Conjugate of polyethylene glycol and anesthetic, and preparation method thereof

Abstract

A conjugate represented by general formula (I), wherein R.sub.0 is a C.sub.1-6 alkyl, B is an anesthetic, and A is a linking group, and a quaternary ammonium salt is formed at the linking position between B and R.sub.0. The conjugate has a prolonged analgesic effect, and can be used in postoperative analgesia or treatment for chronic pain.

Claims

1. A conjugate of polyethylene glycol and anesthetic having the structure of formula (I):
PEG-A-R.sub.0-B (I) wherein, PEG is polyethylene glycol residue with a molecular weight of 1-100 KDa; R.sub.0 is a C.sub.1-6 alkyl; B is an anesthetic; a quaternary ammonium salt with a structure of ##STR00092## is formed at the linking position between B and R.sub.0, R.sup.s0- is selected from the group consisting of F, Cl, Br, I, methanesulfonate, ethanesulfonate, benzenesulfonate, citrate, lactate, succinate, fumarate, glutamate, citrate, salicylate, and maleate; and, A is a linking group selected from the group consisting of the structures shown by the following formula A.sub.1 or A.sub.2: ##STR00093## wherein, R.sub.1 and R.sub.6 are independently selected from C.sub.1-6 alkyl, or R.sub.1 and R.sub.6 are independently selected from (CH.sub.2).sub.iNHCO(CH.sub.2).sub.j, and (CH.sub.2).sub.iCONH(CH.sub.2).sub.j, R.sub.2 is selected from the group consisting of CO, CS, O or S; R.sub.3 and R.sub.7 are independently selected from the group consisting of O or S; R.sub.4 or R.sub.5 is independently selected from the group consisting of H, C.sub.1-6 alkyl or halogen; R.sub.8 or R.sub.9 is independently selected from the group consisting of H, C.sub.1-6 alkyl or O(CO)(CH.sub.2).sub.iCH.sub.3; i and j are integer independently selected from 0 to 6; and, R.sub.20 is ##STR00094## R.sup.s1 is selected from the group consisting of H or C.sub.1-6 alkyl.

2. The conjugate of claim 1, wherein the R.sup.s0- is selected from the group consisting of F, Cl, Br, I, methanesulfonate, ethanesulfonate, and benzenesulfonate; and/or, the R.sub.1 and R.sub.6 are independently selected from the group consisting of methyl, ethyl, or propyl; and/or, the R.sub.4 or R.sub.5 is independently selected from the group consisting of H, methyl or ethyl; and/or, the R.sub.8 or R.sub.9 is independently selected from the group consisting of H, methyl, ethyl, propyl, acetoxy, propionyloxy, and butyryloxy; and/or, the i and j are integer independently selected from 1, 2, or 3; and/or, the R.sup.s1 is selected from the group consisting of H, methyl, ethyl or propyl.

3. The conjugate of claim 1, wherein the PEG has a molecular weight of 10 to 40 KDa.

4. The conjugate of claim 1, wherein the PEG is a linear, double-ended, Y-type, 4-arm, 6-arm or 8-arm polyethylene glycol residue.

5. The conjugate of claim 4, wherein the PEG is a linear polyethylene glycol residue having the structure of formula (II-1): ##STR00095## wherein m is an integer of 20-2000.

6. The conjugate of claim 5, wherein m is an integer of 200-1000.

7. The conjugate of claim 4, wherein the PEG is a 4-arm or 8-arm polyethylene glycol residue having the structures of formula (II-3) to formula (II-5): ##STR00096## wherein, q is an integer selected from 5 to 500; s, t, u, v, x, and y are integer independently selected from 2 to 250.

8. The conjugate of claim 7, wherein q is an integer selected from 50 to 250; and/or, s, t, u, v, x, and y are integer independently selected from 25 to 125.

