Multi-targeted Ubenimex prodrug derivative and preparation method and use thereof

Abstract

The present invention relates to the design, synthesis, and biological study of multi-targeted Ubenimex pro-drug derivative. More particularly, provided in the present invention is a compound as shown by general structural formula (I) (wherein the definition of R is shown in the description). The derivative is a multi-targeted compound obtained by binding an aminopeptidase (APN/CD13) inhibitor, Ubenimex, with some anti-tumor drugs already on the market through an ester bond or amide bond, and is suitable for use as an anti-tumor drug for the treating various malignant tumors, and is especially suitable for treating various solid tumors. ##STR00001##

Claims

1. A multi-targeted Ubenimex pro-drug derivative represented by general structural formula (I), as well as an enantiomer, a diastereoisomer, a racemate or a pharmaceutically acceptable salt or solvate thereof: ##STR00069## wherein, R is selected from: ##STR00070## ##STR00071## wherein n is 1-6; wherein X is NH or O; wherein R.sub.1 is H, CH.sub.3 or CH.sub.2CH.sub.3; and wherein R.sub.2 is selected from H, CH.sub.3, CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2CH(CH.sub.3).sub.2, CH.sub.3CH.sub.2CH(CH.sub.3), CH.sub.2OH, CH.sub.3CH(OH), CH.sub.3SCH.sub.2CH.sub.2, CH.sub.2Ph, or OH-p-PhCH.sub.2.

2. The multi-targeted Ubenimex pro-drug derivative according to claim 1, wherein R is: ##STR00072## wherein, R.sub.1 is H, CH.sub.3 or CH.sub.2CH.sub.3.

3. The multi-targeted Ubenimex pro-drug derivative according to claim 1, R is selected from: ##STR00073##

4. The multi-targeted Ubenimex pro-drug derivative according to claim 1, wherein R is selected from: ##STR00074## wherein n is 1-6.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the tumor size of control mice and combination of ubenimex and 5-FU treated mice.

(2) FIG. 2 illustrates the tumor volume of Kunming mice.

(3) FIG. 3 illustrates the body weight changing curve of Kunming mice by oral administration.

(4) FIG. 4 illustrates the tumor weight of Kunming mice by oral administration.

(5) FIG. 5 illustrates the tumor inhibitory ratio of compounds by oral administration.

(6) FIG. 6 illustrates the photographs of tumors of every groups by oral administration.

(7) FIG. 7 illustrates the body weight changing curve of Kunming mice by intravenous injection.

(8) FIG. 8 illustrates the tumor weight of Kunming mice by intravenous injection.

(9) FIG. 9 illustrates the tumor inhibitory ratio of compounds by intravenous injection.

(10) FIG. 10 illustrates the photographs of tumors of every groups by intravenous injection.

(11) FIG. 11 illustrates the tumor weight and its deviation, wherein, *signed groups were treated by oral gavage, the other were treated by tail intravenous injection.

(12) FIG. 12 illustrates the inhibitory ratio of compounds, wherein, *signed groups were treated by oral gavage, the other were treated by tail intravenous injection.

(13) FIG. 13 illustrates animal weight changing curve, wherein, *signed groups were treated by oral gavage, the other were treated by tail intravenous injection.

(14) FIG. 14 illustrates the photographs of tumors obtained in experiments, wherein, *signed groups were treated by oral gavage, the other were treated by intravenous injection, besides, the O means no tumor detected, while the X means animal dead.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(15) The following examples are served to exemplify the present invention, but not to limit the scope of the invention.

EXAMPLE 1

Preparation of 5-fluoro-1-hydroxymethylpyrimidine-2,4(1H,3H)-dione (2)

(16) 5-FU (0.26 g, 2 mmol) was dissolved in 1 mL of 37% formaldehyde solution and the mixture was reacted at 60 C. for 2 h under oil bath. After evaporating the solvent under reduced pressure, the residue was dried under vacuum to get colorless viscous oil 2 (0.3 g, yield: 94%).

EXAMPLE 2

Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((tert-butoxycarbonyl)amino)-4-methylpentanoate (3)

(17) 2 (0.3 g, 1.9 mmol) was dissolved in acetonitrile, followed by adding Boc-L-leucine (0.7 g, 2.8 mmol), DCC (0.6 g, 2.8 mmol) and DMAP (0.03 g) into it while stirring under ice bath. After the ice bath was removed, the obtained mixture was stirred at room temperature for 12 h. Filtered and evaporated the solvent, extracted with ethyl acetate, the organic phase was washed with water, 1 M citric acid, saturated sodium bicarbonate and saturated sodium chloride solution in turn. Dried with anhydrous sodium sulfate, filtered it and evaporated the solvent to get colorless oil 3 (0.42 g, 60%).

EXAMPLE 3

Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-(amino)-4-methylpentanoate hydrochloride (4)

(18) 3 (0.37 g, 1.0 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and the solution was reacted at room temperature for 2 h. Filtered the solution then to get white powder 4 (0.26 g, 83%).

EXAMPLE 4

Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate (5)

(19) Boc-AHPA (0.3 g, 1.0 mmol) was dissolved in anhydrous dichloromethane, and followed by the addition of EDCI (0.3 g, 1.5 mmol) and HOBt (0.2 g, 1.5 mmol) under ice bath, after stirring for 0.5 h, 4 (0.3 g, 1 mmol) and 0.2 mL of triethylamine were added into the solution. The mixture was reacted for 5 h at room temperature after the ice bath was removed. After the reaction was accomplished, the organic layer was washed with water, 1 M citric acid, saturated sodium bicarbonate and saturated sodium chloride solution respectively, then dried with anhydrous sodium sulfate, filtered it and evaporated the solvent to get white solid 5 (0.33 g, 55%).

