Alkynyl multi-arm polyethylene glycol derivative

Abstract

The present invention provides an alkynyl multi-arm polyethylene glycol derivative having a structure of a general formula I or general formula X VIII. In the derivative, X1, X2, X3 and X4 are linking groups, F1, F2, F3 and F4 are end groups, the end groups may be the same or may also be different, and are selected from: hydroxy, carboxyl, ester group, amino, alkynyl or the like, at least one of the end groups is alkynyl, and PEG is the same or different (CH.sub.2CH.sub.2O).sub.m, wherein m is an integer ranging from 3 to 250, and l is an integer greater than or equal to 1. The multi-arm polyethylene glycol derivatives have stronger application flexibility, and have good application prospect in aspects such as organic synthesis, medicine synthesis and medical instruments. ##STR00001##

Claims

1. An alkynyl multi-arm polyethylene glycol active derivative having a structure of a general formula I or XVII ##STR00035## wherein: PEG is the same or different (CH.sub.2CH.sub.2O).sub.m, m is an integer of average value of 3-250; l is an integer 1; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are linking groups independently selected from the group consisting of C.sub.1-12 alkyl, aryl alkyl, ester group, carbonate group, amide group, amide ester group, ether group, and urethane group; F.sub.1, F.sub.2, F.sub.3 and F.sub.4 are end groups independently selected from the group consisting of the following groups: NH.sub.2, COOH, OCH.sub.3, ##STR00036## and CCH, in the alkynyl multi-arm polyethylene glycol derivative of general formula I, only one or two or three of the F.sub.1, F.sub.2, F.sub.3 and F.sub.4 is (are) CCH; and in the alkynyl multi-arm polyethylene glycol derivative of general formula XVIII, only one or two of the F.sub.1, F.sub.2 and F.sub.4 is (are) CCH.

2. The alkynyl multi-arm polyethylene glycol active derivative of claim 1, wherein, the 1 is 1, 2, 3, 4, 5 or 6.

3. An alkynyl multi-arm polyethylene glycol active derivative having a structure of a general formula I or XVIII ##STR00037## wherein: PEG is the same or different (CH.sub.2CH.sub.2O).sub.m, m is an integer of average value of 3-250; l is an integer 1; X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are independently selected from the group consisting of (CH.sub.2).sub.i, (CH.sub.2).sub.iNHCO(CH.sub.2).sub.j, (CH.sub.2).sub.iCONH(CH.sub.2).sub.j, (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 in X.sub.1, X.sub.2, X.sub.3 and X.sub.4; j is an integer of 1-10 in X.sub.1, X.sub.2, X.sub.3 and X.sub.4; F.sub.1, F.sub.2, F.sub.3 and F.sub.4 are end groups independently selected from the group consisting of the following groups: NH.sub.2, COOH, OCH.sub.3, ##STR00038## and CCH, in the alkynyl multi-arm polyethylene glycol derivative of general formula I, only one or two or three of the F.sub.1, F.sub.2, F.sub.3 and F.sub.4 is (are) CCH; and in the alkynyl multi-arm polyethylene glycol derivative of general formula XVIII, only one or two of the F.sub.1, F.sub.2 and F.sub.4 is (are) CCH.

4. The alkynyl multi-arm polyethylene glycol active derivative of claim 2, wherein, X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are independently selected from the group consisting of (CH.sub.2).sub.i, (CH.sub.2).sub.iNHCO(CH.sub.2).sub.j, (CH.sub.2).sub.iCONH(CH.sub.2).sub.j, i is 1, 2, 3, 4 or 5 in X.sub.1, X.sub.2, X.sub.3 and X.sub.4; and j is 1, 2, 3, 4 or 5 in X.sub.1, X.sub.2, X.sub.3 and X.sub.4.

5. The alkynyl multi-arm polyethylene glycol active derivative of claim 1, wherein, the multi-arm polyethylene glycol derivative has a molecular weight of 1,000-80,000 Da.

6. The alkynyl multi-arm polyethylene glycol active derivative of claim 5, wherein, the multi-arm polyethylene glycol derivative has a molecular weight of 3,000-20,000 Da.