9. The conjugate of claim 1, wherein the linking group A is selected from the group consisting of the structures shown in the following formulas A.sub.3-A.sub.6: ##STR00097## wherein, R.sub.1 is methyl, ethyl, propyl or (CH.sub.2).sub.iNHCO(CH.sub.2).sub.j, (CH.sub.2).sub.iCONH(CH.sub.2).sub.j; R.sub.2 is CO, O or S; R.sub.3 is O or S; R.sub.8 is acetoxy, propionyloxy or butyryloxy; R.sub.9 is H or methyl; R.sub.20 is ##STR00098## i and j are independently selected from 1, 2 or 3; and, P is 1, 2 or 3.

10. The conjugate of claim 1, wherein the anesthetic is a local anesthetic of amides having the structure of formula (III): ##STR00099## wherein, R.sub.10 and R.sub.11 are independently selected from the group consisting of H and C.sub.1-6 alkyl; and, R.sub.12, R.sub.13 and R.sub.14 are independently selected from the group consisting of H and C.sub.1-6 alkyl; or R.sub.13 is selected from the group consisting of H and C.sub.1-6 alkyl, N together with R.sub.12 and R.sub.14 forms a 5- to 8-membered ring.

11. The conjugate of claim 10, wherein R.sub.10 and R.sub.11 are independently selected from the group consisting of H, methyl, ethyl or propyl; and, R.sub.12, R.sub.13 and R.sub.14 are independently selected from the group consisting of H, methyl, ethyl, propyl or butyl; or R.sub.13 is selected from the group consisting of H and C.sub.1-6 alkyl, N together with R.sub.12 and R.sub.14 forms a 6-membered ring.

12. The conjugate of claim 10, wherein the anesthetic is lidocaine, prilocaine, bupivacaine, ropivacaine, mepivacaine or etidocaine.

13. The conjugate of claim 1, wherein the conjugate is selected from the group consisting of the structures shown in the formula (IV) to formula (X): ##STR00100##

14. The conjugate of claim 13, wherein the PEG has a molecular weight of 10 to 40 KDa.

15. The conjugate of claim 14, wherein the PEG is a linear, double-ended, Y-type or multi-branched polyethylene glycol residue.

16. The conjugate of claim 11, wherein the conjugate is selected from the group consisting of the structures shown below: ##STR00101## ##STR00102##

17. The conjugate of claim 1, wherein the conjugate is selected from the group consisting of the structures shown in the formula (XI) to formula (XXXI): ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109## wherein, a quaternary ammonium salt with a structure of ##STR00110## is formed at the linking position between B and CH.sub.2, R.sup.s0- is selected from the group consisting of F, Cl, Br, or I, q is an integer selected from 5 to 500, and s, t, u, v, x, and y are integer independently selected from 2 to 250.

18. A method for analgesic or the treatment of chronic pain, comprising administering an effective amount of the conjugate of claim 1.

19. A preparation method of the conjugate of claim 1 comprising the following steps: (1) obtaining a linking group with one end group of alkynyl or azido at one end and one halogenated or sulfonated end group at one end with a structure represented by the following formula M.sub.1 or M.sub.2; (2) reacting the linking group obtained in step (1) with an anesthetic to form a quaternary ammonium salt; (3) reacting the alkynyl or azido of the quaternary ammonium salt obtained in step (2) with PEG modified with end group modified by azido or alkynyl to form the structure; ##STR00111## wherein: PEG is polyethylene glycol residue with a molecular weight of 1-100 KDa; n is an integer of 1-6, preferably 1, 2 or 3; R.sub.21 is selected from alkynyl or azido; R.sup.s0- is selected from the group consisting of F, Cl, Br, I, methanesulfonate, ethanesulfonate, benzenesulfonate, citrate, lactate, succinate, fumarate, glutamate, citrate, salicylate, and maleate, preferably, R.sup.s0- is selected from the group consisting of F, Cl, Br, I, methanesulfonate, ethanesulfonate, and benzenesulfonate; wherein, R.sub.1 and R.sub.6 are independently selected from C.sub.1-6 alkyl, preferably methyl, ethyl, propyl, or R.sub.1 and R.sub.6 are independently selected from (CH.sub.2).sub.iNHCO(CH.sub.2).sub.j, and (CH.sub.2).sub.iCONH(CH.sub.2).sub.j, i and j are integer independently selected from 0 to 6, preferably 1, 2, or 3; R.sub.2 is selected from the group consisting of CO, CS, O or S; R.sub.3 and R.sub.7 are independently selected from the group consisting of O or S; R.sub.4 or R.sub.5 is independently selected from the group consisting of H, C.sub.1-6 alkyl or halogen, preferably H, methyl or ethyl; R.sub.8 or R.sub.9 is independently selected from the group consisting of H, C.sub.1-6 alkyl or O(CO)(CH.sub.2).sub.iCH.sub.3, i is an integer of 0-6, preferably H, methyl, ethyl, propyl, acetoxy, propionyloxy, or butyryloxy; and, the H atom of the alkynyl end group may be substituted with R.sup.s1, R.sup.s1 is selected from the group consisting of H or C.sub.1-6 alkyl, preferably H, methyl, ethyl or propyl.

