8). The toxicity of vinca alkaloid dipeptide derivatives was far below vinca alkaloids and hydrazinolyzed vinca alkaloids at the dosage of 1 mg/kg. Besides, The vinca alkaloid dipeptide derivatives groups did not lead to significantly body weight lose and organs damage. But the body weight of vinca alkaloids groups and hydrazinolyzed vinca alkaloids groups declined dramatically, and the damage of liver, spleen and other organs appeared.
(60) TABLE-US-00005 TABLE 4 Inhibitory effects of vinca alkaloid derivatives on MDA-MB-231 xenografts in nude mice Anti- Body weight (g) Tumor tumor Compound Dosage Pre-dose Post-dose weight (g) rate (%) Saline 20.3 2.3 25.5 3.6 2.61 0.73 VLB 1 mg/kg 20.7 1.4 18.3 2.4* 1.22 0.35 53.26** JJ-CCJ 1 mg/kg 20.3 0.7 16.4 1.7* 0.32 0.03 87.74** BX-CCJ 1 mg/kg 19.8 1.0 21.2 2.1 0.20 0.12 92.34** JJ-CCRB 1 mg/kg 21.0 2.7 16.7 1.4* 0.59 0.18 77.39** BX-CCRB 1 mg/kg 19.1 1.7 20.1 0.9 0.37 0.07 85.83** JJ-CCFN 1 mg/kg 19.7 2.1 16.3 0.7* 0.65 0.13 75.10** BX-CCFN 1 mg/kg 20.2 0.7 20.6 1.6 0.31 0.11 88.13** JJ-CCXJ 1 mg/kg 21.1 2.5 17.3 1.2* 0.63 0.15 75.86** BX-CCXJ 1 mg/kg 19.5 1.4 21.9 2.9 0.23 0.09 91.18** *P < 0.05, **P < 0.01 vs control
(61) TABLE-US-00006 TABLE 5 Inhibitory effects of vinca alkaloid derivatives on HepG2 xenografts in nude mice Anti- Body weight (g) Tumor tumor Compound Dosage Pre-dose Post-dose weight (g) rate (%) Saline 21.8 1.0 27.3 1.9 2.55 0.68 VLB 1 mg/kg 21.9 0.7 18.2 1.8* 1.46 0.27 42.75* JJ-CCJ 1 mg/kg 21.7 1.2 16.1 0.3** 0.57 0.7 77.65** BX-CCJ 1 mg/kg 22.0 1.7 22.1 1.3 0.34 0.21 86.67** JJ-CCRB 1 mg/kg 21.9 1.6 17.0 0.8* 0.63 0.29 75.29** BX-CCRB 1 mg/kg 22.2 1.5 22.6 0.5 0.37 0.09 85.49** JJ-CCFN 1 mg/kg 22.3 1.7 16.5 0.4** 0.62 0.19 75.69** BX-CCFN 1 mg/kg 21.9 1.3 22.1 1.2 0.30 0.17 88.24** JJ-CCXJ 1 mg/kg 22.0 2.1 17.8 0.9** 0.53 0.12 79.21** BX-CCXJ 1 mg/kg 22.2 1.2 21.9 2.1 0.29 0.13 88.63** *P < 0.05, **P < 0.01 vs control
(62) TABLE-US-00007 TABLE 6 Inhibitory effects of vinca alkaloid derivatives on LOVO xenografts in nude mice Anti- Body weight (g) Tumor tumor Compound Dosage Pre-dose Post-dose weight (g) rate (%) Saline 22.9 0.9 26.2 1.0 2.87 0.47 VLB 1 mg/kg 23.1 1.4 19.6 1.5* 1.59 0.51 44.60* JJ-CCJ 1 mg/kg 22.8 1.7 17.3 1.2** 0.64 0.21 77.71** BX-CCJ 1 mg/kg 23.8 1.9 24.4 1.6 0.43 0.38 85.02** JJ-CCRB 1 mg/kg 23.2 2.1 16.4 0.7** 0.87 0.16 69.69** BX-CCRB 1 mg/kg 23.6 1.2 24.1 2.3 0.57 0.23 80.14** JJ-CCFN 1 mg/kg 22.7 1.5 18.9 2.1* 0.68 0.13 76.31** BX-CCFN 1 mg/kg 23.6 1.3 24.5 1.5 0.47 0.31 83.63** JJ-CCXJ 1 mg/kg 22.9 2.3 17.5 1.3** 0.76 0.16 73.52** BX-CCXJ 1 mg/kg 23.8 1.0 24.2 1.7 0.59 0.33 79.45** *P < 0.05, **P < 0.01 vs control