EXAMPLE 5

Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((2S,3R)-3-(amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate hydrochloride (6(BC-01))

(20) 5 (0.33 g, 0.55 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and the solution was reacted at room temperature for 2 h. Filtered it to get white solid 6(BC-01) (0.23 g, 85%). ESI-MS m/z:451.6 (M+H).sup.+, .sup.1H-NMR (600 MHz DMSO): 0.84-0.87 (m, 6H), 1.53 (m, 1H), 1.60-1.68 (m, 2H), 2.89-2.95 (m, 2H), 3.99-4.03 (m, 2H), 4.25 (m, 1H), 5.56-5.61 (m, 2H), 6.80 (s, 1H), 7.26-7.35 (m, 5H), 8.04 (s, 3H), 8.13 (d, J=6.6 Hz, 1H), 8.47 (d, J=7.2 Hz, 1H), 12.01 (s, 1H). mp: 128-130 C.

EXAMPLE 6

Preparation of (S)-benzyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate (7)

(21) Boc-AHPA (6 g, 20.3 mmol) was dissolved in dichloromethane, and followed by the addition of HOBt (3 g, 22.3 mmol) and EDCI (4.5 g, 22.3 mmol) under ice bath. And after 0.5 h, L-leucine benzyl ester toluene-4-sulfonate (8.5 g, 22.3 mmol) and 3.1 mL of triethylamine were added into the solution. The mixture was reacted for 5 h after the ice bath was removed. The reactant solution was washed with 10% citric acid, saturated NaHCO.sub.3 and saturated NaCl for 3 times respectively, then dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to achieve a yellowish white solid 7 (4.8 g, 47.5%).

EXAMPLE 7

Preparation of (2S)-benzyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-4-phenyl-2-(2-(2H)-tetrahydropyranoxy)butyryl)-4-methylpentanoate (8)

(22) 7 (12 g, 24 mmol) was dissolved in dry dichloromethane, followed by addition of PPTS (0.6 g, 2.4 mmol) and DHP (3.6 g, 43.2 mmol) dropwise. The mixture was reacted at 35 C. for 24 h until TLC test gave the reaction was accomplished, 0.5 g of K.sub.2CO.sub.3 were added, and the mixture was stirred for 20 min, then the solution was washed with saturated NaCl for three times. After dried with anhydrous sodium sulfate, filtered it and evaporated the solvent under reduced pressure to get yellowish white solid 8 (12.6 g, 90.8%).

EXAMPLE 8

Preparation of (2S)-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-4-phenyl-2-(2-(2H)-tetrahydropyranoxy)butyryl)-4-methylpentanoate (9)

(23) 8 (12.6 g, 21.6 mmol) was dissolved in methanol, then 1.3 g Pd/C (10%) was added in several times. The air in the reactant bottle was removed out and was filled with hydrogen by a hydrogen balloon, after being reacted for 12 h, filtered the mixture with two layers of filter papers. The solvent was evaporated until the residue was dried to get colorless foam like solid 9 (10.2 g, 96.0%).

EXAMPLE 9

Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-4-phenyl-2-(2-(2H)-tetrahydropyranoxy)acyl)-4-methylpentanoate (10)

(24) 9 (6 g, 20.3 mmol) was dissolved in re-distilled dichloromethane, followed by addition of HOBt (3.6 g, 26.92 mmol) and EDCI (5.2 g, 26.92 mmol) under ice bath. After 0.5 h, 2 (4.3 g, 26.92 mmol) in acetonitrile solution and 3.7 mL of triethylamine were added into the solution, and the mixture was reacted for 20 h after the ice bath was removed. The reactant solution was washed with 10% citric acid, saturated NaHCO.sub.3 and saturated NaCl for three times, and dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to get yellow oil, and separated the oil by using flash column chromatography to obtain colorless oil 10 (8.8 g, 67%).

EXAMPLE 10

Preparation of (S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((2S,3R)-3-(amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate hydrochloride (6(BC-01))

(25) 10 (8.8 g, 13.9 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2 h. Filtered it to get white solid 6(BC-01) (5.94 g, 88%). ESI-MS m/z:451.5 (M+H).sup.+, .sup.1H-NMR (600 MHz DMSO): 0.85-0.89 (m, 6H), 1.51-1.56 (m, 1H), 1.59-1.66 (m, 2H), 2.87-2.94 (m, 2H), 4.01-4.05 (m, 2H), 4.24-5.28 (m, 1H), 5.60-5.63 (m, 2H), 6.75 (s, 1H), 7.23-7.32 (m, 5H), 8.09 (s, 3H), 8.16 (d, J=6.6 Hz, 1H), 8.48 (d, J=7.2 Hz, 1H), 12.05 (s, 1H). mp: 128-129 C.

EXAMPLE 11

Preparation of 2-((tert-butoxycarbonyl)amino)acetic acid (12)

(26) L-Glycine (2.3 g, 30 mmol) was dissolved in 1 mol/L NaOH, and (Boc).sub.2O (7.2 g, 33 mmol) in tetrahydrofuran solution was added into the mixture dropwise while stirring mechanically under ice bath. After finishing adding and for 0.5 h, the ice bath was removed. The pH of the reactant solution was retained at 10 by using 2 mol/L NaOH, and then reacted overnight. After tetrahydrofuran was evaporated, the residue was extracted with petroleum ether for 3 times, and the pH of water phase was adjusted to 2-3 by using 3 mol/L HCl, and then extracted it with ethyl acetate, dried with anhydrous sodium sulfate. After filtering, evaporating and being dried for 24 h in vacuum, a white solid 12 (5.1 g, 96%) was obtained.

EXAMPLE 12

Preparation of (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1-(2H))methyl-2-((tert-butoxycarbonyl)amino)acetate (13)

(27) 12 (1.1 g, 6.3 mmol) was dissolved in re-distilled dichloromethane, followed by addition of HOBt (1.2 g, 8.8 mmol) and EDCI (1.7 g, 8.8 mmol) under ice bath. After 0.5 h, 2 (1.4 g, 8.8 mmol) in acetonitrile solution and 1.2 mL of trimethylamine were added into the solution, and the mixture was reacted for 20 h after the ice bath was removed. The reactant solution was washed with 10% citric acid, saturated NaHCO.sub.3 and saturated NaCl for three times, and dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to get yellow oil, and separated the oil by using flash column chromatography to obtain colorless oil 13 (1.3 g, 67%).