20. The preparation method of claim 19, wherein the linking group in step (1) is selected from the group consisting of the structures shown in M.sub.3-M.sub.6 below: ##STR00112## wherein: R.sub.21 is selected from the group consisting of alkynyl or azido; R.sup.s0- is selected from the group consisting of F, Cl, Br, and I; R.sub.1 is methyl, ethyl, propyl or (CH.sub.2).sub.iNHCO(CH.sub.2).sub.j, (CH.sub.2).sub.iCONH(CH.sub.2).sub.j, i and j are independently selected from 1, 2 or 3; R.sub.2 is CO, O or S; R.sub.3 is O or S; R.sub.8 is acetoxy, propionyloxy or butyryloxy; R.sub.9 is H or methyl; and, P is 1, 2 or 3.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The following examples are provided to illustrate the present invention, but are not intended to limit the present invention.

(2) The polyethylene glycol used in the examples is provided by Beijing JenKem Technology Co., Ltd., unless specified, the molecular weight is 20K. Other reagents are commercially available.

Example 1 Synthesis of Linking Chain (L1)

(3) ##STR00076##

(4) 4-pentynoic acid (2.0 g, 20 mmol) and N, N-dicyclohexylcarbodiimide (DCC, 5.2 g, 2.5 mmol) were added to dichloromethane (50 mL), cooled in an ice-water bath, then p-hydroxybenzaldehyde (2.68 g, 22 mmol) was added, the ice-water bath was removed after completion of addition, and the resulting mixture was reacted at room temperature overnight, and filtered, the filter cake was washed with ethyl acetate and the filtrate was evaporated to dryness to obtain a crude product, which was purified by column chromatography to give 3.5 g of product 1a.

(5) The compound 1a (3.23 g, 16 mmol) was added to anhydrous methanol (35 mL), cooled to 0 C., then sodium borohydride (365 mg, 9.6 mmol) was added, the reaction was carried out at the same temperature for 10 min and then quenched with 1 M HCl, the resulting mixture was subjected to rotary evaporation to remove methanol, and further added with ethyl acetate and saturated brine, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried, filtered and concentrated to give a crude product, which was purified by column chromatography to give 1.8 g of product 1b.

(6) Iodine (3.35 g, 13.2 mmol), triphenylphosphine (3.46 g, 13.2 mmol) and imidazole (0.9 g, 13.2 mmol) were added to dichloromethane (30 mL), stirred at 0 C. for 20 min, a solution of product 1b (1.8 g, 8.8 mmol) in dichloromethane (7.5 mL) was added, and the resulting mixture was reacted at 0 C. for 30 min, and then washed with 2N hydrochloric acid, saturated sodium bisulfite solution and brine, respectively, dried and evaporated to dryness to obtain a crude product, which was purified by column chromatography to give 2.4 g of product L1. 1H NMR: (CDCl3): 2.03 (s, 1H), 2.61 (t, 2H), 2.77 (t, 2H), 4.44 (s, 2H), 7.01 (d, 2H), 7.39 (d, 2H).

