Multi-arm polyethylene glycol-azido derivative

Abstract

Provided is a multi-arm polyethylene glycol-azido derivative of general formula I, wherein R is a central molecule, which is selected from a polyhydroxy structure, a polyamino structure or a polycarboxyl structure; n is the number of branches or arms, n3; PEG is the same or different (CH.sub.2CH.sub.2O).sub.m, the average value of m being an integer from 3 to 250; X is a linking group of a azido end group; k is the number of the branches having the azido end group; F is selected from the group consisting of amino, carboxyl, sulfhydryl, ester group, maleic imide group and acrylic group; and Y is a linking group of an end group F. ##STR00001##

Claims

1. A multi-arm polyethylene glycol-adizo derivative having a structure of a general formula I: ##STR00036## wherein: R is a central molecule and R is pentaerythritol or polypentaerythritol structure, methyl glucoside, sucrose, glycerol or polyglycerol structure; n is the number of branches or the number of arms, n3; PEG is the same or different (CH.sub.2CH.sub.2O).sub.m, m is an integer of average value of 3-250; X is a linking group of a azido end group, selected from the group consisting of C.sub.1-12 alkyl, aryl alkyl, ester group, carbonate group, amide group, amide ester group, ether group, urethane group; k is the number of the branches having the azido end group, 2kn; F is an active end group different from the azido, selected from the group consisting of amino, carboxyl, sulfhydryl, ester group, maleic imide group and acrylic group; Y is a linking group of an end group F, selected from the group consisting of (CH.sub.2).sub.i, (CH.sub.2).sub.iNH, (CH.sub.2).sub.iOCOO, (CH.sub.2).sub.iOCONH, (CH.sub.2).sub.iNHCOO, (CH.sub.2).sub.iNHCONH, OC(CH.sub.2).sub.iCOO, (CH.sub.2).sub.iCOO, (CH.sub.2).sub.iCONH; i is an integer of 1-10.

2. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, the multi-arm polyethylene glycol-azido derivative having a structure of a general formula II:
R-|-PEG-XN.sub.3].sub.k(II).

3. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, the multi-arm polyethylene glycol-azido derivative having a structure of a general formula III: ##STR00037##

4. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, R is ##STR00038## ##STR00039## wherein 1 is an integer of 1 and 10.

5. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, 3n22.

6. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, 3n6.

7. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, 2k16.

8. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, k is 2, 4, 6, 8, 10, 12, 14 or 16.

9. The multi-arm polyethylene glycol-adizo derivative of claim 4, wherein, l is 1, 2, 3, 4, 5 or 6.

10. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, m is an integer of average value of 68-227.

11. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, X is selected from the group consisting of (CH.sub.2).sub.i, (CH.sub.2).sub.iNH, (CH.sub.2).sub.iOCOO, (CH.sub.2).sub.iOCONH, (CH.sub.2).sub.iNHCOO, (CH.sub.2).sub.iNHCONH, OC(CH.sub.2).sub.iCOO, (CH.sub.2).sub.iCOO, (CH.sub.2).sub.iCONH, (CH.sub.2).sub.iCOO.

12. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, Y is selected from the group consisting of (CH.sub.2).sub.i, (CH.sub.2).sub.iNH, (CH.sub.2).sub.iCOO, (CH.sub.2).sub.iCO.

13. The multi-arm polyethylene glycol-adizo derivative of claim 11, wherein, i is 1, 2, 3, 4 or 5.

14. The multi-arm polyethylene glycol-adizo derivative of claim 12, wherein, i is 1, 2, 3, 4 or 5.

15. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, F is NH.sub.2, COOH, OCH.sub.3, ##STR00040##

16. The multi-arm polyethylene glycol-adizo derivative of claim 1, wherein, the multi-arm polyethylene glycol-adizo derivative is shown as: ##STR00041## ##STR00042##

17. A multi-arm polyethylene glycol-adizo derivative, wherein, the multi-arm polyethylene glycol-adizo derivative having a structure of following general formula: ##STR00043## wherein, l is 1, 2, 3, 4, 5 or 6; X is a linking group of an azido end group, selected from the group consisting of C.sub.1-12 alkyl, aryl alkyl, ester group, carbonate group, amide group, amide ester group, ether group, urethane group.

