Multi-arm single molecular weight polyethylene glycol, active derivative thereof, and preparation and application thereof

Abstract

Being used for drug modification, the multi-arm single molecular weight polyethylene glycol and an active derivative thereof provided herein can effectively improve the solubility, stability, and immunogenicity of the drugs, improve the absorption of the drugs in vivo, prolong the half-life of the drugs, and increase bioavailability, enhance efficacy, and reduce toxic and side effects of the drugs. A gel formed from the active derivative of the multi-arm single molecular weight polyethylene glycol provided herein can be used for the preparation of controlled release drugs so as to prolong the action time of the drugs, thereby reducing the number of administrations and improving patient compliance.

Claims

1. A multi-arm single molecular weight polyethylene glycol having the following structure:
AX.sub.1—R—X.sub.2-PEG-H).sub.n  (IV) wherein A is a core structure, and is a polyol group selected from the group consisting of: residues of pentaerythritol, oligo-pentaerythritol, glycerol and oligoglycerol, and glyceryl ether groups thereof, X.sub.1 is a linking group selected from any one or a combination of two or more of the group consisting of: —(CH.sub.2).sub.i—, —(CH.sub.2).sub.iO—, —(CH.sub.2).sub.iNHCO—, —(CH.sub.2).sub.iCONH—, —(CH.sub.2).sub.iOCO—, and —(CH.sub.2).sub.iCOO—, and i is an integer from 1 to 10, R is a linking group selected from any one or a combination of two or more of the group consisting of: —NHCO—, —CONH—, —OCO—, —COO—, —O—, ##STR00054## X.sub.2 is a linking group selected from any one or a combination of two or more of the group consisting of: —(CH.sub.2).sub.j—, —(CH.sub.2).sub.jO—, —(CH.sub.2).sub.jCO—, —(CH.sub.2).sub.jNH—, —(CH.sub.2).sub.jNHCO—, —(CH.sub.2).sub.jCONH—, —(CH.sub.2).sub.jOCO— and —(CH.sub.2).sub.jCOO—, and j is an integer from 0 to 10, and X.sub.1—R—X.sub.2— is not —CH.sub.2CH.sub.2O—, PEG has the following structure: —(CH.sub.2CH.sub.2O).sub.m—, m is an integer from 4 to 200, and n is an integer from 3 to 24.

2. The multi-arm single molecular weight polyethylene glycol according to claim 1, wherein the A has the following structure: ##STR00055## wherein B has the following structure: ##STR00056## r is an integer from 1 to 5, a, b, c and d are integers and each independently selected from 0 and 1, s is an integer from 1 to 5, e, f and g are integers and each independently selected from 0 and 1.

3. The multi-arm single molecular weight polyethylene glycol according to claim 1, wherein the A is selected from the following structures: ##STR00057##

4. The multi-arm single molecular weight polyethylene glycol according to claim 1, wherein the X.sub.1 is selected from any one or a combination of two or more of the group consisting of: —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CONHCH.sub.2— and —CH.sub.2CONHCH.sub.2CH.sub.2—; and/or the R is selected from any one or a combination of two or more of the group consisting of: —NHCO—, —CONH—, ##STR00058## and/or the X.sub.2 is selected from any one or a combination of two or more of the group consisting of: a single bond, —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CONHCH.sub.2— and —CH.sub.2CONHCH.sub.2CH.sub.2—; and/or the m is an integer from 4 to 100.

5. The multi-arm single molecular weight polyethylene glycol according to claim 4, wherein the multi-arm single molecular weight polyethylene glycol is selected from the following structures: ##STR00059## ##STR00060##

6. A conjugate of the multi-arm single molecular weight polyethylene glycol according to claim 2 or the active derivative thereof and a drug molecule.

7. A pharmaceutical composition comprising the conjugate of claim 6 and a pharmaceutically acceptable additive.

8. A process for the preparation of a multi-arm single molecular weight polyethylene glycol according to claim 1, which comprises the step of reacting compound AX.sub.1—Y).sub.n with W—X.sub.2-PEG-PG to link them together, and the reaction formula is as follows:
AX.sub.1—Y).sub.n+W-X.sub.2-PEG-PG.fwdarw.AX.sub.1—R—X.sub.2-PEG-PG).sub.n wherein W is a terminal group selected from any one of the group consisting of: hydrogen, hydroxyl, carboxyl, ester group, ketone group, amino, mercapto group, maleimide group, alkynyl, and azido, and PG is a hydroxyl protecting group, A is a core structure, and is a polyol group selected from the group consisting of: residues of pentaerythritol, oligo-pentaerythritol, glycerol and oligoglycerol, and glyceryl ether groups thereof, X.sub.1 is a linking group selected from any one or a combination of two or more of the group consisting of: —(CH.sub.2).sub.i—, —(CH.sub.2).sub.iO—, —(CH.sub.2).sub.iNHCO—, —(CH.sub.2).sub.iCONH—, —(CH.sub.2).sub.iOCO—, and —(CH.sub.2).sub.iCOO—, and i is an integer from 1 to 10, Y is a terminal group selected from any one of the group consisting of: hydrogen, hydroxyl, carboxyl, ester group, ketone group, amino, mercapto group, maleimide group, alkynyl, and azido, and n is an integer from 3 to 24.