7. An alkynyl multi-arm polyethylene glycol active derivative, wherein, having following general structure: ##STR00039## ##STR00040## ##STR00041## ##STR00042## wherein: PEG is the same or different (CH.sub.2CH.sub.2O).sub.m, m is an integer of average value of 3-250; l is an integer 1; X.sub.1 is selected from of the groups consisting of (CH.sub.2).sub.i, (CH.sub.2).sub.iNHCO(CH.sub.2).sub.j, (CH.sub.2).sub.iCONH(CH.sub.2).sub.j, (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 in X.sub.1; j is an integer of 1-10 in X.sub.1; and k is an integer of 1-10.

8. The alkynyl multi-arm polyethylene glycol active derivative of claim 7, wherein, X.sub.1 is selected from the group consisting of (CH.sub.2).sub.i, (CH.sub.2).sub.iNHCO(CH.sub.2).sub.j, and (CH.sub.2).sub.iCONH(CH.sub.2).sub.j, i is 1, 2, 3, 4 or 5 in X.sub.1; and j is 1, 2, 3, 4 or 5 in X.sub.1.

9. The alkynyl multi-arm polyethylene glycol active derivative of claim 7, wherein, k is 1, 2, 3, 4 or 5 in the alkynyl multi-arm polyethylene glycol active derivative.

10. The alkynyl multi-arm polyethylene glycol active derivative of claim 7, wherein, the alkynyl multi-arm polyethylene glycol derivative has a molecular weight of 1,000-80,000 Da.

11. The alkynyl multi-arm polyethylene glycol active derivative of claim 10, wherein, the alkynyl multi-arm polyethylene glycol derivative has a molecular weight of 3,000-20,000 Da.

12. A method of preparing the alkynyl multi-arm polyethylene glycol active derivative having a structure of a general formula I or XVIII of claim 1, wherein, comprising: dissolving a multi-arm polyethylene glycol in a solvent, adding sodium hydride and reaction at room temperature, adding propargyl bromide and potassium iodide and reaction, obtaining a multi-arm polyethylene glycol-propynyl after separation; or, dissolving a multi-arm polyethylene glycol-to-end acetic acid and N-hydroxy succinimide (NHS) in a solvent, adding N,N-dicyclohexyl-carbodiimide and reaction, adding propargyl amine and reaction, obtaining a multi-arm polyethylene glycol-propynyl acetamide after separation; or, dissolving a multi-arm polyethylene glycol-to-end hydroxyl-to-end acetic acid methyl ester in a solvent, adding sodium hydride and reaction at room temperature, adding propargyl bromide and potassium iodide and reaction, obtaining a multi-arm polyethylene glycol-propynyl-acetic acid methyl ester; hydrolyzing the multi-arm polyethylene glycol-propynyl-acetic acid methyl ester, obtaining a polyethylene glycol-propynyl-acetic acid derivative; or, dissolving a multi-arm polyethylene glycol-to-end hydroxyl-to-end acetic acid methyl ester in a solvent, adding sodium hydride and reaction at room temperature, adding propargyl bromide and potassium iodide and reaction, obtaining a multi-arm polyethylene glycol-propynyl-acetic acid methyl ester; hydrolyzing the multi-arm polyethylene glycol-propynyl-acetic acid methyl ester, obtaining a polyethylene glycol-propynyl-acetic acid derivative; dissolving the polyethylene glycol-propynyl-acetic acid derivative in a solvent, adding N-hydroxy succinimide and N,N-dicyclohexyl-carbodiimide and reaction, obtaining a multi-arm polyethylene glycol-propynyl-acetic acid NHS ester; or, dissolving a multi-arm polyethylene glycol-to-end hydroxyl-to-end acetic acid methyl ester in a solvent, adding sodium hydride and reaction at room temperature, adding propargyl bromide and potassium iodide and reaction, obtaining a multi-arm polyethylene glycol-propynyl-acetic acid methyl ester; hydrolyzing the multi-arm polyethylene glycol-propynyl-acetic acid methyl ester, obtaining a polyethylene glycol-propynyl-acetic acid derivative; dissolving the polyethylene glycol-propynyl-acetic acid derivative in a solvent, adding N-hydroxy succinimide and N,N-dicyclohexyl-carbodiimide and reaction, adding maleimido-ethylene-diamine and reaction, obtaining a multi-arm polyethylene glycol-to-end propynyl-to-end acetic acid MAL ester; or, dissolving a multi-arm polyethylene glycol-to-end acetic acid-to-end ethyl amine in a solvent, adding triethylamine and di-tert-butyl dicarbonate and reaction, obtaining multi-arm polyethylene glycol-acetic acid-Boc amide; after the reaction, dissolving the multi-arm polyethylene glycol-acetic acid-Boc amide and N-hydroxy succinimide in a solvent, adding N,N-dicyclohexyl-carbodiimide and reaction, adding propargyl amine and reaction, obtaining a multi-arm polyethylene glycol-alkynyl-Boc amide; dissolving the multi-arm polyethylene glycol-alkynyl-Boc amide in a solvent, adding trifluoro acetic acid and reaction, obtaining a multi-arm polyethylene glycol-to-end alkynyl-to-end ethyl amine; or, dissolving a multi-arm polyethylene glycol-to-end acetic acid-to-end ethyl amine in a solvent, adding triethylamine and di-tert-butyl dicarbonate and reaction, obtaining a multi-arm polyethylene glycol-acetic acid-Boc amide; after the reaction, dissolving the multi-arm polyethylene glycol-acetic acid-Boc amide and N-hydroxy succinimide in a solvent, adding N,N-dicyclohexyl-carbodiimide and reaction, adding propargyl amine and reaction, obtaining a multi-arm polyethylene glycol-alkynyl-Boc amide; dissolving the multi-arm polyethylene glycol-alkynyl-Boc amide in a solvent, adding trifluoro acetic acid and reaction, obtaining a multi-arm polyethylene glycol-to-end alkynyl-to-end ethyl amine; dissolving the multi-arm polyethylene glycol-to-end alkynyl-to-end ethyl amine in a solvent, adding 3-maleimidopropionic acid N-hydroxysuccinimide ester and reaction, obtaining a multi-arm polyethylene glycol-to-end alkynyl-to-end ethyl amine MAL.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Examples below describes the derivatives and the preparation method thereof of the present invention, the examples are not intended to limit the invention, the scope of the invention is limited by the claims of the application.