Example 2 Synthesis of Quaternary Ammonium Salt of Lidocaine (Y1)

(7) ##STR00077##

(8) Lidocaine (0.50 g, 2.13 mmol) and compound L1 (1.0 g, 3.19 mmol) were added to acetonitrile (20 mL) and reacted at 50 C. overnight. TLC monitoring showed that the lidocaine was reacted completely, and the reaction liquid was concentrated to give a crude product, which was purified by column chromatography to give 1.2 g of product Y1. 1H-NMR: (CDCl3): 1.59 (m, 6H), 2.06 (s, 2H), 2.31 (s, 6H), 2.64 (m, 2H), 2.87 (m, 2H), 3.49 (m, 2H), 3.71 (m, 2H), 4.89 (s, 2H), 4.93 (s, 2H), 7.06 (m, 3H), 7.28 (d, 2H), 7.62 (d, 2H), 9.79 (s, 1H).

Example 3 Synthesis of Conjugate 1 of Lidocaine (mPEG-Lidocaine, 20 K)

(9) ##STR00078##

(10) mPEG-N3 (20 K, 2 g, 0.1 mmol), compound Y1 (65.8 mg, 0.12 mmol), vitamin C (52.8 mg, 3 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (30 mg, 0.12 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.9 g of product. 1H NMR: (CDCl3): 1.42 (m, 6H), 2.19 (s, 6H), 3.02 (m, 4H), 3.23 (m, 4H), 3.31 (s, 3H), 3.50 (m, 1800H), 3.80 (m, 2H) (m, 2H), 4.20 (m, 2H), 4.50 (s, 2H), 4.82 (s, 2H), 7.12 (m, 3H), 7.30 (d, 2H), 7.64 (d, 2H) s, 1H), 10.28 (s, 1H).

Example 4 Synthesis of Conjugate 2 of Lidocaine (4-Arm-PEG-Lidocaine, 10 K)

(11) ##STR00079##

(12) 4-arm-PEG-N3 (10 K, 2 g, 0.2 mmol), compound Y1 (548.5 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.8 g of product. 1H NMR: (CDCl3): 1.43 (m, 24H), 2.22 (s, 24H), 3.07 (m, 16H), 3.25 (m, 16H), 3.34 (s, 12H), 3.50 (m, 900H), 3.83 (m, 8H), 4.22 (m, 8H), 4.53 (s, 8H), 4.85 (s, 8H), 7.13 (m, 12H), 7.30 (d, 8H), 7.65 (d, 8H), 7.92 (s, 4H), 10.26 (s, 4H).

Example 5 Synthesis of Conjugate 3 of Lidocaine (8-Arm-PEG-Lidocaine, 20 K)

(13) ##STR00080##

(14) 8-arm-PEG-N3 (20 K, 2 g, 0.1 mmol), compound Y1 (548.5 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.6 g of product. 1H NMR: (CDCl3): 1.44 (M, 48H), 2.22 (s, 48H), 3.09 (m, 32H), 3.23 (m, 32H), 3.37 (s, 24H), 3.51 (m, 1800H), 3.84 (m, 16H) (m, 16H), 4.23 (m, 16H), 4.54 (s, 16H), 4.86 (s, 16H), 7.12 (m, 24H), 7.31 (d, 16H), 7.66 (d, 16H), 7.93 (s, 8H), 10.27 (s, 8H).

Example 6 Synthesis of Quaternary Ammonium Salt of Bupivacaine (Y2)

(15) ##STR00081##

(16) Bupivacaine (0.50 g, 1.74 mmol) and compound L1 (1.0 g, 3.19 mmol) were added to acetonitrile (20 mL) and reacted at50 C. overnight. TLC monitoring showed that the bupivacaine was reacted completely, and the reaction liquid was concentrated to give a crude product, which was purified by column chromatography to give 1.1 g of product Y2. 1H NMR: (CDCl3): 1.09 (m, 3H), 1.46 (m, 3H), 1.85 (m, 4H), 2.19 (m, 1H), 2.30 (s, 6H), 2.61 (m, 2H), 2.76 (m, 2H), 2.93 (s, 1H), 3.30 (m, 2H), 3.39 (m, 2H), 3.71 (m, 2H), 4.91 (s, 2H), 4.96 (s, 2H), 7.18 (m, 3H), 7.26 (d, 2H), 7.42 (d, 2H), 10.15 (s, 1H).