18. A multi-arm polyethylene glycol-adizo derivative, wherein, the multi-arm polyethylene glycol-adizo derivative having a structure of following general formula: ##STR00044## wherein, l is 1, 2, 3, 4, 5 or 6; X is a linking group of an azido end group, selected from the group consisting of C.sub.1-12 alkyl, aryl alkyl, ester group, carbonate group, amide group, amide ester group, ether group, urethane group.

Description

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Preparation of Four-Arm Polyethylene Glycol-Azido Derivative

(1) ##STR00018##

(2) 10.0 g of four-arm polyethylene glycol (molecular weight of 5,000) was dissolved in 100 mL of toluene, under nitrogen atmosphere, heated and evaporated 20 mL of toluene, down to room temperature, 10 mL of methylene chloride and 1.45 mL of triethylamine were added, stirred for 10 minutes, 742 L of methanesulfonyl chloride was added, a sealed reaction overnight, 2 mL of absolute ethanol was added, stirred for 15 minutes, filtered, concentrated to a viscous at 60 C., 150 mL of isopropyl alcohol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol, dried under vacuum to obtain a four-arm polyethylene glycol sulfonic acid ester.

(3) 5.0 g of above four-arm polyethylene glycol sulfonate and 0.52 g of sodium azide (NaN.sub.3) were dissolved in 25 mL of N,N-dimethylformamide (DMF), heated to 90 C. and reacted for 2 hours, down to room temperature, 25 mL of water and 5 g of sodium chloride were added, extracted with 25 mL of methylene chloride for three times, combined organic phases, dried with anhydrous sodium sulfate and filtered, concentrated at 50 C., 100 mL of diethyl ether was added to precipitate, the precipitate was collected by filtration and dried under vacuum, the resulting four-arm polyethylene glycol-azido derivative.

(4) IR: 2110 cm.sup.1 (NNN)

Example 2: Preparation of Eight-Arm Polyethylene Glycol-Azido Derivative

(5) ##STR00019##

(6) 10.0 g of eight-arm polyethylene glycol (molecular weight of 10,000) was dissolved in 100 mL of toluene, under nitrogen atmosphere, heated and evaporated 20 mL of toluene, down to room temperature, 10 mL of methylene chloride and 1.45 mL of triethylamine were added, stirred for 10 minutes, 742 L of methanesulfonyl chloride was added, a sealed reaction overnight, 2 mL of absolute ethanol was added, stirred for 15 minutes, filtered, concentrated to a viscous at 60 C., 150 mL of isopropyl alcohol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol, dried under vacuum to obtain the eight-arm polyethylene glycol sulfonic acid ester.

(7) 5.0 g of above eight-arm polyethylene glycol sulfonate and 0.52 g of sodium azide (NaN.sub.3) were dissolved in 25 mL of N,N-dimethylformamide (DMF), heated to 90 C. and reacted for 2 hours, down to room temperature, 25 mL of water and 5 g of sodium chloride were added, extracted with 25 mL of methylene chloride for three times, combined organic phases, dried with anhydrous sodium sulfate and filtered, concentrated at 50 C., 100 mL of diethyl ether was added to precipitate, the precipitate was collected by filtration and dried under vacuum, the resulting eight-arm polyethylene glycol-azido derivative.