Example 1

Preparation of Four-Arm Polyethylene Glycol (10,000 Da)-Propynyl

(2) ##STR00022##

(3) 10.0 g of a four-arm polyethylene glycol (10,000 Da) was dissolved in 150 mL of tetrahydrofuran, lowered the temperature to 0 C. under nitrogen, 0.48 g of sodium hydride was added, reacted at room temperature for 0.5 hours, 22.4 mL of propargyl bromide (80% solution in toluene) was added, 0.09 g of potassium iodide was added, heated to reflux for 2 hours protected from light, cooled, 100 mL of water was added, out of the tetrahydrofuran, extracted with methylene chloride for three times, dried with anhydrous sodium sulfate, filtered, and concentrated to a viscous at 45 C., precipitated with 100 mL of ethyl ether, the precipitate was collected by filtration and dried under vacuum. The resulting 8.7 g of four-arm polyethylene glycol-propynyl.

(4) 1H-NMR (DMSO) : 3.08 (s, CCH, 4H)

Example 2

Preparation of Eight-Arm Polyethylene Glycol (20,000 Da)-Propynyl Acetamide

(5) ##STR00023##

(6) 10.0 g of a eight-arm polyethylene glycol (20,000 Da)-acetic acid and 0.69 g of N-hydroxy succinimide (NHS) was dissolved in 100 mL of methylene chloride under nitrogen, 1.32 g of N,N-dicyclohexyl-carbodiimide (DCC) was added, reaction for 4 h, 0.9 mL of propargyl amine was added, the reaction was allowed to proceed overnight protected from light, filtered, concentrated at 40 C., 150 mL of isopropyl alcohol was heated to dissolve and precipitated with an ice-water bath, filtered, washed the filter cake with isopropanol twice, dried under vacuum. The resulting 9.0 g of eight-arm polyethylene glycol-propynyl acetamide.

(7) 1H-NMR (DMSO) : 3.08 (s, CCH, 4H), 7.66 (t, CONH, 4H)

Example 3

Preparation of Four-Arm Polyethylene Glycol (5,000 Da)-Alkynyl- to Mono-Acetic Acid

(8) ##STR00024##

(9) 10.0 g of a four-arm polyethylene glycol (5,000 Da)-hydroxy-mono-acetic acid methyl ester was dissolved in 150 mL of tetrahydrofuran, lowered the temperature to 0 C. under nitrogen atmosphere, 0.72 g of sodium hydride was added, reacted at room temperature for 0.5 hours, 33.6 mL of propargyl bromide (80% solution in toluene) was added, 0.135 g of potassium iodide was added, heated to reflux for 2 hours protected from light, cooled, 100 mL of water was added, out of the tetrahydrofuran, extracted with methylene chloride for three times, dried with anhydrous sodium sulfate, filtered, and concentrated to a viscous at 45 C., 100 mL of ethyl ether was added for precipitation, the precipitate was collected by filtration and dried under vacuum. The resulting 8.2 g of four-arm polyethylene glycol-propynyl mono-acetic acid methyl ester.