Example 7 Synthesis of Conjugate 4 of Bupivacaine (mPEG-Bupivacaine, 20 K)

(17) ##STR00082##

(18) mPEG-N3 (20 K, 2 g, 0.1 mmol), compound Y2 (89.7 mg, 0.12 mmol), vitamin C (52.8 mg, 3 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (30 mg, 0.12 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.7 g of product. 1H NMR: (CDCl3): 1.09 (m, 3H), 1.25 (m, 1H), 1.37 (m, 3H), 1.75 (m, 4H), 1.97 (m, 1H), 2.16 (s, 6H), 2.23 (m, 1H), 2.57 (m, 2H), 2.85 (m, 2H), 3.27 (m, 4H), 3.32 (s, 3H), 3.56 (m, 1800H), 3.95 (m, 2H), 4.10 (m, 2H), 4.52 (s, 2H), 4.60 (s, 2H), 7.19 (m, 3H), 7.27 (d, 2H), 7.30 (d, 2H), 7.69 (s, 1H), 10.19 (s, 1H).

Example 8 Synthesis of Conjugate 5 of Bupivacaine (4-Arm-PEG-Bupivacaine, 10 K)

(19) ##STR00083##

(20) 4-arm-PEG-N3 (10 K, 2 g, 0.2 mmol), compound Y2 (747.7 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.8 g of product. 1H NMR: (CDCl3): 1.08 (m, 12H), 1.26 (m, 4H), 1.38 (m, 12H), 1.74 (m, 8H), 1.98 (m, 4H), 2.17 (s, 24H), 2.25 (m, 4H), 2.56 (m, 8H), 2.83 (m, 8H), 3.27 (m, 16H), 3.31 (s, 12H), 3.54 (m, 900H), 3.95 (m, 8H), 4.07 (m, 8H), 4.50 (s, 8H), 4.58 (s, 8H), 7.21 (m, 12H), 7.29 (d, 8H), 7.33 (d, 8H), 7.65 (s, 4H), 10.15 (s, 4H).

Example 9 Synthesis of Conjugate 6 of Bupivacaine (8-Arm-PEG-Bupivacaine, 20K)

(21) ##STR00084##

(22) 8-arm-PEG-N3 (20 K, 2 g, 0.1 mmol), compound Y2 (747.7 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.6 g of product. 1H NMR: (CDCl3): 1H NMR: (CDCl3): 1.09 (m, 24H), 1.26 (m, 8H), 1.39 (m, 24H), 1.73 (m, 16H), 1.97 (m, 8H), 2.17 (s, 48H), 2.24 (m, 8H), 2.57 (m, 16H), 2.81 (m, 16H), 3.27 (m, 32H), 3.32 (s, 24H), 3.51 (m, 1800H), 3.93 (m, 16H), 4.09 (m, 16H), 4.51 (s, 16H), 4.57 (s, 16H), 7.20 (m, 24H), 7.27 (d, 16H), 7.32 (d, 16H), 7.63 (s, 8H), 10.16 (s, 8H).

Example 10 Synthesis of Linking Chain (L2)

(23) ##STR00085##

(24) 3,4-dihydroxybenzaldehyde (25 g, 181 mmol) was added to N, N-dimethylformamide (180 mL), cooled to 0 C., sodium hydride (7.2 g, 181 mmol) was added in batches, after completion of addition, the resulting mixture was reacted for 10 minutes, then heated to room temperature, acetic anhydride (18.7 g, 183 mmol) was added, and after completion of addition, the resulting mixture was reacted for 4 hours. Ice water and 10% hydrochloric acid were added to the reaction liquid, and the resulting mixture was extracted with ethyl acetate, the organic phases were combined, washed with saturated sodium bicarbonate solution, water and saturated brine, respectively, and dried. The resulting solution was concentrated, and then the resulting residue was crystallized with chloroform to give 19.8 g of product 2a.

(25) 3-hydroxy-4-acetoxybenzaldehyde (3.7 g, 30 mmol) and sodium carbonate (12.4 g, 90 mmol) were added to N, N-dimethylformamide (50 mL), the resulting mixture was heated to 60 C. and reacted for half of an hour and then cooled to room temperature, a solution of propargyl bromide in toluene (80%, 4.1 mL) was added, after the completion of addition, the resulting mixture was continued to react at the same temperature, after completion of the reaction, the mixture was poured into ice water and the resulting solution was extracted with diethyl ether, the organic phases were combined, washed with water, dried and concentrated to give 5.3 g of product 2b.