(8) IR: 2110 cm.sup.1 (NNN)

Example 3: Preparation of Four-Arm Polyethylene Glycol-Azido-Mono-Acetic Acid Derivative

(9) ##STR00020##

(10) 6.0 g of four-arm polyethylene glycol sulfonate-mono-acetic acid methyl ester (molecular weight of 5,000) and 0.468 g of sodium azide (NaN.sub.3) were dissolved in 30 mL of N,N-dimethylformamide (DMF), heated to 90 C. and reacted for 2 hours, down to room temperature, 30 mL of water and 7 g of sodium chloride were added, extracted with 30 mL of methylene chloride for three times, combined organic phases, dried with anhydrous sodium sulfate and filtered, concentrated at 50 C., 120 mL of diethyl ether was added to precipitate, the precipitate was collected by filtration and dried under vacuum, the resulting four-arm polyethylene glycol-azido-mono-acetic acid methyl ester.

(11) 5.0 g of above four-arm polyethylene glycol azido-mono-acetic acid methyl ester was dissolved in 50 mL of degassed water, 0.5 N aqueous sodium hydroxide to mediate pH 12.0, reacted for 2-2.5 hours at room temperature, 1 N aqueous hydrochloric acid to mediate pH 2-3, 6.0 g of sodium chloride was added, extracted with 50 mL of methylene chloride for three times, combined organic phase, dried with anhydrous sodium sulfate, filtered, concentrated to a viscous at 45 C., 75 mL of diethyl ether was added to precipitate, the precipitate was collected by filtration and dried under vacuum. The resulting four-arm polyethylene glycol-azido-mono-acetic acid derivative.

(12) IR: 2110 cm.sup.1 (NNN)

(13) 1H-NMR (DMSO) : 4.01 (s, CH.sub.2COOH, 2H)

Example 4: Preparation of Four-Arm Polyethylene Glycol-Azido-Mono-NHS Ester Derivative

(14) ##STR00021##

(15) Weighed 1.0 g of four-arm polyethylene glycol-azido-mono-acetic acid (molecular weight of 5,000) and 0.0276 g of N-hydroxy succinimide (NHS), dissolved with 10 mL of methylene chloride, under nitrogen atmosphere, 0.0536 g of N,N-dicyclohexyl-carbodiimide (DCC) was added, a sealed reaction overnight, filtered, concentrated to dryness at 40 C., 20 mL of isopropanol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol twice, dried under the vacuum to obtain the four-arm polyethylene glycol-azido-mono-NHS ester derivative.

(16) IR: 2110 cm.sup.1 (NNN)

(17) 1H-NMR (DMSO) : 4.6 (s, CH.sub.2CO, 2H), 2.8 (s, CH.sub.2 ring, 4H)

Example 5: Preparation of Four-Arm Polyethylene Glycol-Azido-Diacetic Acid Derivative

(18) ##STR00022##

(19) The starting composition is a four-arm polyethylene glycol-sulfonic acid-acetate-diacetic acid methyl ester (molecular weight of about 5,000), the synthetic step is as the same as in example 3.

(20) IR: 2110 cm.sup.1 (NNN)

(21) 1H-NMR (DMSO) : 4.01 (s, CH.sub.2COOH, 4H)

Example 6: Preparation of Four-Arm Polyethylene Glycol-Azido-Di-NHS Ester Derivative

(22) ##STR00023##

(23) The starting composition is a four-arm polyethylene glycol-azido-diacetic acid (molecular weight of about 5,000), the synthetic step is as the same as in example 4.

(24) IR: 2110 cm.sup.1 (NNN)

(25) 1H-NMR (DMSO) : 4.6 (s, CH.sub.2CO, 4H), 2.8 (s, CH.sub.2 ring, 8H)

Example 7: Preparation of Four-Arm Polyethylene Glycol-Three-Azido-Mono Amine Derivative

(26) ##STR00024##

(27) 10.0 g of four-arm polyethylene glycol hydroxy-mono-amine (molecular weight of 5,000) was dissolved in 100 mL of methylene chloride, 0.31 mL of triethylamine was added, stirred for 10 minutes, 0.6 mL of di-tert-butyl dicarbonate (Boc.sub.2O) was added, reacted overnight at room temperature, concentrated at 45 C., precipitated with 100 mL of diethyl ether, filtered, and dried under the vacuum to obtain the four-arm polyethylene glycol hydroxy-mono-Boc amide.