EXAMPLE 13

Preparation of (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1-(2H))methyl-2-amino)acetate hydrochloride (14)

(28) 13 (1.3 g, 4.2 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and the solution was reacted at room temperature for 3 h. Filtered it to give a white solid 14 (0.93 g, 88%).

EXAMPLE 14

Preparation of (6R,7S,10S)-(5-fluoro-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-3-yl)methyl-6-benzyl-10-isobutyl-2,2-dimethyl-4,8,11-trioxo-7-(2-2H-tetrahydropyranoxy)-3-oxo-5,9,12-triazatetradecanoate (15)

(29) 9 (1.21 g, 2.45 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.65 g, 3.4 mmol) and HOBt (0.46 g, 3.4 mmol) under ice bath, and after 0.5 h, 14 (0.93 g, 3.4 mmol) and 0.48 mL of triethylamine were added into the solution. The mixture was reacted for 5 h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 15 (0.86 g, 51%).

EXAMPLE 15

Preparation of (5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)acetate hydrochloride (16(BC-02))

(30) 15 (0.86 g, 1.24 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2 h. Filtered it to get a white solid 16(BC-02) (0.57 g, 85%). ESI-MS m/z:508.4 (M+H).sup.+, .sup.1H-NMR (400 MHz DMSO): 0.86-0.89 (m, 6H), 1.49-1.53 (m, 2H), 1.62-1.66 (m, 1H), 2.87-2.97 (m, 2H), 3.55-3.57 (m, 1H), 3.80-3.92 (m, 2H), 4.00-4.06 (m, 1H), 4.31-4.32 (m, 1H), 5.57-5.80 (m, 2H), 6.73 (s, 1H), 7.25-7.37 (m, 5H), 8.02-8.08 (m, 4H), 8.14 (d, J=6.6 Hz, 1H), 8.59-8.62 (m, 1H), 12.01 (s, 1H). mp: 136-137 C.

EXAMPLE 16

(31) The following Compounds were prepared as the general procedure as described in BC-02.

(S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)propanoate hydrochloride (BC-03)

(32) ESI-MS m/z:521.5 (M+H).sup.+, .sup.1H-NMR (400 MHz DMSO): 0.85-0.91 (m, 6H), 1.25-1.28 (m, 3H), 1.43-1.51 (m, 2H), 1.60-1.64 (m, 1H), 2.87-2.94 (m, 2H), 3.53-3.57 (m, 1H), 3.99-4.04 (m, 1H), 4.24-4.31 (m, 2H), 5.54-5.64 (m, 2H), 7.27-7.34 (m, 5H), 7.98-8.00 (m, 4H), 8.12 (d, J=6.6 Hz, 1H), 8.61 (d, J=6.8 Hz, 1H), 11.99 (s, 1H). mp: 111-112 C.

(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-4-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)propanoate hydrochloride (BC-04)

(33) ESI-MS m/z:522.5 (M+H).sup.+, .sup.1H-NMR (400 MHz DMSO): 0.84-0.88 (m, 6H), 1.45-1.59 (m, 4H), 2.86-2.97 (m, 2H), 3.20-3.31 (m, 2H), 3.56-3.57 (m, 1H), 4.00-4.06 (m, 1H), 4.18-4.23 (m, 1H), 5.55-5.60 (m, 2H), 7.26-7.36 (m, 5H), 7.99-8.01 (m, 4H), 8.09-8.11 (m, 1H), 8.21-8.24 (m, 1H), 11.96 (s, 1H). mp: 119-120 C.

(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-6-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)butanoate hydrochloride (BC-05)

(34) ESI-MS m/z:536.4 (M+H).sup.+, .sup.1H-NMR (400 MHz DMSO): 0.85-0.89 (m, 6H), 1.48-1.65 (m, 6H), 2.32-2.36 (m, 2H), 2.89-2.94 (m, 2H), 3.02-3.07 (m, 2H), 3.56-3.57 (m, 1H), 4.01-4.04 (m, 1H), 4.20-4.26 (m, 1H), 5.56-5.62 (m, 2H), 7.27-7.35 (m, 5H), 7.99-8.04 (m, 5H), 8.12-8.15 (m, 1H), 11.96 (s, 1H). mp: 117-118 C.

(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-4-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)hexanoate hydrochloride (BC-06)

(35) ESI-MS m/z:564.5 (M+H).sup.+, .sup.1H-NMR (400 MHz DMSO): 0.86-0.89 (m, 6H), 1.16-1.24 (m, 2H), 1.36-1.38 (m, 2H), 1.46-1.52 (m, 5H), 2.28-2.32 (m, 2H), 2.89-3.02 (m, 4H), 3.55 (s, 1H), 3.99-4.04 (m, 1H), 4.22-4.26 (m, 1H), 5.56 (s, 2H), 7.27-7.35 (m, 5H), 7.99-8.15 (m, 6H), 11.96 (s, 1H). mp: 98-99 C.

(S)-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl-1(2H))methyl-1-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutanamido)-4-methylpentanamido)-2-tetrahydropyrrolidine carboxylate hydrochloride (BC-07)

(36) ESI-MS m/z:548.4 (M+H).sup.+, .sup.1H-NMR (400 MHz DMSO): 0.87-0.92 (m, 6H), 1.39-1.41 (m, 1H), 1.46-1.49 (m, 1H), 1.65-1.71 (m, 1H), 1.85-1.99 (m, 2H), 2.16-2.17 (m, 1H), 2.89-2.91 (m, 2H), 3.48-3.53 (m, 4H), 3.70-3.72 (m, 1H), 3.96-4.04 (m, 1H), 4.31-4.34 (m, 1H), 4.46-4.53 (m, 1H), 5.54 (d, J=10.3 Hz, 1H), 5.67 (d, J=10.3 Hz, 1H), 6.71 (s, 1H), 7.27-7.36 (m, 5H), 8.02-8.03 (m, 3H), 8.11-8.14 (m, 2H), 12.00 (s, 1H). mp: 140-141 C.