(26) The compound 2b (4.4 g, 20 mmol) was added to anhydrous methanol (50 mL), and cooled to 0 C., then sodium borohydride (456 mg, 12 mmol) was added, the reaction was carried out at the same temperature for 10 min and then quenched with 1 M HCl, the resulting mixture was subjected to rotary evaporation to remove methanol, and further added with ethyl acetate and saturated brine, the aqueous phase was extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried, filtered and concentrated to give a crude product, which was purified by column chromatography to give 3.3 g of product 2c.

(27) Iodine (3.8 g, 15 mmol), triphenylphosphine (3.9 g, 15 mmol) and imidazole (1.0 g, 15 mmol) were added to dichloromethane (30 mL) and stirred at 0 C. for 20 min, a solution of product 2c (2.2 g, 10 mmol) in dichloromethane (10 mL) was added, the resulting mixture was reacted at 0 C. for 30 min, and then washed with 2N hydrochloric acid, saturated sodium bisulfite solution and brine, respectively, dried and evaporated to dryness to obtain a crude product, which was purified by column chromatography to give 2.4 g of product L2. 1H NMR: (CDCl3): 2.35 (s, 3H), 3.37 (s, 1H), 4.51 (s, 2H), 4.72 (s, 2H), 6.77 (d, 1H), 7.02 (s, 1H), 7.09 (d, 1H).

Example 11 Synthesis of Quaternary Ammonium Salt of Lidocaine (Y3)

(28) ##STR00086##

(29) Lidocaine (0.5 g, 2.1 mmol) and compound L2 (1.8 g, 3.2 mmol) were added to acetonitrile (20 mL), and reacted at 50 C. overnight. TLC monitoring showed that the lidocaine was reacted completely, and the reaction liquid was concentrated to give a crude product, which was purified by column chromatography to give 1.4 g of product Y3. 1H NMR: (CDCl3): 1.05 (m, 6H), 2.13 (s, 2H), 2.30 (s, 3H), 2.42 (m, 4H), 3.31 (s, 2H), 3.35 (s, 1H), 3.63 (m, 2H), 4.71 (s, 2H), 6.92 (d, 1H), 7.08 (d, 1H), 7.12 (s, 1H), 7.19 (m, 3H), 9.82 (s, 1H).

Example 12 Synthesis of Conjugate 7 of Lidocaine (4-Arm-PEG-Lidocaine, 10K)

(30) ##STR00087##

(31) 4-arm-PEG-N3 (10 K, 2 g, 0.2 mmol), compound Y3 (564.5 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.8 g of product. 1H NMR: (CDCl3): 1.05 (m, 24H), 2.13 (s, 24H), 2.30 (s, 12H), 2.42 (m, 16H), 3.50 (s, 8H), 3.53 (m, 900H), 3.86 (m, 8H), 3.91 (m, 8H), 4.55 (s, 8H), 4.81 (s, 8H), 5.23 (s, 8H), 6.91 (d, 4H), 7.06 (s, 4H), 7.20 (m, 12H), 7.68 (s, 4H), 10.26 (s, 4H).

Example 13 Synthesis of Conjugate 8 of Lidocaine (8-Arm-PEG-Lidocaine, 20K)

(32) ##STR00088##

(33) 8-arm-PEG-N3 (20 K, 2 g, 0.1 mmol), compound Y3 (618.5 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.7 g of product. 1H NMR: (CDCl3): 1H NMR: (CDCl3): 1.07 (m, 48H), 2.14 (s, 48H), 2.31 (s, 24H), 2.41 (m, 32H), 3.52 (s, 16H), 3.54 (m, 1800H), 3.79 (m, 16H), 3.92 (m, 16H), 4.53 (s, 16H), 4.82 (s, 16H), 5.22 (s, 16H), 6.90 (d, 8H), 7.09 (s, 8H), 7.28 (m, 24H), 7.69 (s, 8H), 10.27 (s, 8H).