Description

SPECIFIC EMBODIMENTS

(1) In the present invention, the term “protecting group” means a substituent which is generally used to block or protect a specific functional group when other functional groups of the compound react. For example, “hydroxy protecting group” refers to a substituent attached to a hydroxyl group that blocks or protects a hydroxyl functional group. Commonly used hydroxyl protecting groups include acetyl, trialkylsilyl, and the like. For general description of protecting groups and their uses, see T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

(2) The term “pharmaceutically acceptable” means a drug is physiologically compatible after administration to a human, and does not cause gastrointestinal disorders, allergic reactions such as dizziness, or the likes.

(3) The term “prevention/preventing” or “treatment/treating” includes therapeutic or prophylactic treatment or measures with the goal of preventing or ameliorating a targeted pathological condition or disorder. The disease of a subject is successfully “prevented” or “treated” if, after receiving a therapeutic amount of the fusion protein of the invention according to the method of the invention, the subject exhibits an observable and/or measurable decrease or disappearance of one or more signs and symptoms of a particular disease.

(4) The technical solutions of the present invention will be described clearly and completely below accompanying with the Examples of the invention. It is obvious that the described embodiments are only part of the embodiments of the invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without creative efforts are within the scope of the present invention.

(5) The compounds used in the invention are either commercially available or can be prepared according to the disclosed preparation methods, and they do not limit the therapeutic range of the invention.

Example 1: Synthesis of Tetraacrylic Acid Substituted Pentaerythritol

(6) The tetraacrylic acid substituted pentaerythritol having the following structure (Ia) was synthesized:

(7) ##STR00046##

(8) Pentaerythritol (1 mol), potassium t-butoxide (0.01 mol), and DMF were added to a three-necked flask, and the mixture was stirred. Then, tert-butyl acrylate (5 mol) was added dropwise to the reaction solution to react at room temperature overnight. After the completion of the reaction, the mixture was filtered, then the reaction mixture was spin-dried, and a silica gel column was used to obtain a pure product of tert-butyl tetraacrylate substituted pentaerythritol.

(9) The pure product of tert-butyl tetraacrylate substituted pentaerythritol was dissolved in a dichloromethane solution containing 50% TFA, reacting at room temperature overnight. After the completion of the reaction, the reaction solution was spin-dried, and then acetonitrile was recrystallized to obtain a pure product of tetraacrylic acid substituted pentaerythritol.

(10) .sup.1H-NMR (DMSO-d.sub.6): 2.32-2.36 (t, 8H), 3.20 (s, 8H), 3.50-3.54 (t, 8H).

Example 2: Synthesis of Octaacrylic Acid Substituted Pentaerythritol Tetraglyceryl Ether

(11) The octaacrylic acid substituted pentaerythritol tetraglyceryl ether having the following structure (Ib) was synthesized:

(12) ##STR00047##

(13) Pentaerythritol tetraglyceryl ether (1 mol), potassium t-butoxide (0.01 mol), and DMF were added to a three-necked flask, and the mixture was stirred. Then, tert-butyl acrylate (10 mol) was added dropwise to the reaction solution to react at room temperature overnight. After the completion of the reaction, the mixture was filtered, then the reaction mixture was spin-dried, and a silica gel column was used to obtain a pure product of tert-butyl octaacrylate substituted pentaerythritol glycerol ether.

(14) The pure product of tert-butyl octaacrylate substituted pentaerythritol glyceryl ether was dissolved in a dichloromethane solution containing 50% TFA, reacting at room temperature overnight. After the completion of the reaction, the reaction solution was spin-dried, and then acetonitrile was recrystallized to obtain a pure product of octaacrylic acid substituted pentaerythritol glyceryl ether.

(15) .sup.1H-NMR (DMSO-d.sub.6): 2.39-2.45 (m, 16H), 3.29-3.40 (m, 24H), 3.51-3.55 (m, 4H), 3.57-3.61 (m, 8H), 3.67-3.72 (m, 8H); MALDI-TOF (1031.0, M+Na).