(63) TABLE-US-00008 TABLE 7 Inhibitory effects of vinca alkaloid derivatives on K562 xenografts in nude mice Anti- Body weight (g) Tumor tumor Compound Dosage Pre-dose Post-dose weight (g) rate (%) Saline 21.8 1.1 25.5 1.7 1.68 0.32 VLB 1 mg/kg 21.9 0.7 17.4 0.4* 0.86 0.10 49.81* JJ-CCJ 1 mg/kg 22.1 1.9 16.5 0.8** 0.53 0.19 68.45** BX-CCJ 1 mg/kg 22.3 1.0 23.1 1.4 0.39 0.27 76.79** JJ-CCRB 1 mg/kg 21.8 1.4 18.2 0.5* 0.68 0.20 59.52** BX-CCRB 1 mg/kg 22.1 0.5 23.4 1.2 0.46 0.29 72.62** JJ-CCFN 1 mg/kg 21.5 2.1 17.4 0.9* 0.71 0.21 57.74** BX-CCFN 1 mg/kg 22.9 1.3 23.0 0.8 0.57 0.13 66.08** JJ-CCXJ 1 mg/kg 22.4 1.7 17.1 0.3** 0.67 0.22 60.12** BX-CCXJ 1 mg/kg 21.2 0.9 22.5 1.1 0.42 0.14 75.00** *P < 0.05, **P < 0.01 vs control
(64) TABLE-US-00009 TABLE 8 Inhibitory effects of vinca alkaloid derivatives on HL-60 xenografts in nude mice Anti- Body weight (g) Tumor tumor Compound Dosage Pre-dose Post-dose weight (g) rate (%) Saline 19.9 1.8 22.7 1.6 2.08 0.38 VLB 1 mg/kg 19.4 1.0 16.2 0.7* 0.98 0.25 52.89** JJ-CCJ 1 mg/kg 20.1 1.7 16.1 0.4* 0.64 0.17 69.23** BX-CCJ 1 mg/kg 18.8 1.1 20.1 1.0 0.41 0.19 81.29** JJ-CCRB 1 mg/kg 19.5 1.3 17.1 0.3* 0.72 0.21 65.38** BX-CCRB 1 mg/kg 19.0 1.2 19.2 1.3 0.53 0.28 74.52** JJ-CCFN 1 mg/kg 19.2 1.9 17.3 0.1* 0.83 0.13 60.10** BX-CCFN 1 mg/kg 18.7 1.4 18.6 1.4 0.61 0.35 70.68** JJ-CCXJ 1 mg/kg 19.7 1.5 16.3 0.6* 0.84 0.13 59.62** BX-CCXJ 1 mg/kg 19.3 1.1 19.7 0.9 0.58 0.17 72.12** *P < 0.05, **P < 0.01 vs control
Example 8. Inhibitory Effects of Vinca Alkaloid Derivatives on the Invasion Capacity of HUVECs
(65) Experimental method: The HUVECs at logarithmic phase were digested, centrifuged and counted. Then, cells (510.sup.6/mL) were added to the transwell system coated with Matrigel (BD Bioscience), with each well 0.1 mL. After 24 h of incubation at 37 C. with 5% CO.sub.2, blank control group and drug groups were set up, the drug groups were added vinca alkaloids and vinca alkaloid derivatives respectively. Final concentration of drugs was 100 pmol/L. After 24 h, medium was removed and cells were washed with PBS, fixed with 4% (W/V) paraformaldehyde for 15 min and stained with Giemsa Stain for 30 min, followed by once washed with PBS. Images were recorded and cell numbers were counted. The results were shown in
(66) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all inhibited the invasion of HUVECs. Besides, compared with vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly suppressed the invasion of HUVECs.