(10) 5.0 g of a four-arm polyethylene glycol-propynyl 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 at room temperature for 2-2.5 hours, 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 ethyl ether was added for precipitation, the precipitate was collected by filtration and dried under vacuum. The resulting 3.6 g of four-arm polyethylene glycol-propynyl-mono acetic acid.

(11) 1H-NMR (DMSO) : 3.08 (s, CCH, 4H), 4.01 (s, CH.sub.2COOH, 8H)

Example 4

Preparation of Four-Arm Polyethylene Glycol (5,000 Da)-Alkynyl-Mono Acetic Acid Active NHS Ester

(12) ##STR00025##

(13) Weighed 1.0 g of four-arm polyethylene glycol (5,000 Da)-propynyl mono-acetic acid and 0.0276 g of N-hydroxy succinimide (NHS), dissolved with 10 mL of methylene chloride, under nitrogen, 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-propynyl-mono-acetic acid NHS ester.

(14) 1H-NMR (DMSO) : 3.08 (s, CCH, 3H), 4.6 (s, CH.sub.2CO, 2H), 2.8 (s, CH.sub.2 ring, 4H)

Example 5

Preparation of Four-Arm Polyethylene Glycol (5,000 Da)-Alkynyl Mono-Acetic Acid MAL

(15) ##STR00026##

(16) Weighed 1.0 g of four-arm polyethylene glycol (5,000 Da)-propynyl-mono-acetic acid and 0.035 g of n-hydroxy succinimide (NHS), dissolved with 10 mL of methylene chloride, under nitrogen atmosphere, 0.066 g of N,N-dicyclohexyl-carbodiimide (DCC) was added and dissolved with 10 mL of methylene chloride, reaction for 4 h, 0.115 g of maleic anhydride ethylene-amine was added, the 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-propynyl-mono-acetic acid MAL.

(17) 1H-NMR (DMSO) : 3.08 (s, CCH, 3H), 2.32 (t, CH.sub.2N, 2H), 7.0 (s, CH ring, 2H)

Example 6

Preparation of Four-Arm Polyethylene Glycol (20,000 Da)-Dialkynyl-Diethylamine

(18) ##STR00027##

(19) 10.0 g of four-arm polyethylene glycol-diacetic acid-diethylamine (molecular weight of 20,000) was dissolved in 100 mL of methylene chloride, 0.15 mL of triethylamine was added, stirred for 10 minutes, 0.3 mL of di-tert-butyl dicarbonate (Boc.sub.2O) was added, reacted at room temperature overnight, concentrated at 45 C., precipitated with 100 mL of diethyl ether, filtered, dried under the vacuum to obtain 9.7 g of four-arm polyethylene glycol-di-acetic acid-di-Boc amide.

(20) 5 g of four-arm polyethylene glycol-diacetic acid-di-Boc amide and 0.086 g of N-hydroxy succinimide (NHS) were dissolved in 50 mL of methylene chloride, under nitrogen atmosphere, 0.165 g of N,N-dicyclohexyl-carbodiimide (DCC) was added, reaction for 4 h, 0.11 mL of propargyl amine was added, a sealed reaction overnight and filtered, concentrated to dryness at 40 C., 75 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 4.3 g of four-arm polyethylene glycol-di alkynyl-di-BOC amide.

(21) 3.0 g of four-arm polyethylene glycol-di-alkynyl-di-BOC amide was dissolved in 21 mL of methylene chloride, 9 mL of trifluoroacetic acid was added, reaction for 3 hours, concentrated at 45 C., precipitated with 60 mL of diethyl ether, filtered, dried under the vacuum to obtain 2.3 g of four-arm polyethylene glycol-di-alkynyl-diethylamine.

(22) 1H-NMR (DMSO) : 3.08 (s, CCH, 2H), 3.0 (m, CH.sub.2N, 4H)

Example 7

Preparation of Four-Arm Polyethylene Glycol (20,000 Da)-Dialkynyl-Diethylamine-MAL

(23) ##STR00028##

(24) 1.0 g of four-arm polyethylene glycol (20,000 Da)-dialkynyl-diethyl amine was dissolved in 10 mL of methylene chloride, 0.035 g of 3-maleimidopropionic acid hydroxysuccinimide ester was added, the 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-dialkynyl-diethylamine MAL.