(28) 8.0 g of above four-arm polyethylene glycol hydroxy-mono-BOC amide was dissolved in 80 mL of toluene, under the nitrogen atmosphere, heated and evaporated 15 mL of toluene, down to room temperature, 8 mL of methylene chloride and 0.31 mL of triethylamine were added, stirred for 10 minutes, 0.16 mL of methanesulfonyl chloride was added, a sealed reaction overnight, 0.5 mL of absolute ethanol was added, stirred for 15 minutes, filtered, concentrated to a viscous at 60 C., 120 mL of isopropyl alcohol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol, dried under vacuum to obtain four-arm polyethylene glycol sulfonate-mono-BOC amide.

(29) 5.0 g of above four-arm polyethylene glycol sulfonate-mono-BOC amide and 0.39 g of sodium azide (NaN.sub.3) were dissolved in 25 mL of N,N-dimethylformamide (DMF), heated to 90 C. and reacted for 2 hours, down to room temperature, 25 mL of water and 5 g of sodium chloride were added, extracted with 25 mL of methylene chloride for three times, combined organic phases, dried with anhydrous sodium sulfate and filtered, concentrated at 50 C., 100 mL of diethyl ether was added to precipitate, the precipitate was collected by filtration and dried under vacuum, the resulting four-arm polyethylene glycol-azido-mono-BOC amide.

(30) 3.0 g of above four-arm polyethylene glycol-azido-mono-BOC amide was dissolved in 21 mL of methylene chloride, 9 mL of trifluoroacetic acid was added, reacted for 3 hours, concentrated at 45 C., precipitated with 60 mL of diethyl ether, filtered, dried the vacuum to obtain four-arm polyethylene glycol-azido-mono-amine derivative.

(31) IR: 2110 cm.sup.1 (NNN)

(32) 1H-NMR (DMSO) : 3.0 (m, CH.sub.2NH.sub.2, 2H)

Example 8: Preparation of Four-Arm Polyethylene Glycol-Azido-Mono-Maleimide Derivative

(33) ##STR00025##

(34) 1.0 g of four-arm polyethylene glycol-azido-mono-amine (molecular weight of 5,000) was dissolved in 10 mL of methylene chloride, under nitrogen atmosphere, 0.031 mL of triethylamine was added, stirred for 10 minutes, 0.074 g of MAL-NHS was added, reaction was allowed to proceed overnight protected from light, concentrated to dryness at 40 C., 20 mL of isopropanol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol twice, dried under the vacuum to obtain the four-arm polyethylene glycol-azido-mono-maleimide derivative.

(35) IR: 2110 cm.sup.1 (NNN)

(36) 1H-NMR (DMSO) : 2.32 (t,

(37) ##STR00026##
2H), 7.0 (s, CH ring, 2H)

Example 9: Preparation of Four-Arm Polyethylene Glycol-Diazido-Diamino Derivative

(38) ##STR00027##

(39) The starting composition is a four-arm polyethylene glycol-hydroxy-diamine (molecular weight of about 5,000), the synthetic step is as the same as in example 7.

(40) IR: 2110 cm.sup.1 (NNN)

(41) 1H-NMR (DMSO) : 3.0 (m, CH.sub.2NH.sub.2, 4H)

Example 10: Preparation of Four-Arm Polyethylene Glycol-Diazido-Dimaleimide Derivative

(42) ##STR00028##

(43) The starting composition is a four-arm polyethylene glycol-azido-diamine (molecular weight of about 5,000), the synthetic step is as the same as in example 8.