Example 9. Inhibitory Effects of Vinca Alkaloid Derivatives on Migration Capacity of HUVECs
(67) Experimental method: The HUVECs at logarithmic phase were centrifuged and resuspended in serum-free medium after being washed with PBS. Then, cells (210.sup.6/mL) were added to the transwell system, with each well 100 L. Blank control group and drug groups were set up, the drug groups were added with vinca alkaloid Vinblastine (CCJ), Vinorelbine (CCRB), Vinflunine (CCFN), Vincristine (CCXJ) and vinca alkaloid derivatives hydrazinolyzed Vinblastine (JJ-CCJ), Vinblastine dipeptide (BX-CCJ), hydrazinolyzed Vinorelbine (JJ-CCRB), Vinorelbine dipeptide (BX-CCRB), hydrazinolyzed Vinflunine (JJ-CCFN), Vinflunine dipeptide (BX-CCFN), hydrazinolyzed Vincristine (JJ-CCXJ), Vincristine dipeptide (BX-CCXJ) respectively. Final concentration of drugs was 100 pmol/L. 700 L RPMI-1640 medium containing 10% new bovine serum was added to the lower chamber. After 24 h of incubation at 37 C. with 5% CO.sub.2, the medium was removed and the cells were fixed with 4% paraformaldehyde for 30 min at 4 C., washed with water slowly twice and stained with Giemsa Stain for 30 min Afterward, cells on the surface of the membrane were gently scraped away with cotton swabs. The remaining cells were washed with water slowly. Images were recorded and cell numbers were counted. The results were shown in
(68) Results: Compared with vinca alkaloids, vinca alkaloid derivatives which obtained from Example 1 significantly suppressed the migration of HUVECs. Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all inhibited the migration of HUVECs. What's more, compared with vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives suppressed the migration of HUVECs more significantly.
Example 10. Inhibitory Effects of Vinca Alkaloid Derivatives on Tube Formation of HUVECs
(69) Experimental method: Matrigel was added into a pre-cooled 48 well plates, with each well 100 L, and then incubated at 37 C. for 20 min. After the Matrigel solidified, 110.sup.5 cells/well were seeded onto the Matrigel, and the medium were pre-added with vinca alkaloids, vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ, BX-CCXJ (final concentration 100 pmol/L) and VEGF (final concentration 100 ng/mL). The well without drugs and VEGF was taken as blank control. After 8 h of incubation, the formation of tubeular structures were observed and photographed under inverted microscope. The results were shown in
(70) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all inhibited the VEGF-mediated tube formation of HUVECs. Besides, compared with vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly suppressed the tube formation of HUVECs.
Example 11. Disruptive Effects of Vinca Alkaloid Derivatives on Preformed Tubular Structures of HUVECs
(71) Experimental method: According to the literature [Zhi-Ting Deng, et al. Biochemical Pharmacology 2011, 82:1832-42], and to optimize. Matrigel was added into a pre-cooled 48 well plates, with each well 100 L, and then incubated at 37 C. for 20 min After the Matrigel solidified, 110.sup.5 cells/well were seeded onto the Matrigel and incubated 4 h. When the tubuelar structures formed, the medium was changed with which containing vinca alkaloids, vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ, BX-CCXJ (final concentration 100 pmol/L) and VEGF (final concentration 100 ng/mL) respectively. The well without drugs and VEGF was taken as blank control. After 8 h of incubation, the formation of tubuelar structures were observed and photographed under inverted microscope. The results were shown in
(72) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all destroyed the preformed tubuelar structures of HUVECs induced by VEGF. What's more, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly destroyed the preformed tubuelar structures of HUVECs induced by VEGF.
Example 12. Inhibitory Effects of Vinca Alkaloid Derivatives on Vessel Growth of Chick Embryo Chorioallantoic Membrane (CAM)
(73) Experimental method: According to the literature [Cho S G, et al. Cancer Res. 2009, 69(17): 7062-7070], 60 five-day-old embryonic eggs were divided randomly into PBS group, vinca alkaloids group, vinca alkaloid derivatives JJ-CCJ group, BX-CCJ group, JJ-CCRB group, BX-CCRB group, JJ-CCFN group, BX-CCFN group, JJ-CCXJ group and BX-CCXJ group, with 10 eggs in each group. Embryonic eggs were placed in 37 C. incubator after sterilizing with the air chamber up. After 8 days of incubation, a 1.5 cm1.5 cm window was opened at the air chamber of the eggs and the shell membrane was removed to expose the CAM. Then, filter paper with drugs were placed on the chick chorioallantoic membrane (CAM) where vessels are less. The window was sealed and the eggs were returned to the incubator. After further incubation for one day, remove the sealing membrane. The chick chorioallantoic membrane (CAM) was fixed with methanol:acetone (1:1, V/V) for 15 min at room temperature. After the vessels were solidified, the membrane was cut by centering around the filter paper, and transferred to culture dish, which contained a small amount of water, then unfolded over the filter paper. After that, the filter paper, along with the membrane, was taken out from the water. Vessels within the scope of 5 mm of experimental part edge was observed, counted and photographed. The results were shown in
(74) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all inhibited the vessel growth of CAM. Moreover, compared with vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly suppressed the vessel growth of CAM.