EXAMPLE 17

Preparation of (S)-5 fluorophenyl-2-((tert-butoxycarbonyl)amino)-4-methy-pentanoate (18)

(37) Boc-L-leucine (1.17 g, 5 mmol) was dissolved in anhydrous tetrahydrofuran, followed by addition of pentafluorophenol (1.01 g, 5.5 mmol) and EDCI (1.05 g, 5.5 mmol), and the mixture was reacted at room temperature for 12 h. Evaporated the solvent and its residue was separated by column chromatography to get colorless oil 18 (1.7 g, 86%).

EXAMPLE 18

Preparation of (S)-1-((2-(tert-butoxycarbonylamino)-4-methylvaleryl)oxy)urea (19)

(38) Hydroxyurea (0.35 g, 4.3 mmol) was dissolved in DMF, followed by addition of 0.52 mL of N-Methylmorpholine and 18 (1.7 g, 4.3 mmol) was added dropwise in the reactant solution, then the mixture was reacted for 12 h at room temperature. Evaporated the solvent and its residue was separated by column chromatography to get white solid 19 (0.75 g, 60%).

EXAMPLE 19

Preparation of (S)-1-((2-amino-4-methylvaleryl)oxy)urea hydrochloride (20): 19

(39) (0.75 g, 2.58 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2 h. Filtered it to get a white solid 20 (0.47 g, 81%).

EXAMPLE 20

Preparation of (2R,3S)-3-(tert-butoxycarbonylamino)-2-hydroxy-N((S)-4-methylvaleryl)oxy)ureido)-4-phenylbutanamide (21)

(40) Boc-AHPA (1 g, 3.38 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.71 g, 3.72 mmol) and HOBt (0.5 g, 3.72 mmol), and after 0.5 h, 20 (0.84 g, 3.72 mmol) and 0.54 mL of triethylamine were added into the solution. The mixture was reacted for 5 h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 21 (0.71 g, 45%).

EXAMPLE 21

Preparation of (2S,3R)-3-amino-2-hydroxy-N((S)-4-methylvaleryl)oxy)ureido)-4-phenylbutanamide hydrochloride (22)

(41) 21 (0.71 g, 1.52 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2 h. Filtered it to get a white solid 22 (0.48 g, 78%). ESI-MS m/z:367.3 (M+H).sup.+, .sup.1H-NMR (600 MHz DMSO): 0.80-0.92 (m, 6H), 1.56-1.75 (m, 3H), 2.90-3.05 (m, 2H), 4.01-4.06 (m, 2H), 4.53 (m, 1H), 6.48-6.55 (m, 2H), 6.88 (s, 1H), 7.26-7.38 (m, 5H), 8.02-8.09 (m, 3H), 8.63 (d, J=7.2 Hz, 1H), 9.77 (m, 1H). mp: 110-112 C.

EXAMPLE 22

Preparation of Boc-Epirubicin (24)

(42) 23 (1.09 g, 2 mmol) was dissolved in anhydrous dichloromethane, followed by addition of 0.84 mL of trimethylamine and (Boc).sub.2O (0.52 g, 2.4 mmol) in dichloromethane solution was added into the mixture dropwise, the mixture was reacted overnight at room temperature until TLC test gave the reaction was accomplished. After the reaction was accomplished, the reactant solution was washed with 10% citric acid and saturated NaCl for 3 times, dried with anhydrous sodium sulfate. Filtered it and evaporated the solvent to get compound 24 (1.09 g, 85%).

EXAMPLE 23

Preparation of (S)-2-((2S,4S)-4-((2S,4S,5R,6S)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-6-methyl-2(2H)-tetrahydropyranyloxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-2-(1,2,3,4,6,11-hexahydrotetracenyl))-2-ethoxy-2-(benzyloxycarbonylamino)-4-methylpentanoate (25)

(43) Cbz-L-leucine (0.5 g, 1.87 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.36 g, 1.87 mmol) and HOBt (0.25 g, 1.87 mmol) under ice bath, and after 0.5 h, 24 (1.09 g, 1.7 mmol) was added into the solution. The mixture was reacted for 5 h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 25 (0.76 g, 50%).

EXAMPLE 24

Preparation of (S)-2-((2S,4S)-4-((2S,4S,5R,6S)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-6-methy-2(2H)-tetrahydropyranyloxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-2-(1,2,3,4,6,11-hexahydrotetracenyl))-2-ethoxy-2-amino-4-methylpentanoate (26)

(44) 25 (0.76 g, 0.85 mmol) was dissolved in methanol, then 0.1 g Pd/C (10%) was added in several times. The air in the reactant bottle was removed out and was filled with hydrogen by a hydrogen balloon, after being reacted for 12 h, filtered the mixture with two layers of filter papers. The solvent was evaporated until the residue was dried to get colorless foam like solid 26 (0.6 g, 93.0%).

EXAMPLE 25

Preparation of (S)-2-((2S,4S)-4-((2S,4S,5R,6S)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-6-methyl-2(2H)-tetrahydropyranyloxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-2-(1,2,3,4,6,11-hexahydrotetracenyl))-2-ethoxy-2-42S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate (27)

(45) Boc-AHPA (0.27 g, 0.88 mmol) was dissolved in anhydrous dichloromethane followed by addition of EDCI (0.17 g, 0.88 mmol) and HOBt (0.27 g, 0.88 mmol) under ice bath, and after 0.5 h, 26 (0.6 g, 0.8 mmol) was added into the solution. The mixture was reacted for 5 h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 27 (0.47 g, 57%).