(44) IR: 2110 cm.sup.1 (NNN)

(45) 1H-NMR (DMSO) : 2.32 (t,

(46) ##STR00029##
4H), 7.0 (s, CH ring, 4H)

Example 11: Preparation of Eight-Arm Polyethylene Glycol-Seven Azido-Mono-Acetic Acid Derivative

(47) ##STR00030##

(48) 10.0 g of eight-arm polyethylene glycol sulfonate-mono-acetic acid methyl ester (molecular weight of 10,000) and 0.91 g of sodium azide (NaN.sub.3) were dissolved in 50 mL of N,N-dimethylformamide (DMF), heated to 90 C. and reacted for 2 hours, down to room temperature, 50 mL of water and 12 g of sodium chloride were added, extracted with 50 mL of methylene chloride for three times, combined organic phases, dried with anhydrous sodium sulfate and filtered, concentrated at 50 C., 200 mL of diethyl ether was added to precipitate, the precipitate was collected by filtration and dried under vacuum, the resulting eight-arm polyethylene glycol-azido-mono-acetic acid methyl ester.

(49) 5.0 g of above eight-arm polyethylene glycol-azido-mono methyl acetate was dissolved in 50 mL of degassed water, 0.5 N aqueous sodium hydroxide to mediate pH 12.0, reacted for 2-2.5 hours at room temperature, 1 N aqueous hydrochloric acid to mediate pH 2-3, 6.0 g of sodium chloride was added, extracted with 50 mL of methylene chloride for three times, combined organic phase, dried with anhydrous sodium sulfate, filtered, concentrated to a viscous at 45 C., 75 mL of diethyl ether was added to precipitate, the precipitate was collected by filtration and dried under vacuum. The resulting eight-arm polyethylene glycol-azido-mono-acetic acid derivative.

(50) IR: 2110 cm.sup.1 (NNN)

(51) 1H-NMR (DMSO) : 4.01 (s, CH.sub.2COOH, 2H)

Example 12: Preparation of Eight-Arm Polyethylene Glycol-Seven-Azido-Mono-NHS Ester Derivative

(52) ##STR00031##

(53) Weighed 2.0 g of eight-arm polyethylene glycol-azido-mono-acetic acid (molecular weight of 10,000) and 0.03 g of N-hydroxy succinimide (NHS), dissolved with 20 mL of methylene chloride, under nitrogen, 0.058 g of N,N-dicyclohexyl-carbodiimide (DCC) was added, a sealed reaction overnight, filtered, concentrated to dryness at 40 C., 40 mL of isopropanol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol twice, dried under the vacuum to obtain the eight-arm polyethylene glycol-seven-azido-mono-NHS ester derivative.

(54) IR: 2110 cm.sup.1 (NNN)

(55) 1H-NMR (DMSO) : 4.6 (s, CH.sub.2CO, 2H), 2.8 (s, CH.sub.2 ring, 4H)

Example 13: Preparation of Eight-Arm Polyethylene Glycol-Seven Azido-Mono-Amine Derivative

(56) ##STR00032##

(57) 10.0 g of eight-arm polyethylene glycol hydroxy-mono-amine (molecular weight 10,000) was dissolved in 100 mL of methylene chloride, 0.16 mL of triethylamine was added, stirred for 10 minutes, 0.3 mL of di-tert-butyl dicarbonate (Boc.sub.2O) was added, reacted overnight at room temperature, concentrated at 45 C., precipitated with 150 mL of diethyl ether, filtered, and dried under the vacuum to obtain the four-arm polyethylene glycol hydroxy-mono-Boc amide.

(58) 8.0 g of above eight-arm polyethylene glycol hydroxy-mono-BOC amide was dissolved in 80 mL of toluene, under the nitrogen atmosphere, heated and evaporated 15 mL of toluene, down to room temperature, 8 mL of methylene chloride and 0.16 mL of triethylamine were added, stirred for 10 minutes, 0.08 mL of methanesulfonyl chloride was added, a sealed reaction overnight, 0.5 mL of absolute ethanol was added, stirred for 15 minutes, filtered, concentrated to a viscous at 60 C., 120 mL of isopropyl alcohol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol, dried under vacuum to obtain eight-arm polyethylene glycol sulfonate-mono-BOC amide.