Example 13. Disruptive Effects of Vinca Alkaloid Derivatives on Preformed Vessel of Chick Embryo Chorioallantoic Membrane (CAM)
(75) Experimental method: 60 five-day-old embryonic eggs were divided randomly into vinca alkaloids group, vinca alkaloid derivatives JJ-CCJ group, BX-CCJ group, JJ-CCRB group, BX-CCRB group, JJ-CCFN group, BX-CCFN group, JJ-CCXJ group and BX-CCXJ group, with 10 eggs in each group. Embryonic eggs were placed in 37 C. incubator after sterilizing with the air chamber up. After 8 days of incubation, a 1.5 cm1.5 cm window was opened at the air chamber of the eggs and the shell membrane was removed to expose the CAM. Then, a filter paper with VEGF (100 nmol) were placed on the CAM where vessels are less. The window was sealed and the eggs were returned to the incubator. After incubating for another one day, remove the sealing membrane. Vinca alkaloids, vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ and BX-CCXJ (1 nmol for each) were added onto the filter papers respectively. Then the window was sealed again. After further incubation for one day, the sealing film was removed. The CAM was fixed with methanol:acetone (1:1, V/V) for 15 min at room temperature. After the vessels were solidified, the membrane was cut by centering around the filter paper, and transferred to culture dish, which contained a small amount of water, then unfolded over the filter paper. Afterward, the filter paper, along with the membrane, was taken out from the water. Vessels within the scope of 5 mm of experimental part edge was observed, counted and photographed. The results were shown in
(76) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all destroyed preformed vessel of CAM. What's more, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly destroyed the preformed vessel of CAM.
Example 14. Inhibitory Effects of Vinca Alkaloid Derivatives on Microvessel Growth of Rat Aortic Ring
(77) Experimental method: According to the literature method [Srinivas Reddy Boreddy, et al. PLoS One. 2011, 6(10): e25799], 100 L pre-cooled and well-dissolved Matrigel was added into the pre-cooled 48 well plates and then incubated at 37 C. for 30 min until the Matrigel solidified. During this period, Sprague Dawley rat was put into the 75% alcohol for 3 min after CO.sub.2 euthanasia. Then split the mouse thorax on bacteria-free operating bench and separated the thoracic aorta. After washing with PBS, the aortas isolated from rat were transferred to DMEM/F12 complete medium, cleaned off connective tissues and periadventitial fat, and then cut it into 1 mm long rings with ophthalmic scissors. The rings were placed on the Matrigel and covered with an additional 100 L of pre-cooled Matrigel. After 30 min of incubation at 37 C., blank control group and drug groups were set up, the drug groups were added with DMEM-F12 complete medium containing 2 nmol/L vinca alkaloids and vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ, BX-CCXJ (containing 100 ng/mL VEGF) respectively. The medium was replaced every two days, after the fourth replacement, the microvessel growth was photographed under inverted microscope and quantified. The results were shown in
(78) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all inhibited the microvessel growth of rat aortic ring. Besides, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly inhibited the microvessel growth of rat aortic ring.
Example 15. Disruptive Effects of Vinca Alkaloid Derivatives on Preformed Microvessel of Rat Aortic Ring
(79) Experimental method: According to the literature [Deng Z T, et al. Biochemical Pharmacology 2011, 82:1832-42], and to optimize. 100 L pre-cooled and well dissolved Matrigel was added to the pre-cooled 48 well plates and then incubated at 37 C. for 30 min until the Matrigel solidified. During this period, SD rat was put into the 75% alcohol for 3 min after CO.sub.2 euthanasia. Then split the mouse thorax on bacteria-free operating bench and separated the thoracic aorta. After washing with PBS, the aortas isolated from rat were transferred to complete DMEM/F12 medium, cleaned off connective tissues and periadventitial fat with scissors and tweezers, and then cut it into 1 mm long rings with ophthalmic scissors. The rings were placed on the Matrigel and covered with an additional 100 L of pre-cooled Matrigel. After 30 min of incubation at 37 C., complete DMEM-F12 medium were added (containing 100 ng/mL VEGF). The medium was replaced every two days, when the microvessel formed, DMEM-F12 complete medium containing 2 nmol/L vinca alkaloids and vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ and BX-CCXJ were added respectively. 24 h later, the microvessel growth was photographed under inverted microscope and quantified by manual counting. The results were shown in
(80) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all destroyed the preformed microvessel of rat aortic ring. What's more, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly destroyed the preformed microvessel of rat aortic ring.