Example 14 Synthesis of Quaternary Ammonium Salt of Bupivacaine (Y4)

(34) ##STR00089##

(35) Bupivacaine (0.5 g, 1.7 mmol) and compound L2 (1.8 g, 3.2 mmol) were added to acetonitrile (20 mL) and reacted at 50 C. overnight. TLC monitoring showed that bupivacaine was reacted completely, and the reaction liquid was concentrated to give a crude product, which was purified by column chromatography to give 1.5 g of product Y4. 1H NMR: (CDCl3): 1.09 (m, 3H), 1.25 (m, 1H), 1.33 (m, 3H), 1.72 (m, 3H), 1.95 (m, 1H), 2.13 (s, 6H), 2.20 (m, 1H), 2.30 (s, 6H), 3.20 (m, 1H), 3.25 (m, 2H), 3.29 (m, 1H), 3.34 (s, 1H), 4.57 (s, 2H), 4.69 (s, 2H), 4.75 (s, 2H), 6.90 (d, 1H), 7.05 (d, 1H), 7.09 (d, 1H), 7.18 (m, 3H), 10.07 (s, 1H).

Example 15 Synthesis of Conjugate 9 of Bupivacaine (4-Arm-PEG-Bupivacaine, 10 K)

(36) ##STR00090##

(37) 4-arm-PEG-N3 (10 K, 2 g, 0.2 mmol), compound Y4 (747.7 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution (4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.8 g of product. 1H NMR: (CDCl3): 1.09 (m, 12H), 1.25 (m, 4H), 1.33 (m, 12H), 1.72 (m, 12H), 1.95 (m, 4H), 2.13 (s, 24H), 2.20 (m, 4H), 2.30 (s, 24H), 3.20 (m, 4H), 3.25 (m, 8H), 3.28 (m, 4H), 3.50 (s, 8H), 3.54 (m, 900H), 3.86 (m, 8H), 3.91 (m, 8H), 4.52 (s, 8H), 4.59 (s, 8H), 5.23 (s, 8H), 6.82 (d, 4H), 7.03 (d, 4H), 7.09 (s, 4H), 7.18 (m, 12H), 7.65 (s, 4H), 10.13 (s, 4H).

Example 16 Synthesis of Conjugate 10 of Bupivacaine (8-Arm-PEG-Bupivacaine, 20K)

(38) ##STR00091##

(39) 8-arm-PEG-N3 (20 K, 2 g, 0.1 mmol), compound Y2 (747.7 mg, 1 mmol), vitamin C (440 mg, 2.5 mmol) were added to N, N-dimethylformamide (20 mL), the resulting mixture was rapidly stirred to dissolve, then added with an aqueous solution 4.4 mL, 2.2 mL/g PEG) of copper sulfate pentahydrate (250 mg, 1 mmol), the resulting mixture was reacted at room temperature overnight and precipitated with isopropanol to give 1.6 g of product. 1H NMR: (CDCl3): 1H NMR: (CDCl3): 1.08 (m, 24H), 1.24 (m, 8H), 1.34 (m, 24H), 1.73 (m, 24H), 1.93 (m, 8H), 2.14 (s, 48H), 2.21 (m, 8H), 2.31 (s, 48H), 3.21 (m, 8H), 3.25 (m, 16H), 3.28 (m, 8H), 3.50 (s, 16H), 3.53 (m, 1800H), 3.85 (m, 16H), 3.92 (m, 16H), 4.53 (s, 16H), 4.59 (s, 16H), 5.22 (s, 16H), 6.81 (d, 8H), 7.05 (d, 8H), 7.09 (s, 8H), 7.19 (m, 24H), 7.67 (s, 8H), 10.12 (s, 8H).

Example 17 Study on the Efficacy of Compounds

(40) 1. Material

(41) 60 SD rats, male, with weight of 260-280 grams; lidocaine hydrochloride injection; bupivacaine hydrochloride injection; four-arm and eight-arm lidocaine and bupivacaine (2, 3, 5, and 6) injections.