(25) 1H-NMR (DMSO) : 3.08 (s, CCH, 2H), 2.32 (t, CH.sub.2N, 4H), 7.0 (s, CH ring, 4H)

Example 8

Preparation of Eight-Arm Polyethylene Glycol (10,000 Da)-Mono-Alkynyl-Seven-Acetic Acid

(26) ##STR00029##

(27) 14.7 g of 4-alkynyl-acid was dissolved in 100 mL of methylene chloride, 1.89 g of N-hydroxy succinimide (NHS) was added, under nitrogen atmosphere, 3.75 g of N,N-dicyclohexyl-carbodiimide (DCC) was added, a sealed reaction overnight at room temperature, 10 g of eight arm polyethylene glycol (10,000 Da)-mono-hydroxy-seven-acetic acid methyl ester was dissolved in 100 mL of methylene chloride, the above solution was added to the reaction flask, reaction for 4 hours, 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 8.3 g of eight-arm polyethylene glycol-mono-alkynyl-seven-acetic acid methyl ester.

(28) The above eight-arm polyethylene glycol-mono-alkynyl-seven acetic acid methyl ester 5.0 g was dissolved in 50 mL of degassed water, 0.5 N of aqueous sodium hydroxide to mediate pH 12.0, reacted for 2 to 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 the organic phase, dried with anhydrous sodium sulfate, filtered, concentrated to a viscous at 45 C., precipitated with 75 mL of diethyl ether, the precipitate was collected by filtration and dried under vacuum. The resulting 3.3 g of eight-arm polyethylene glycol-alkynyl-seven-acetic acid.

(29) 1H-NMR (DMSO) : 3.08 (s, CCH, 1H), 4.01 (s, CH.sub.2COOH, 14H)

Example 9

Preparation of Eight-Arm Polyethylene Glycol (10,000 Da)-Alkynyl-Seven Acetic Acid NHS Ester

(30) ##STR00030##

(31) 1.0 g of eight-arm polyethylene glycol (10,000 da)-mono-alkynyl-seven acetic acid and 0.12 g of N-hydroxy succinimide (NHS) were dissolved in 10 mL of methylene chloride, under nitrogen atmosphere, 0.23 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 eight-arm polyethylene glycol-mono-alkynyl-seven acetic acid NHS ester.

(32) 1H-NMR (DMSO) : 3.08 (s, CCH, 1H), 4.6 (s, CH.sub.2CO, 14H), 2.8 (s, CH.sub.2 ring, 28H)

Example 10

Preparation of Eight-Arm Polyethylene Glycol (10,000 Da)-Alkynyl-Seven Acetic Acid MAL

(33) ##STR00031##

(34) Weighed 1.0 g of eight-arm polyethylene glycol (10,000 da)-alkynyl-seven acetic acid and 0.12 g of n-hydroxy succinimide (NHS) and dissolved in 10 mL of methylene chloride, under nitrogen atmosphere, 0.23 g of N,N-dicyclohexyl-carbodiimide (DCC) was added, reaction for 4 h, 0.35 g of maleic acid ethylenediamine was added, the 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 (10,000 da)-alkynyl-seven acetic acid MAL.

(35) 1H-NMR (DMSO) : 3.08 (s, CCH, 1H), 2.32 (t, CH.sub.2N, 14H), 7.0 (s, CH ring, 14H)

Example 11

Preparation of Eight-Arm Polyethylene Glycol (20,000 Da)-Seven Alkynyl-Mono-Amine

(36) ##STR00032##

(37) 10.0 g of eight-arm polyethylene glycol (20,000 Da)-seven hydroxy-mono-ethyl amine was dissolved in 100 mL of methylene chloride, 0.077 mL of triethylamine was added, stirred for 10 minutes, 0.15 mL of di-tert-butyl dicarbonate (Boc.sub.2O) was added, reacted overnight at room temperature, concentrated at 40 C., precipitated with 100 mL of diethyl ether, filtered, dried under the vacuum to obtain 9.5 g of eight-arm polyethylene glycol (20,000 Da)-seven hydroxy-mono-Boc amide.