Example 16. Inhibitory Effects of Vinca Alkaloid Derivatives on Vessel Growth of Matrigel Plug
(81) Experimental method: According to the literature method [Nasim Akhtar, et al. Angiogenesis. 2002, 5: 75-80], 500 L, pre-cooled Matrigel:PBS (1:1, V/V) was mixed with 500 ng VEGF and 150 units heparin, and then mixed with vinca alkaloids and vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ, BX-CCXJ (final concentration of drugs was 100 pmol/L) respectively. The Matrigel mixture was injected subcutaneously into the back of 6-week-old BALB/C nu/nu mice. The PBS group was taken as blank control, each group of 6 mice. The mice were euthanasiaed with CO.sub.2 at 14 days, the Matrigel plugs were removed and fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin and stained with hematoxylin and eosin (H&E). The vessel growth was observed, photographed and counted under inverted microscope. The results were shown in
(82) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all inhibited the vessel growth of Matrigel Plug. Besides, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly inhibited the vessel growth of Matrigel Plug.
Example 17. Disruptive Effects of Vinca Alkaloid Derivatives on Preformed Vessel of Matrigel Plug
(83) Experimental method: According to the literature method Nasim Akhtar, et al. Angiogenesis. 2002, 5: 75-801, and to optimize. 500 L pre-cooled Matrigel was mixed with 500 ng VEGF and 150 units heparin and then injected subcutaneously into the back of 6-week-old BALB/C nu/nu mice. One week later, the mice were intravenously (i.v.) injected every two days with 1 mg/kg of vinca alkaloids and vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ, BX-CCXJ. The saline group was taken as blank control, each group of 6 mice. The mice were practiced for CO.sub.2 euthanasia after 7 injection, the matrigel plugs were removed and fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin and stained with H&E. The vessel growth was observed, photographed and counted under inverted microscope. The results were shown in
(84) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all destroyed the preformed vessel of Matrigel Plug. What's more, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly destroy the preformed vessel of Matrigel Plug.
Example 18. Inhibitory Effects of Vinca Alkaloid Derivatives on Vessel Growth of Rat Corneal Micropocket
(85) Experimental method: Rat corneal micropocket model was established according to the literature method [Yi Z F, et al. Int J Cancer. 2009, 124: 843-852]. Briefly, the slow-release pellet containing 200 ng VEGF was made of ulcerlmin and Poly-HEMA. Following anesthesia with an injection of 2% Nembutal, VEGF pellets were implanted into rat corneal micropocket. The cornea was covered with Chloramphenicol hydrochloride after surgery. On the day after operation, rats were divided randomly into PBS group, vinca alkaloids group, vinca alkaloid derivatives JJ-CCJ group, BX-CCJ group, JJ-CCRB group, BX-CCRB group, JJ-CCFN group, BX-CCFN group, JJ-CCXJ group and BX-CCXJ group, with 10 rats in each group. The mice were intravenously (i.v.) injected every two days with these drug at the dose of 1 mg/kg. Both the vascular length (VL) and clocks of neoneovascularization (CN) were observed and recorded after 7th injection. The formula below was used to determine the neoneovascularization area: Area (mm.sup.2)=0.2VL (mm)CN (mm) The results were shown in
(86) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all inhibited the vessel growth of rat corneal micropocket. Besides, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly inhibited the vessel growth of rat corneal micropocket.
Example 19. Disruptive Effects of Vinca Alkaloid Derivatives on Preformed Vessel of Rat Corneal Micropocket
(87) Experimental method: Rat corneal micropocket model was established according to the method from literature [Yi Z F, et al. Int J Cancer. 2009, 124: 843-852], and to optimize. Briefly, the slow-release pellet containing 200 ng VEGF was made of ulcerlmin and Poly-HEMA. Following anesthesia with an injection of 2% Nembutal, VEGF pellets were implanted into rat corneal micropocket. The cornea was covered with Chloramphenicol hydrochloride after surgery. On the day after operation, rats were divided randomly into PBS group, vinca alkaloids group, vinca alkaloid derivatives JJ-CCJ group, BX-CCJ group, JJ-CCRB group, BX-CCRB group, JJ-CCFN group, BX-CCFN group, JJ-CCXJ group and BX-CCXJ group, with 10 rats in each group. One week later when small vessels were formed, 1 mg/kg drugs were given respectively every two days by intravenous injection for 7th. Both the length and clocks of neoneovascularization were observed and recorded. The formula below was used to determine the neoneovascularization area: Area (mm.sup.2)=0.2VL (mm)CN (mm) The results were shown in
(88) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all destroyed the preformed vessel of rat corneal micropocket. What's more, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly destroyed the preformed vessel of rat corneal micropocket.