(42) 2. Method

(43) 2.1. Grouping and Modeling

(44) SD rats with normal pain threshold were randomly divided into 9 groups, with 6 rats in each group. The pain model was established with reference to the Brennan method, and the specific method was: fasting for 6 hours, fasting water for 1 hour before surgery, determining pain threshold by Von Frey method, then placing the rats into a closed anesthesia box, carrying out induced anesthesia with 1.5-2% isoflurane, when the animal consciousness disappeared, taking the animals out, disinfecting their right hind foot with iodophor, making an incision with a length of about 1 cm starting about 5 mm from proximal end of plantar and extending toward the toes; cutting the skin and fascia, elevating the plantar muscles with an ophthalmic forceps and longitudinally cutting (maintaining the starting and ending and attachment of the muscles intact); pressing to stop bleeding, before stitching the skin, injecting the animals in each group with 0.5 ml normal saline, 4 mg/kg bupivacaine hydrochloride, 10 mg/kg lidocaine hydrochloride injection, 4 mg/kg polyethylene glycol bupivacaine (counted by bupivacaine), and 10 mg/kg polyethylene glycol lidocaine (counted by lidocaine) in the incision, respectively; after the completion of administration, stitching the incision with 2-0 thin silk for 2 stitches. During the entire surgery, the rats were anesthetized with the same concentration of isoflurane through a mask.

(45) 2.2 Von Frey Test and Result Statistics

(46) After the animals were awake and their state returned to normal (about 1 hour after surgery), the pain threshold was measured with Von Frey method. Then the pain thresholds of each group of animals were measured at the 3.sup.rd, 6.sup.th, 12.sup.th, 24.sup.th, 48.sup.th and 72.sup.nd hours after surgery, respectively. When the pain threshold was measured, the surgical foot of each model rat was stimulated by a filament with gradually increased force, and the paw withdrawal threshold (g) of animals was recorded, the measurement was repeated 2-3 times and the mean value was taken. The test results were analyzed by SPSS17.0. The number of the result was expressed in xs. The comparison between groups was analyzed by one-way analysis of variance and P<0.05 meant that the differences were statistically significant. The experimental results are shown in Table 1 below.

(47) TABLE-US-00001 TABLE 1 Von Frey Hairs (g) Groups Basline 1 h 3 h 6 h 12 h 24 h 48 h 72 h Sham-operated group 7.4 0.7 7.5 0.6 7.3 0.7 7.8 0.9 7.5 0.8 7.2 0.9 7.4 0.6 7.5 0.7 Vehicle group 7.4 0.8 0.6 0.2 0.7 0.4 1.1 0.3 1.1 0.2 1.1 0.5 2.0 0.4 2.9 0.6 Lidocaine hydrochloride 7.5 0.8 6.6 0.5 6.1 0.4 4.0 0.6 1.5 0.4 1.4 0.2 Bupivacaine 7.3 0.7 4.3 0.7 6.6 0.4 5.3 0.5 3.4 0.9 1.7 0.4 hydrochloride 2 (4a-lidocaine) 7.6 0.7 4.8 0.5 5.3 0.6 4.1 0.7 4.3 0.4 2.7 0.3 2.0 0.5 2.4 0.3 3 (8a-lidocaine) 7.5 0.7 4.2 0.3 4.3 0.6 3.8 0.5 3.2 0.4 2.2 0.2 1.8 0.4 3.1 0.6 5 (4a-bupivacaine) 7.3 0.8 4.9 0.3 5.8 0.4 6.2 0.5 6.1 0.4 6.7 0.8 5.4 0.5 5.7 0.4 6 (8a-bupivacaine) 7.4 0.7 3.6 0.6 4.6 0.5 4.9 0.7 5.3 0.6 4.8 0.6 4.2 0.4 3.9 0.2

(48) 2.3 Discussion of the Results

(49) From the results, it can be seen that bupivacaine hydrochloride takes effect more slowly than lidocaine hydrochloride, but has a relatively long duration, and after PEGylation, the two drugs have significantly prolonged duration, which have significant analgesic effect in 48-72 hours after surgery. Relatively speaking, the four-arm type drug has better analgesic intensity and duration of analgesia than that of eight-arm type drug.