(59) 5.0 g of above eight-arm polyethylene glycol sulfonate-mono-BOC amide and 0.455 g of sodium azide (NaN.sub.3) were dissolved in 25 mL of N,N-dimethylformamide (DMF), heated to 90 C. and reacted for 2 hours, down to room temperature, 25 mL of water and 5 g of sodium chloride were added, extracted with 25 mL of methylene chloride for three times, combined organic phases, dried with anhydrous sodium sulfate and filtered, concentrated at 50 C., 100 mL of diethyl ether was added to precipitate, the precipitate was collected by filtration and dried under vacuum, the resulting eight-arm polyethylene glycol-azido-mono-BOC amide.

(60) 3.0 g of above eight-arm polyethylene glycol-azido-mono-BOC amide was dissolved in 21 mL of methylene chloride, 9 mL of trifluoroacetic acid was added, reacted for 3 hours, concentrated at 45 C., precipitated with 60 mL of diethyl ether, filtered, dried the vacuum to obtain eight-arm polyethylene glycol-seven-azido-mono-amine derivative.

(61) IR: 2110 cm.sup.1 (NNN)

(62) 1H-NMR (DMSO) : 3.0 (m, CH.sub.2NH.sub.2, 2H)

Example 14: Preparation of Eight-Arm Polyethylene Glycol-Seven Azido-Mono-Maleimide Derivative

(63) ##STR00033##

(64) 1.0 g of eight-arm polyethylene glycol-azido-mono-amine (molecular weight 10,000) was dissolved in 10 mL of methylene chloride, under nitrogen atmosphere, 0.016 mL of triethylamine was added, stirred for 10 minutes, 0.037 g of MAL-NHS was added, reaction was allowed to proceed overnight protected from light, concentrated to dryness at 40 C., 20 mL of isopropanol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol twice, dried under the vacuum to obtain eight-arm polyethylene glycol-seven azido-mono-maleimide derivative.

(65) IR: 2110 cm.sup.1 (NNN)

(66) 1H-NMR (DMSO) : 2.32 (t,

(67) ##STR00034##
2H), 7.0 (s, CH ring, 2H)

Example 15: Preparation of Click Gel by using a Four-Arm Polyethylene Glycol-Azido (Molecular Weight of 5,000) and the Drug Release Test In Vitro Thereof

(68) 0.25 g of four-arm polyethylene glycol-azido (molecular weight of about 5,000), 0.25 g of polyethylene glycol-alkynyl derivatives (molecular weight of about 5,000) and 0.05 g of irinotecan-glycine hydrochloride salt were dissolved in 5 mL of water, 0.008 g of sodium ascorbate and 0.004 g of copper acetate were added, stirred for 25 minutes to form a gel at room temperature.

(69) The above gel placed in a dialysis bag (throttle molecular weight of 5000), washed with 20 mL of water for three times, until detecting no UV absorption in aqueous solution with high performance liquid chromatography (HPLC), put into a glass vial, 20 mL of water was added, shaken at 40 C. in thermostatic oscillatorthe, sampled at 30 min, 2 h, 4 h respectively, 0.0025 g/mL of irinotecan-glycine hydrochloride in water as the reference sample, measured the release amount of the gel at individual time points with high performance liquid chromatography.

(70) The released ratio of Irinotecan-glycine is: 21% at 0.5 hour, 36% at 2 hours, 42% at 4 hours.

Example 16: Preparation of Click Gel by using a Four-Arm Polyethylene Glycol-Azido (Molecular Weight of 10,000) and Drug Release Test In Vitro Thereof

(71) 0.25 g of four-arm polyethylene glycol-azido (molecular weight of about 10,000), 0.25 g of polyethylene glycol-alkynyl derivatives (molecular weight of about 10,000) and 0.05 g of irinotecan-glycine hydrochloride salt were dissolved in 5 mL of water, 0.004 g of sodium ascorbate and 0.002 g of copper acetate were added, stirred for 1 hour to form a gel at room temperature.