Example 20. Inhibitory Effects of Vinca Alkaloid Derivatives on the Angiogenesis in Synovial Tissue of CIA Mouse
(89) Experimental method: CIA model (antigen-induced arthritis) in C57 mouse was established according to the method from literature [Campbell I K, et al. Eur J Immunol, 2000, 30: 1568-1575]. Each mouse was injected i.d. at several sites into the base of the tail with 100 L emulsion consisting 100 g type II collagen. Mouse injected i.d. with saline was taken as control group and with the same method as model groups. Mice were divided randomly, with 10 mice in each group. On day one after molding, PBS, vinca alkaloids, vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ and BX-CCXJ (1.0 mg/kg) were administered every other day by tail vein injection respectively. Briefly, mice were killed by CO.sub.2 asphyxiation after consecutive 7 administration. Then, joints were removed, trimmed of the surrounding musculature, and fixed with 4% paraformaldehyde for 24 h. The synovial tissue angiogenesis was tested according to immunohistochemical assay [Yajuan Song, et al. Angiogenesis. 2012, 15: 421-432], with CD31 as marker. The standard to assess microvessel was according to literature [Tatsuta M, et al. Int J Cancer, 1999, 80: 396-399]: brown dying single vascular cells or cell clusters were counted as one vessel. Large vessels with lumen greater than size of eight red blood cells or with thick muscular were not counted. Slices were examined under low power (10) to select three regions of highest vessel density, then microvessels density (MVD) were counted in a 40 field in each of these three regions. The results were shown in
(90) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all inhibited the angiogenesis in synovial tissue of CIA mouse. Besides, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly inhibited the angiogenesis in synovial tissue of CIA mouse.
Example 21. Disruptive Effects of Vinca Alkaloid Derivatives on Preformed Vessel in Synovial Tissue of CIA Mouse
(91) Experimental method: CIA model in C57 mouse was established according to the method from literature [Campbell I K, et al. Eur J Immunol, 2000, 30: 1568-1575], and to optimize. Each mouse was injected i.d. at several sites into the base of the tail with 100 L emulsion consisting 100 g type II collagen. Mouse injected i.d. with saline was taken as control group and followed the method of the model group. Mice were divided randomly, with 10 mice in each group. After 7 days, PBS, vinca alkaloids, vinca alkaloid derivatives JJ-CCJ, BX-CCJ, JJ-CCRB, BX-CCRB, JJ-CCFN, BX-CCFN, JJ-CCXJ and BX-CCXJ (1.0 mg/kg) were intravenously (i.v.) injected every two days. Briefly, mice were killed by CO.sub.2 asphyxiation after a total of 7 administration. Then, joints were removed, trimmed of the surrounding musculature, and fixed with 4% paraformaldehyde for 24 h. The synovial tissue angiogenesis was tested according to immunohistochemical assay [Yajuan Song, et al. Angiogenesis. 2012, 15: 421-432], with CD31 as marker. The standard to assess microvessel according to the literature [Tatsuta M, et al. Int J Cancer, 1999, 80: 396-399]: brown dying single vascular cells or cell clusters were counted as one vessel. Large vessels with lumen greater than size of eight red blood cells or with thick muscular were not counted. Slices were examined under low power (10) to select three regions of highest vessel density, then microvessels density (MVD) were counted in a 40 field in each of these three regions. The results were shown in
(92) Results showed that vinca alkaloids, hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives all destroyed the preformed vessel in synovial tissue of CIA mouse. What's more, compared with vinca alkaloids, the hydrazinolyzed vinca alkaloids and vinca alkaloid dipeptide derivatives more significantly destroyed the preformed vessel in synovial tissue of CIA mouse.
(93) The above mentioned experimental results have confirmed that the vinca alkaloid derivatives of the invention can be applied in the treatment and prevention of the diseases, such as malignant tumor, diabetic retinopathy, rheumatoid arthritis, etc., in vivo and in vitro, particularly, the hydrazinolyzed vinca alkaloids and the vinca alkaloid dipeptide derivatives have better effects on preventing or treating the diseases, such as malignant tumor, diabetic retinopathy, rheumatoid arthritis, etc.
(94) The above mentioned embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited thereto, any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention, which shall be the equivalent replacements, are all included within the scope of the present invention.