(38) 5.0 g of eight-arm polyethylene glycol (20,000 Da)-seven hydroxy-mono-BOC amide was dissolved in 100 mL of tetrahydrofuran, a nitrogen atmosphere for lowering the temperature to 0 C., 0.024 g of sodium hydride was added, reacted for 0.5 hours at room temperature, 1.12 mL of propargyl bromide (80% solution in toluene) was added, 0.0045 g of potassium iodide was added, heated to reflux for 2 hours protected from light, cooled, 50 mL of water was added, out of the tetrahydrofuran, extracted with methylene chloride for three times, dried with anhydrous sodium sulfate, filtered, concentrated, concentrated to a viscous at 45 C., precipitated with 100 mL of ethyl ether, the precipitate was collected by filtration and dried under vacuum. The resulting 3.9 g of eight-arm polyethylene glycol (20,000 Da)-seven propynyl-mono-BOC amide.

(39) 3.0 g of eight-arm polyethylene glycol (20,000 Da)-seven propynyl-mono-BOC amide was dissolved in 21 mL of methylene chloride, 9 mL of trifluoroacetic acid was added, reaction for 3 hours, concentrated at 40 C., precipitated with 60 mL of diethyl ether, filtered, dried under the vacuum to obtain 2.3 g of eight-arm polyethylene glycol (20,000 Da)-seven alkynyl-mono-ethyl amine.

(40) 1H-NMR (DMSO) : 3.08 (s, CCH, 7H), 3.0 (m, CH.sub.2N, 2H)

Example 12

Preparation of Eight-Arm Polyethylene Glycol (20,000 Da)-Seven Alkynyl-Mono-Ethyl Amine MAL

(41) ##STR00033##

(42) 1.0 g of eight-arm polyethylene glycol (20,000 Da)-seven alkynyl-mono-ethyl amine was dissolved in 10 mL of methylene chloride, 0.017 g of 3-maleimidopropionic acid hydroxysuccinimide ester 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 (20,000 Da)-seven alkynyl-mono-ethyl amine MAL.

(43) 1H-NMR (DMSO) : 3.08 (s, CCH, 7H), 2.32 (t, CH.sub.2N, 2H), 7.0 (s, CH ring, 2H)

Example 13

Preparation of Four-Arm Polyethylene Glycol-Alkynyl (Molecular Weight of about 5,000) Gel and Drug Release Test In Vitro Thereof

(44) 0.25 g of four-arm polyethylene glycol-alkynyl (molecular weight of about 5,000), 0.25 g of polyethylene glycol-azido 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.

(45) 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.

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

Example 14

Preparation of Four-Arm Polyethylene Glycol-Alkynyl (Molecular Weight of about 10,000) Gel and Drug Release Test In Vitro Thereof

(47) 0.25 g of four-arm polyethylene glycol-alkynyl (molecular weight of about 10,000), 0.25 g of polyethylene glycol-azido 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.

(48) The above gel placed in a dialysis bag (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.

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

Example 15

Preparation of Eight-Arm Polyethylene Glycol-Alkynyl (Molecular Weight of about 10,000) Gel and Drug Release Test In Vitro Thereof

(50) 0.25 g of eight-arm polyethylene glycol-alkynyl (molecular weight of about 10,000), 0.25 g of polyethylene glycol-azido 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 min to form a gel at room temperature.

(51) The above gel placed in a dialysis bag (having a 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.

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

(53) Examples 13-15 shows that, in the preparation of gel via a multi-arm polyethylene glycol-alkynyl 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-alkynyl derivatives can also be used to control the drug release sustaining process.

Example 16

Preparation of Four-Arm Polyethylene Glycol-Three Alkynyl-Dopamine and Gel Thereof

(54) ##STR00034##

(55) 3.0 g of four-arm polyethylene glycol-propynyl-mono-acetate NHS active ester (molecular weight of 5,000) was dissolved in 30 mL of methylene chloride, 0.12 g of dopamine and 0.11 mL of triethylamine were added, reaction was allowed to proceed overnight protected from light, concentrated to dryness at 40 C., 60 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-three-propynyl-dopamine.

(56) 1H-NMR (DMSO) : 3.08 (s, CCH, 3H), 6.4 (m, ring, 1H), 6.6 (m, ring, 2H), 8.6 (s, OH, 1H), 8.7 (s, OH, 1H)

(57) 0.33 g of four-arm polyethylene glycol-three-propynyl-dopamine (having a molecular weight of about 5,000), 0.25 g of polyethylene glycol-azido derivatives (having a 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.