Example 3: Synthesis of 4ARM-(EG.SUB.24.-OH).SUB.4

(16) The 4ARM-(EG.sub.24-OH).sub.4 having following structure (IIa) was synthesized:

(17) ##STR00048##

(18) Tetraacrylic acid substituted pentaerythritol (Ia, 1 mol, prepared in Example 1) and NHS (4.4 mol) were added to a three-necked bottle, and then dissolved by stirring with DMF, and DCC (5.2 mol) was added to react at room temperature overnight to obtain a reaction solution 1; then NH.sub.2(CH.sub.2CH.sub.2O).sub.24Tr (4.8 mol) and triethylamine (5.2 mol) were dissolved in DMF to obtain a reaction solution 2. Finally, the reaction solution 1 was added dropwise to the reaction solution 2, and the reaction was terminated after reacting at room temperature for 24 hours. After the reaction, the mixture was filtered, and the filtrate was spin-dried, and then a silica gel column was used to obtain a pure product of 4ARM-(EG.sub.24-OTr).sub.4.

(19) The pure product of 4ARM-(EG.sub.24-OTr).sub.4 was dissolved in a dichloromethane solution containing 8% TFA, reacting at room temperature overnight. An appropriate amount of 0.1 g/ml sodium hydroxide solution was added under ice bath, reacting at room temperature for 3 hours. After the reaction, the pH of the reaction solution was adjusted to 6.5-7 with 1N hydrochloric acid, then an appropriate amount of sodium chloride was added, and the mixture was extracted twice with dichloromethane, and the dichloromethane phase was spin-dried; and then an appropriate amount of water was added to dissolve the spin dried substance of the dichloromethane phase. Then the mixture was filtered, and the filtrate was again collected and spin-dried to give a product of 4ARM-(EG.sub.24-OH).sub.4.

(20) .sup.1H-NMR (DMSO-d.sub.6): 2.26-2.30 (m, 8H), 3.17-3.21 (m, 16H), 3.35-3.64 (m, 384H), 4.58-4.62 (t, 4H), 7.90-7.94 (t, 4H);

(21) MALDI-TOF (4671.5, M+Na).

Example 4: Synthesis of 4ARM-(EG.SUB.12.-OH).SUB.4

(22) The 4ARM-(EG.sub.12-OH).sub.4 having the following structure (IIb) was synthesized:

(23) ##STR00049##

(24) Tetraacrylic acid substituted pentaerythritol (Ia, 1 mol, prepared in Example 1) and NHS (4.4 mol) were added to a three-necked bottle, and then dissolved by stirring with DMF, and DCC (5.2 mol) was added to react at room temperature overnight to obtain a reaction solution 1; then NH.sub.2(CH.sub.2CH.sub.2O).sub.12Tr (4.8 mol) and triethylamine (5.2 mol) were dissolved in DMF to obtain a reaction solution 2. Finally, the reaction solution 1 was added dropwise to the reaction solution 2, and the reaction was terminated after reacting at room temperature for 24 hours. After the reaction, the mixture was filtered, and the filtrate was spin-dried, and then a silica gel column was used to obtain a pure product of 4ARM-(EG.sub.12-OTr).sub.4.

(25) The pure product of 4ARM-(EG.sub.12-OTr).sub.4 was dissolved in a dichloromethane solution containing 8% TFA, reacting at room temperature overnight. An appropriate amount of 0.1 g/ml sodium hydroxide solution was added under ice bath, reacting at room temperature for 3 hours. After the reaction, the pH of the reaction solution was adjusted to 6.5-7 with 1N hydrochloric acid, then an appropriate amount of sodium chloride was added, and the mixture was extracted twice with dichloromethane, and the dichloromethane phase was spin-dried; and then an appropriate amount of water was added to dissolve the spin dried substance of the dichloromethane phase. Then the mixture was filtered, and the filtrate was again collected and spin-dried to give a product of 4ARM-(EG.sub.12-OH).sub.4.

(26) .sup.1H-NMR (DMSO-d.sub.6): 2.26-2.30 (m, 8H), 3.17-3.21 (m, 16H), 3.35-3.64 (m, 192H), 4.56-4.59 (t, 4H), 7.88-7.92 (t, 4H);

(27) MALDI-TOF (2557.6, M+Na).