EXAMPLE 26

Preparation of (S)-2-((2S,4S)-4-((2S,4S,5R,6S)-4-((tert-butoxycarbonyl)amino)-5-hydroxy-6-methyl-2(2H)-tetrahydropyranyloxy))-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-2-(1,2,3,4,6,11-hexahydrotetracenyl)-2-ethoxy-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate hydrochloride (28)

(46) 27 (0.47 g, 0.46 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2 h. Filtered it to get white solid 28 (0.36 g, 87%). ESI-MS m/z:833.7 (M+H).sup.+, .sup.1H-NMR (600 MHz DMSO): 0.81-0.94 (m, 6H), 1.23 (d, J=7.2 Hz, 3H), 1.58-1.83 (m, 3H), 1.75-1.79 (m, 1H), 2.07-2.09 (m, 1H), 2.20-2.24 (m, 2H), 2.93-3.18 (m, 4H), 3.44-3.49 (m, 2H), 3.88 (s, 3H), 3.98 (t, J=5.4 Hz, 1H), 4.06-4.09 (m, 2H), 4.55 (m, 1H), 4.58-4.69 (m, 2H), 4.91-4.98 (m, 2H), 5.05-5.09 (m, 1H), 5.28-5.35 (m, 1H), 5.47 (s, 1H), 5.76 (s, 1H), 6.93 (s, 1H), 7.26-7.38 (m, 5H), 7.69 (m, 1H), 7.91-7.98 (m, 2H), 8.11-8.19 (m, 3H), 8.63 (d, J=7.2 Hz, 1H). mp: 136-138 C.

EXAMPLE 27

Preparation of (S)-2-(4-(6-(5-(2-chloro-6-methylphenyl)-2-thiazolylcarbonyl amino)-2-methyl-4-pyrimidinyl)-1-piperazinyl)ethyl-2-(tert-butoxycarbonylamino)-4-methylpentanoate (29)

(47) Boc-L-leucine (0.25 g, 1.1 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.21 g, 1.1 mmol) and HOBt (0.15 g, 1.1 mmol) under ice bath, and after 0.5 h, Dasatinib (0.51 g, 1 mmol) and 0.2 mL of triethylamine were added into the solution. The mixture was reacted for 5 h at room temperature after the ice bath was removed. After the reaction was accomplished, the organic layer was washed with water, 1 M citric acid, saturated sodium bicarbonate and saturated sodium chloride solution respectively, then dried with anhydrous sodium sulfate, filtered it and evaporated the solvent to get white solid 29 (0.28 g, 40%).

EXAMPLE 28

Preparation of (S)-2-(4-(6-(5-(2-chloro-6-methylphenyl)-2-thiazolylcarbonyl amino)-2-methyl-4-pyrimidinyl)-1-piperazinyl)ethyl-2-amino-4-methylpentanoae hydrochloride (30)

(48) 29 (0.7 g, 1 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2 h. Filtered it to get white solid 30 (0.54 g, 80%).

EXAMPLE 29

Preparation of (S)-2-(4-(6-(5-((2-chloro-6-methylphenyl)-2-thiazolylcarbonyl amino)-2-methyl-4-pyrimidinyl)-1-piperazinyl)ethyl-2-((2S,3R)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate (31)

(49) Boc-AHPA (0.16 g, 0.55 mmol) was dissolved in anhydrous dichloromethane, followed by addition of EDCI (0.11 g, 0.55 mmol) and HOBt (0.08 g, 0.55 mmol) under ice bath, and after 0.5 h, 30 (0.34 g, 0.5 mmol) was added into the solution. The mixture was reacted for 5 h at room temperature after the ice bath was removed. Until the reaction was accomplished, evaporated the solvent and its residue was separated by column chromatography to obtain white solid 31 (0.22 g, 51%).

EXAMPLE 30

Preparation of (S)-2-(4-(6-(5-(2-chloro-6-methylphenyl)-2-thiazolylcarbonyl amino)-2-methyl-4-pyrimidinyl)-1-piperazinyl)ethyl-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyryl)-4-methylpentanoate hydrochloride (32)

(50) 31 (0.22 g, 0.25 mmol) was dissolved in ethyl acetate solution saturated by chloride hydrogen, and let the solution react at room temperature for 2 h. Filtered it to get white solid 32 (0.18 g, 84%). ESI-MS m/z:777.5 (M+H).sup.+, .sup.1H-NMR (600 MHz DMSO): 0.83-0.95 (m, 6H), 1.59-1.85 (m, 3H), 2.13 (s, 3H), 2.26 (s, 3H), 2.54-2.58 (m, 6H), 2.90-3.05 (m, 2H), 3.60-3.70 (m, 6H), 4.01-4.06 (m, 2H), 4.53-4.58 (m, 1H), 6.09 (s, 1H), 6.88 (s, 1H), 7.26-7.38 (m, 7H), 7.48 (d, J=7.2 Hz, 1H), 8.02-8.09 (m, 3H), 8.22 (s, 1H), 8.63 (d, J=7.2 Hz, 1H), 9.57-9.59 (m, 2H), 9.77-9.78 (m, 2H). mp: 174-176 C.

EXAMPLE 31

Inhibition Activity Against Enzyme In Vitro

(51) 1: Materials and Methods

(52) Aminopeptidase N and L-Leu-p-nitroanilide as substrate were purchased from Sigma cooperation.

(53) Preparation of Buffer Solution: 12.89 g Na.sub.2HPO.sub.4.12H.sub.2O and 2.18 g NaH.sub.2PO.sub.4.2H.sub.2O were dissolved in a 1000 mL volumetric flask, then the mixture was diluted to 1000 mL with fresh distilled water to obtain 50 mM Phosphate Buffer solution (PBS, pH 7.2), which was kept at room temperature to spare.

(54) Aminopeptidase N was dissolved in the buffer solution to obtain 0.1 IU/mL solution.

(55) The substrate was dissolved in DMSO to obtain a 16 mmol/mL solution, which was kept in refrigerator to spare.