(72) The above gel placed in a dialysis bag (throttle molecular weight of 5,000), washed with 20 mL of water for three times, until detecting no UV absorption in aqueous solution with high performance liquid chromatography (HPLC), put into a glass vial, 20 mL of water was added, shaken at 40 C. in thermostatic oscillatorthe, sampled at 30 min, 2 h, 4 h respectively, 0.0025 g/mL of irinotecan-glycine hydrochloride in water as the reference sample, measured the release amount of the gel at individual time points with high performance liquid chromatography.

(73) The released ratio of Irinotecan-glycine is: 35% at 0.5 hour, 54% at 2 hours, 69% at 4 hours.

Example 17: Preparation of Click Gel by Using a Eight-Arm Polyethylene Glycol-Azido (Molecular Weight of 10,000) and Drug Release Test In Vitro Thereof

(74) 0.25 g of eight-arm polyethylene glycol-azido (molecular weight of about 10,000), 0.25 g of polyethylene glycol-alkynyl derivatives (molecular weight of about 10,000) and 0.05 g of irinotecan-glycine hydrochloride salt were dissolved in 5 mL of water, 0.008 g of sodium ascorbate and 0.004 g of copper acetate were added, stirred for 10 minutes to form a gel at room temperature.

(75) The above gel placed in a dialysis bag (throttle molecular weight of 5,000), washed with 20 mL of water for three times, until detecting no UV absorption in aqueous solution with high performance liquid chromatography (HPLC), put into a glass vial, 20 mL of water was added, shaken at 40 C. in thermostatic oscillatorthe, sampled at 30 min, 2 h, 4 h respectively, 0.0025 g/mL of irinotecan-glycine hydrochloride in water as the reference sample, measured the release amount of the gel at individual time points with high performance liquid chromatography.

(76) The released ratio of Irinotecan-glycine is: 24% at 0.5 hour, 48% at 2 hours, 60% at 4 hours.

(77) Examples 15-17 shows that, in the preparation of gel via a multi-arm polyethylene glycol-azido derivatives reacted with other polyethylene glycol derivatives, the molecular weight and the number of branches of the polyethylene glycol can be used to affect or control the time of gel formation, the lower the molecular weight, the higher the number of the branch, the shorter the time of gel formation, and an increase in the branch number of poly ethylene glycol may be more effective in increasing the gel formation rate. At the same time, the molecular weight and the branch number also have a significant effect on the in vitro drug release time, therefore, to prepare a multi-arm polyethylene glycol-azido derivatives can also be used to control the drug release sustaining process.

Example 18: Preparation of Four-Arm Polyethylene Glycol-Three-Azido-Adamantine and Gel Thereof

(78) ##STR00035##

(79) 2.0 g of four-arm polyethylene glycol-three-azido-mono-amine (molecular weight of 5,000) was dissolved in 20 mL of methylene chloride, 0.12 mL of triethylamine was added, stirred for 10 minutes, 0.23 g of the adamantane-1-carboxamide was added, reaction was allowed to proceed overnight protected from light, concentrated to dryness at 45 C., 40 mL of isopropanol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol twice, dried under the vacuum to obtain four-arm polyethylene glycol-three azido-adamantane.

(80) IR: 2110 cm.sup.1 (NNN)

(81) 1H-NMR (DMSO) : 1.6 (m, ring, 6H), 1.7 (m, ring, 6H), 1.9 (m, ring, 3H)

(82) 0.33 g of four-arm polyethylene glycol-three-azido-adamantyl (molecular weight of about 5,000) and 0.25 g of polyethylene glycol alkynyl derivatives (molecular weight of about 5,000) were dissolved in 6 mL of water, 0.008 g of sodium ascorbate and 0.004 g of copper acetate were added, stirred at room temperature for 30 minutes to form a gel.