Example 5: Synthesis of 8ARM-(EG.SUB.4.-OH).SUB.8

(28) The 8ARM-(EG.sub.4-OH).sub.8 having the following structure 8 (IIc) was synthesized:

(29) ##STR00050##

(30) Octaacrylic acid substituted pentaerythritol tetraglyceryl ether (Ib, 1 mol, prepared in Example 2) and NHS (8.8 mol) were added to a three-necked bottle, and then dissolved by stirring with DMF, and DCC (10.4 mol) was added to react at room temperature overnight to obtain a reaction solution 1; then NH.sub.2(CH.sub.2CH.sub.2O).sub.4Tr (9.6 mol) and triethylamine (10.4 mol) were dissolved in DMF to obtain a reaction solution 2. Finally, the reaction solution 1 was added dropwise to the reaction solution 2, and the reaction was terminated after reacting at room temperature for 24 hours. After the reaction, the mixture was filtered, and the filtrate was spin-dried, and then a silica gel column was used to obtain a pure product of 8ARM-(EG.sub.4-OTr).sub.4.

(31) The pure product of 8ARM-(EG.sub.4-OTr).sub.4 was dissolved in a dichloromethane solution containing 8% TFA, reacting at room temperature overnight. An appropriate amount of 0.1 g/ml sodium hydroxide solution was added under ice bath, reacting at room temperature for 3 hours. After the reaction, the pH of the reaction solution was adjusted to 6.5-7 with 1N hydrochloric acid, then an appropriate amount of sodium chloride was added, and the mixture was extracted twice with dichloromethane, and the dichloromethane phase was spin-dried; and then an appropriate amount of water was added to dissolve the spin dried substance of the dichloromethane phase. Then the mixture was filtered, and the filtrate was again collected and spin-dried to give a product of 8ARM-(EG.sub.4-OH).sub.4.

(32) .sup.1H-NMR (DMSO-d.sub.6): 2.27-2.33 (m, 16H), 3.16-3.20 (m, 16H), 3.28-3.44 (m, 24H), 3.48-3.50 (m, 116H), 3.55-3.60 (m, 8H), 3.66-3.70 (m, 8H), 4.56-4.59 (t, 8H), 7.87-7.90 (t, 8H);

(33) MALDI-TOF (2433.4, M+Na).

Example 6: Synthesis of Four-Arm Dodecaethylene Glycol Tetraglycidyl Ether (IIIa)

(34) The four-arm dodecaethylene glycol tetraglycidyl ether having the following structure (IIIa) was synthesized:

(35) ##STR00051##

(36) 4ARM-(EG.sub.12-OH).sub.4 (0.1 mol, prepared in Example 4), tetrahydrofuran (100 mL) and potassium hydroxide (0.8 mol) were added to a three-necked flask, stirring in a water bath; then epichlorohydrin (ECH, 1.2 mol) was added dropwise to the reaction system, and the reaction temperature was controlled to not exceed 35° C., reacting at room temperature overnight. After the reaction, the reaction solution was filtered, and the residue was washed with dichloromethane. Then the filtrate was collected, and a crude product was obtained by removing the dichloromethane through rotary evaporation. The crude product was purified by a silica gel column to obtain a pure product of dodecaethylene glycol tetraglycidyl ether.

(37) .sup.1H-NMR (DMSO-d.sub.6): 2.26-2.30 (m, 8H), 2.54-2.55 (m, 4H), 2.72-2.73 (m, 4H), 3.09-3.10 (m, 4H), 3.17-3.28 (m, 20H), 3.35-3.64 (m, 192H), 3.70-3.71 (m, 4H), 7.88-7.92 (t, 4H);

(38) MALDI-TOF (2780.3, M+Na).

Example 7: Synthesis of Four-Arm Tetracosethylene Glycol Tetraglycidyl Ether (IIIb)

(39) The four-arm tetracosethylene glycol tetraglycidyl ether having the following structure (IIIb) was synthesized:

(40) ##STR00052##

(41) 4ARM-(EG.sub.24-OH).sub.4 (0.1 mol, prepared in Example 3), tetrahydrofuran (100 mL) and potassium hydroxide (0.8 mol) were added to a three-necked flask, stirring in a water bath; then epichlorohydrin (ECH, 1.2 mol) was added dropwise to the reaction system, and the reaction temperature was controlled to not exceed 35° C., reacting at room temperature overnight. After the reaction, the reaction solution was filtered, and the residue was washed with dichloromethane. Then the filtrate was collected, and a crude product was obtained by removing the dichloromethane through rotary evaporation. The crude product was purified by a silica gel column to obtain a pure product of tetracosethylene glycol tetraglycidyl ether.

(42) .sup.1H-NMR (DMSO-d.sub.6): 2.26-2.30 (m, 8H), 2.54-2.55 (m, 4H), 2.72-2.73 (m, 4H), 3.09-3.10 (m, 4H), 3.17-3.28 (m, 20H), 3.35-3.64 (m, 384H), 3.70-3.71 (m, 4H), 7.88-7.92 (t, 4H);

(43) MALDI-TOF (4665.9, M+Na).