(56) 2: Experimental Procedure

(57) TABLE-US-00001 Aminopeptidase substrate buffer N solution solution solution No. (L) (L) (L) Inhibitor 100% control 10 5 185 0 group Blank control 0 5 195 0 group Inhibitor group 10 5 145 40

(58) The aminopeptidase N solution 10 L, the substrate solution 5 L and compound with different concentration 40 L were added into a 96-well cell plate, and then adjusted to 200 L with phosphate buffer saline solution (pH 7.2). 100% group did not include inhibitor. And the substrate solution 5 L of blank group was adjusted to 200 L with buffer solution. The mixture was incubated at 37 C. for 0.5 h, and absorbance was then determined at 405 nm wavelength Inhibitory ratio can be calculated as follows:

(59) $\mathrm{Inhibitory}\mathrm{ratio}\left(\%\right)=\frac{\mathrm{absorbance}\mathrm{of}100\%-\mathrm{absorbance}\mathrm{of}\mathrm{compound}}{\mathrm{absorbance}\mathrm{of}100\%-\mathrm{absorbance}\mathrm{of}\mathrm{blank}\mathrm{group}}100\%$

(60) According to concentration of compound and corresponding inhibitory ratio, the IC.sub.50 value was calculated by using a fitting curve of Origin 7.5 software.

(61) 3: Experimental Results:

(62) The inhibition activity against aminopeptidase N of the compound represented by formula (I) of the present invention and ubenimex as positive control drug are shown in the following table:

(63) TABLE-US-00002 IC.sub.50(M) Compd(compound) APN 6(BC-01) 6.98 16(BC-02) 0.38 BC-03 0.36 BC-04 1.30 BC-05 1.39 BC-06 4.66 BC-07 0.15 22 2.92 28 5.56 32 7.88 ubenimex 5.12

(64) The result of inhibition activity against enzyme in vitro showed that, the target compounds BC-01, BC-02, BC-03, BC-04, BC-05, BC-06, BC-07, 22, 28 and 32 represented by formula (I) of the present invention all established an inhibitory activity against aminopeptidase N, wherein Compounds BC-02, BC-03, BC-04, BC-05, BC-06 and BC-07 were much more potent than the positive control drug ubenimex, especially, BC-07 showed great activity with 0.15 M. Compounds BC-01, 22, 28 and 32 showed a comparable activity with ubenimex. This indicated that synergetic fragment linked at carboxyl group of ubenimex may increase its activity against enzyme without destroying inhibition activity against enzyme of the whole structure.

EXAMPLE 32

Pharmacodynamic Experiment In Vitro (MTT Method)

(65) Human leukemia cell line K562, clear-cell ovarian carcinoma cell line ES-2, human prostate cancer cell line PC-3, human breast cancer cell line MCF-7, cell line Hela, human liver cancer cell line H7402 and human ovarian cancer cell line 3-AO were transferred into a culture flask and were cultured in a medium at 37 C. in a 5% CO.sub.2 and saturation humidity. One bottle of cells in logarithmic growth phase was picked out and was blew and beaten to be uniform with a pipette, then took some cell suspension solution to prepare blood counting plate smear to count cells under inverted microscope, and then adjusted the cell number to be 110.sup.5/mL by adding some culture medium. Cells were seeded on a 96-well plate on which also drug tests performed. Blank control group, negative control group, positive control group and drug test group were established on the plate while the surrounding wells of the plate were not used (but filled with sterile PBS), wherein the blank control groups were prepared by addition of culture medium 150 L per well, the negative control groups were prepared by addition of cell suspension solution 100 L per well and culture medium 50 L per well, the positive control groups were prepared by addition of cell suspension solution 100 L per well and positive control drug solution 50 L per well, the drug test groups were prepared by addition of cell suspension solution 100 L per well and test compound solution 50 L per well, and there were 5 different concentrations of drug in the positive control group and the drug test group respectively: 0.01, 0.1, 1, 10, 100 mol.Math.L.sup.1, each concentration was established with 3 parallel wells. After the drug was added, the 96-well plate was incubated for 48 h at 37 C. in a CO.sub.2 incubator of 5% CO.sub.2 and saturation humidity. Upon completion of the incubation, 20 L MTT solution (concentration was 5 mg/mL) was added to each well and incubated for an additional 4 h, and after the 96-well plate was centrifuged at 2000 rpm for 30 min, medium of each well was suck out carefully and removed and 100 L DMSO was added instead, followed by vibrating on a tablet shaker for 15 min to re-dissolve formazan crystals completely. OD value of each well was measured at 570 nm wavelength using an enzyme-linked immunosorbent assay reader to calculate cellular proliferation inhibitory ratio of each drug in different concentrations. And half-inhibitory concentration (IC.sub.50) was calculated using statistical software SPSS 16.0, wherein the cellular proliferation inhibitory ratio can be calculated as following equation:

(66) $\mathrm{cellular}\mathrm{proliferation}\mathrm{inhibitory}\mathrm{ratio}=\frac{\begin{array}{c}\mathrm{OD}\mathrm{mean}\mathrm{value}\mathrm{in}\mathrm{well}\mathrm{of}\mathrm{negative}\mathrm{control}-\\ \mathrm{OD}\mathrm{mean}\mathrm{value}\mathrm{in}\mathrm{well}\mathrm{of}\mathrm{drug}\mathrm{test}\end{array}}{\begin{array}{c}\mathrm{OD}\mathrm{mean}\mathrm{value}\mathrm{in}\mathrm{well}\mathrm{of}\mathrm{negative}\mathrm{control}-\\ \mathrm{OD}\mathrm{mean}\mathrm{value}\mathrm{in}\mathrm{well}\mathrm{of}\mathrm{blank}\mathrm{control}\end{array}}100\%$

(67) The results of proliferation inhibition activity of target compounds BC-01, BC-02, 22, 28, 32 and positive control drugs ubenimex and 5-fluorouracil (5-FU) against clear-cell ovarian carcinoma cell line ES-2, human leukemia cell line K562, human prostate cancer cell line PC-3, human breast cancer cell line MCF-7, cell line Hela, human liver cancer cell line H7402 and human ovarian cancer cell line 3-AO are shown in following table:

(68) TABLE-US-00003 EC.sub.50 (M) Compd ES-2 K562 PC-3 MCF-7 Hela H7402 3-AO 6(BC-01) 2.53 5.82 10.71 1.35 0.73 1.86 2.59 BC-02 1.34 ND 3.45 ND ND 10.98 ND 22 5.66 6.52 13.34 6.74 5.55 7.98 9.06 28 3.34 4.35 7.67 2.55 1.99 4.02 3.02 32 6.45 6.33 14.13 3.65 3.23 6.75 4.89 5-FU ND 16.03 15.70 76.92 132.99 385.38 60.81 Uubenimex >400 >400 >400 32.70 79.59 188.54 >400 ND: not determined.

(69) The results of bioactivities showed that, the target compounds BC-01, BC-02, 22, 28 and 32 represented by formula (I) of the present invention established an obvious anti-proliferation effect against all the tumor cells above. By comparing with the positive drugs ubenimex and 5-fluorouracil (5-FU), BC-01 showed much higher proliferation inhibition activity against cell line Hela, and had an obvious proliferation inhibition activity against clear-cell ovarian carcinoma cell line ES-2, human leukemia cell line K562, human prostate cancer cell line PC-3, human breast cancer cell line MCF-7, human liver cancer cell line H7402 and human ovarian cancer cell line 3-AO as well; BC-02 established a great proliferation inhibition activity against clear-cell ovarian carcinoma ES-2 cell line. Compounds 22, 28 and 32 were much more potent than 5-FU on proliferation inhibition activity.

(70) The results of cells test in vitro showed that, the target compounds BC-01, BC-02, 22, 28 and 32 represented a great proliferation inhibition activity against 7 cell lines.

EXAMPLE 33

Inhibition Assay of the Target Compounds Against Liver Cancer H22

(71) 1: Establishment of Transplanting Tumor Bearing Mice Models

(72) Extracted the ascites from H22 ascites tumour bearing mice, washed the ascites with sterile PBS for three times and then resuspended the cells with sterile PBS (cells counts: 3.7510.sup.7 cells/mL), inoculated it into right oxter of Kunming mice 100 uL per mouse. After getting rid of the Kunming mice overweight or underweight, the tumor-bearing mice were randomized to several groups, and then the mice was began to administrate drug based on administration strategy, henceforth administrated once a day with two days off when having administrated for 5 days until two cycles one of which had 7 days (the volume of administration was: 200 uL/20 g once per mouse, the method of administration was: oral gavage). The body weight of mouse was recorded at the beginning and ending of every cycle, and the average volume of administration was 200 uL/20 g once per mouse; the method of administration was: oral gavage, and administrated once a day.

(73) 2: Pharmacodynamic Experiment

(74) 2.1 Inhibition Assay of Oral Administration Against Liver Cancer H22

(75) The H22 tumor bearing mice were randomized to 4 groups (10 mice per group) after being weighted. (1) the negative control: PBS; (2) Xeloda group: 100 mg/kg/d; (3) TFU group: 70 mg/kg/d; (4) BC-01 group: 100 mg/kg/d. And administrated once a day with two days off when having administrated for 5 days until two cycles one of which had 7 days (the volume of administration was: 200 uL/20 g once per mouse, the method of administration was: oral gavage). The size of tumor was measured by using a vernier calliper and weight of mouse by using an electronic balance at the beginning and ending of every cycle, then calculated their mean value. The mice were sacrificed after 13 days and the tumors were taken out and weighted. Tumor volume and inhibitory ratio were calculated as the following equation (L and W referred to length and width of tumor respectively):

(76) $\mathrm{Tumor}\mathrm{volume}=\frac{1}{2}{\mathrm{LW}}^2$$\mathrm{Inhibitory}\mathrm{ratio}\mathrm{of}\mathrm{tumor}\mathrm{weight}\left(100\%\right)=\left(1-\frac{\mathrm{mean}\mathrm{tumor}\mathrm{weight}\mathrm{of}\mathrm{test}\mathrm{group}}{\mathrm{mean}\mathrm{tumor}\mathrm{weight}\mathrm{of}\mathrm{control}\mathrm{group}}\right)100\%$$\mathrm{Inhibitory}\mathrm{ratio}\mathrm{of}\mathrm{tumor}\mathrm{volume}\left(100\%\right)=\left(1-\frac{\mathrm{mean}\mathrm{tumor}\mathrm{volume}\mathrm{of}\mathrm{test}\mathrm{group}}{\mathrm{mean}\mathrm{tumor}\mathrm{volume}\mathrm{of}\mathrm{control}\mathrm{group}}\right)100\%$
2.2: Inhibition Assay of Intravenous Administration Against Liver Cancer H22

(77) The H22 tumor-bearing mice were randomized to 4 groups (7 mice per group) after being weighted. (1) the negative control: PBS; (2) 5-FU group: 20 mg/kg/d; (3) BC-01 group: 50 mg/kg/d; (4) BC-01 group: 70 mg/kg/d; (5) BC-02 group: 70 mg/kg/d; (6) combination group of ubenimex and 5-FU: 5-FU: 15 mg/kg/d (intravenous injection), ubenimex: 30 mg/kg/d (oral administration). And administrated once a day with two days off when having administrated for 5 days until two cycles one of which had 7 days (the volume of administration was: 200 uL/20 g once per mouse, the method of administration was: tail intravenous injection). The size of tumor was measured by using a vernier calliper and weight of mouse by using an electronic balance at the beginning and ending of every cycle, then calculated their mean value. The mice were sacrificed after 13 days and the tumors were taken out and weighted. Tumor volume and inhibitory ratio were calculated as the following equation (L and W referred to length and width of tumor respectively):