Example 8: Synthesis of Eight-Arm Tetraethylene Glycol Octaglycidyl Ether (IIIc)

(44) The eight-arm tetraethylene glycol octaglycidyl ether having the following structure (IIIc) was synthesized:

(45) ##STR00053##

(46) 8ARM-(EG.sub.4-OH).sub.8 (0.1 mol, prepared in Example 5), tetrahydrofuran (100 mL) and potassium hydroxide (1.6 mol) were added to a three-necked flask, stirring in a water bath; then epichlorohydrin (ECH, 2.4 mol) was added dropwise to the reaction system, and the reaction temperature was controlled to not exceed 35° C., reacting at room temperature overnight. After the reaction, the reaction solution was filtered, and the residue was washed with dichloromethane. Then the filtrate was collected, and a crude product was obtained by removing the dichloromethane through rotary evaporation. The crude product was purified by a silica gel column to obtain a pure product of eight-arm tetraethylene glycol octaglycidyl ether.

(47) .sup.1H-NMR (DMSO-d.sub.6): 2.27-2.33 (m, 16H), 2.54-2.55 (m, 8H), 2.72-2.73 (m, 8H), 3.09-3.10 (m, 8H), 3.16-3.26 (m, 24H), 3.28-3.44 (m, 24H), 3.48-3.50 (m, 116H), 3.55-3.60 (m, 8H), 3.66-3.71 (m, 16H), 7.87-7.90 (t, 8H);

(48) MALDI-TOF (2880.8, M+Na).

Example 9: Synthesis of Four-Arm Tetracosethylene Glycol-Monoacetic Acid (IIId) and Four-Arm Tetracosethylene Glycol-Diacetic Acid (IIIe)

(49) The four-arm tetracosethylene glycol-monoacetic acid having the following structure (IIId) was synthesized:
[H(OCH.sub.2CH.sub.2).sub.24NHCOCH.sub.2CH.sub.2OCH.sub.2].sub.3C[CH.sub.2OCH.sub.2CH.sub.2CONH(CH.sub.2CH.sub.2O).sub.24CH.sub.2COOH]  (IIId),

(50) and four-arm tetracosethylene glycol-diacetic acid having the following structure (IIIe) was synthesized:
[H(OCH.sub.2CH.sub.2).sub.24NHCOCH.sub.2CH.sub.2OCH.sub.2].sub.2C[CH.sub.2OCH.sub.2CH.sub.2CONH(CH.sub.2CH.sub.2O).sub.24CH.sub.2COOH].sub.2   (IIIe)

(51) 4ARM-(EG.sub.24-OH).sub.4 (prepared in Example 3) was taken to remove water with toluene, then distilling off the remaining toluene; tetrahydrofuran and potassium t-butoxide was added to react at room temperature for 2 hours, and t-butyl bromoacetate was added dropwise to react at room temperature overnight, filtering the mixture, and concentrating the filtrate through rotary evaporation. Then NaOH solution (1 mol/L) was added for alkaline hydrolysis at 80° C. for 2 hours, adjusting the pH to 2-3 with 2N hydrochloric acid, then adding NaCl and extracting three times with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate, the mixture was filtered, and the filtrate was concentrated through rotary evaporation. The crude product was separated by DEAE anion exchange resin column, and different fractions were respectively collected to obtain a fraction of four-arm tetracosethylene glycol-monoacetic acid and a fraction of four-arm tetracosethylene glycol-diacetic acid. The structures of the products were determined by .sup.1H-NMR.

(52) IIId: .sup.1H-NMR (DMSO-d.sub.6): 2.26-2.30 (m, 8H), 3.17-3.21 (m, 16H), 3.35-3.64 (m, 384H), 4.01 (s, 2H), 4.58-4.62 (t, 3H), 7.90-7.94 (t, 4H);

(53) MALDI-TOF (4729.4, M+Na).

(54) IIIe: .sup.1H-NMR (DMSO-d.sub.6): 2.27-2.30 (m, 8H), 3.17-3.20 (m, 16H), 3.36-3.64 (m, 384H), 4.02 (s, 4H), 4.59-4.62 (t, 2H), 7.92-7.94 (t, 4H);

(55) MALDI-TOF (4787.5, M+Na).

(56) The above are only the preferred examples of the invention, and are not intended to limit the present invention. Any modifications, equivalent substitutions, etc. within the spirit and principles of the invention, should be included in the scope of the invention.