(78) $\mathrm{Tumor}\mathrm{volume}=\frac{1}{2}{\mathrm{LW}}^2$$\mathrm{Inhibitory}\mathrm{ratio}\mathrm{of}\mathrm{tumor}\mathrm{weight}\left(100\%\right)=\left(1-\frac{\mathrm{mean}\mathrm{tumor}\mathrm{weight}\mathrm{of}\mathrm{test}\mathrm{group}}{\mathrm{mean}\mathrm{tumor}\mathrm{weight}\mathrm{of}\mathrm{control}\mathrm{group}}\right)100\%$$\mathrm{Inhibitory}\mathrm{ratio}\mathrm{of}\mathrm{tumor}\mathrm{volume}\left(100\%\right)=\left(1-\frac{\mathrm{mean}\mathrm{tumor}\mathrm{volume}\mathrm{of}\mathrm{test}\mathrm{group}}{\mathrm{mean}\mathrm{tumor}\mathrm{volume}\mathrm{of}\mathrm{control}\mathrm{group}}\right)100\%$
3: Experimental Results
3.1: Experimental Results of Inhibition Assay of Oral Administration Against Liver Cancer H22 (Shown in FIGS. 3, 4, 5 and 6)

(79) TABLE-US-00004 Ctrl (control group) Xeloda TFU BC-01 tumor weight 0.79 0.60 0.68 0.26 SD 0.52 0.25 0.45 0.13 inhibitory ratio (%) 24.53 13.38 66.67
3.2: Experimental Results of Inhibition Assay of Intravenous Administration Against Liver Cancer H22 (Shown in FIGS. 7, 8, 9 and 10)

(80) TABLE-US-00005 Num- inhibi- ber of Body weight(g) tory mice begin- Tumor ratio Compd alive ning(g) ending(g) weight(g) (%) Ctrl(control 7 31.60 4.33 33.61 4.21 1.37 0.27 group) 5-FU 7 31.37 4.05 31.92 4.98 0.84 0.19 38.83 BC-01(50) 7 30.32 1.57 31.53 4.71 0.58 0.12 57.66 BC-01(70) 7 30.00 1.15 31.05 1.78 0.37 0.08 73.00 BC-02 7 31.81 4.99 32.83 4.64 0.63 0.01 54.31 5-FU + Bes 7 32.96 1.75 34.54 1.18 0.47 0.19 65.55

EXAMPLE 34

Evaluation of Activities of Compounds in H22 Tumor Cells Bearing Kunming Mice Models

(81) 1. Grouping and Dosage:

(82) TABLE-US-00006 Method of Number Group Administration dosage administration of mice Ctrl group Equivalent PBS solution Intravenous 8 injection Xeloda 108 mg/kg/d (0.3 mmol/kg/d) Oral gavage 8 BC-01 120 mg/kg/d (0.25 mmol/kg/d) Oral gavage 8 BC-02 130 mg/kg/d (0.25 mmol/kg/d) Oral gavage 8 5-FU 20 mg/kg/d (0.15 mmol/kg/d) Intravenous 8 injection BC-01 50 mg/kg/d (0.1 mmol/kg/d) Intravenous 8 injection BC-07 58.3 mg/kg/d (0.1 mmol/kg/d) Intravenous 8 injection BC-02 55 mg/kg/d (0.1 mmol/kg/d) Intravenous 8 injection BC-02 79 mg/kg/d (0.15 mmol/kg/d) Intravenous 8 injection

(83) Wherein, d represents day.

(84) 2. Experimental Procedure

(85) The ascites was extracted from H22 tumour bearing mice with well growth, followed by adding sterile PBS to dilute the ascites to a concentration of 8.510.sup.7 cells/mL. 100 uL of the cells solution was suck up by a 1 mL sterile injector to inoculate into right oxter of mice. After 3 days, the mice were weighted and randomized to several groups (almost 8 mice per group, and a bit more in control group). These mice were administrated a predetermined dosage by intravenous or oral gavage with two days off when having administrated for 5 days until two cycles one of which had 7 days. The body weight of mouse was recorded at the beginning and ending of every cycle,

(86) After being administrated for two cycles, the mice were weighted and recorded, then sacrificed by cervical dislocation to get lung, liver and spleen weighted respectively.

(87) Overall difference was calculated by the function of one-way analysis of variance (One-Way ANOVA) in Origin 7.5 software; and t-test was used for pair-comparison between drug administrated group and blank group, and the inhibitory ratio of drug in each group was calculated based on the following equation: inhibitory ratio (100%)=(mean tumor weight of control groupmean tumor weight of drug administrated group)/mean tumor weight of control group100.

(88) 3. Experimental Results (Shown in FIGS. 11, 12, 13 and 14)

(89) TABLE-US-00007 Tumor Num- growth ber of Body weight(g) Tumor inhibi- mice Begin- weight tion Groups alive ning(g) Ending(g) (g) (%) Ctrl group 8 29.66 3.60 31.37 4.97 0.60 0.20 *Xeloda 7 33.00 1.81 31.42 3.56 0.35 0.09 40.95 *BC-01(50) 7 27.87 2.55 29.50 2.15 0.12 0.16 79.52 *BC-02(70) 7 27.98 3.73 27.49 4.94 0.06 0.11 89.76 5-FU 4 26.89 2.06 25.51 4.50 0.54 0.06 10.41 BC-01(50) 8 31.52 3.63 31.78 5.76 0.09 0.14 84.38 BC-07 6 32.17 3.86 31.44 5.01 0.34 0.24 43.61 BC-02 7 25.67 2.62 28.35 3.71 0.06 0.11 89.52 BC-02 7 30.32 4.42 31.13 3.55 0.0 0.0 100 In this table, *signed groups were treated by oral gavage

(90) As observed in FIGS. 11-14, compounds BC-01 and BC-02 all showed excellent anti-tumor growth activity, what's more, at a dosage of 0.15 mmol/kg/d of intravenous administrated BC-02, no tumor appeared until the end of the experiment.

(91) At the end of experiments, there is no significant changes in visceral organs by visual observation of dissected mice, it indicates that compounds at administration dosage have no evident toxicity.