WO2006099794A1 - Dérivés de la n-acétylcystéine et utilisation de ceux-ci - Google Patents

Dérivés de la n-acétylcystéine et utilisation de ceux-ci Download PDF

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WO2006099794A1
WO2006099794A1 PCT/CN2006/000422 CN2006000422W WO2006099794A1 WO 2006099794 A1 WO2006099794 A1 WO 2006099794A1 CN 2006000422 W CN2006000422 W CN 2006000422W WO 2006099794 A1 WO2006099794 A1 WO 2006099794A1
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peg
solution
mpeg
nac
acetyl cysteine
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Yonghai Yu
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Yonghai Yu
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C327/00—Thiocarboxylic acids
    • C07C327/20—Esters of monothiocarboxylic acids
    • C07C327/32—Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C07C327/34—Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by carboxyl groups with amino groups bound to the same hydrocarbon radicals
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/334—Polymers modified by chemical after-treatment with organic compounds containing sulfur
    • C08G65/3348—Polymers modified by chemical after-treatment with organic compounds containing sulfur containing nitrogen in addition to sulfur

Definitions

  • This invention relates to N-acetyl cysteine derivatives and their use. Background technique
  • Protein drugs such as insulin and interferon have the characteristics of specificity and high efficiency, and play an important role in the treatment of human diseases. With the completion of the Human Genome Project and the full development of proteomics research, more and more protein drugs will be used for disease treatment. However, protein drugs have poor stability, are prone to antigen-antibody reaction and protease degradation, have high renal elimination rate, high plasma clearance rate, and short half-life in vivo, thus limiting the clinical application of protein drugs.
  • PEGylation Polyethylene glycol
  • PEG Polyethylene glycol
  • the "PEGylation” technique is the attachment of polyethylene glycol molecules to the surface of protein molecules to protect proteins from the immune system and enzymes. Compared with proteins, the immunogenicity and antigenicity of PEG-protein conjugates are reduced, plasma clearance is reduced, and half-life in vivo is prolonged.
  • PEG polyethylene glycol
  • proteins such as insulin and enzymes to obtain a conjugate in which the protein is less immunogenic and retains most of the physiological activity
  • U.S. Patent 4,179,337 The binding of PEG to islet activating proteins to reduce their side effects and immunogenicity is disclosed in U.S. Patent No. 4,791,192 to Nakagawa et al. Abuchowski et al., "Enzymes as Drugs" (1981), describe that PEG-modified peptides can significantly reduce immunogenicity and antigenicity, prolong half-life in vivo, and modified polypeptides such as enzymes will be used in a variety of therapeutic areas. Play a role.
  • the modification should be highly specific to avoid the production of low or inactive PEG-protein conjugates; the modification step should be as simple as possible and the purification steps should be as small as possible.
  • Aldehyde is mainly used for the modification of the amino terminus of a polypeptide with high specificity, but usually requires a two-step reaction, ie, a modification reaction and a reduction reaction;
  • the ester group has high activity and mild reaction conditions, and is the most widely used active group.
  • the disadvantage is that although the amino group on the modified polypeptide is dominant, it can also modify the imidazole group and the tyrosine on the histidine. Hydroxyl and the like.
  • N-acetyl cysteine derivative provided by the present invention has the structure of formula I,
  • POLY is a polydecyl diol having a molecular weight ranging from 1000 Da to 500,000 Da, and a binary copolymer, a terpolymer, a mixed copolymer thereof, including polyethylene glycol, polypropylene glycol, and ethylene glycol propylene glycol.
  • Binary copolymer a polydecyl diol having a molecular weight ranging from 1000 Da to 500,000 Da
  • the structural formula of the N-acetyl cysteine derivative is of formula II,
  • n is a positive integer between 25 and 4000;
  • Y is a ⁇ —, an S—, an HCO—, a CONH—, a COO_, an OCO—, an NHCO 2 —, or an O 2 CNH —; is 11 or methyl;
  • R 2 is H or methyl;
  • m is a positive integer between 0 and 5.
  • n in the formula II is preferably a positive integer between 113 and 1500; preferably H, R 2 is preferably H; R is preferably a methyl group; Y is preferably an O-; m is a positive integer between 0 and 5, m More preferably, it is a positive integer between 1 and 5, and more effectively, m is 2.
  • the N-acetyl cysteine derivative of the present invention may also be a compound of the formula:
  • ni and n 2 are each a positive integer between 25 and 4000, preferably a positive integer of 113 to 1500; mj and m 2 are each a positive integer of 0-5, preferably a positive integer between 1 and 5, More preferably, m is 2.
  • the N-acetyl cysteine derivatives of the present invention can be synthesized by a conventional method in the art, for example, mPEG-based N-acetyl cysteine derivatives (R is a methyl group, and R ⁇ W is mainly prepared. include:
  • mPEG-OH activation of mPEG-OH, for example by reacting methanesulfonyl chloride with mPEG-OH, to activate it to form mPEG-methanesulfonyl ester;
  • mPEG-acid is reacted with thionyl chloride in an organic solvent such as toluene or dichloromethane to form mPEG-acyl chloride;
  • mPEG-NAC can be obtained by reacting mPEG-acid chloride with N-acetylcysteine (NAC).
  • NAC N-acetylcysteine
  • another synthetic route can be used: 1) mPEG-OH or 111?£0- ⁇ 3 ⁇ 4 triphosgene, acryloyl chloride, bisacyl chloride compound, etc., directly obtain mPEG-acid chloride;
  • mPEG-NAC can be obtained by reacting mPEG-acid chloride with N-acetylcysteine (NAC).
  • NAC N-acetylcysteine
  • Figure 1 is an electropherogram of mPEG 2()k -(CH 2 ) 2 -CO-NAC modified interferon a -2a.
  • Figure 2 is an electrophoresis pattern of mPEG 5k -(CH 2 ) 2 -CO-NAC modified bovine hemoglobin.
  • the precipitate was dissolved in a mixture of 10 ml of toluene and 15 ml of dichloromethane, and 0.33 g of N-acetylcysteine (NAC) was added thereto, and 0.3 ml of triethylamine was added dropwise thereto, and the mixture was stirred at room temperature overnight. After filtering the reaction mixture, the filtrate was concentrated to dryness, and 50 ml of isopropanol was added thereto, and the mixture was heated until the solution was transparent, poured into a beaker, and stirred to cool and crystallize. The crystals were filtered off, washed twice with isopropanol and air dried, to give mPEG 5k -0-CO-NAC.
  • NAC N-acetylcysteine
  • the mPEG-O-iCH ⁇ COOH prepared in the above step was dissolved in 320 ml of toluene, and 10 ml of toluene was distilled off under a nitrogen atmosphere. To the reflux liquid were added 3.0 mmol of anhydrous pyridine and 3.0 mmol of thionyl chloride, and the mixture was refluxed for 6 hours and then stirred overnight. The reaction solution was vacuumed under reduced pressure, concentrated and added to 360 ml. Precipitation of cold ether. The precipitate was collected, dissolved in 180 ml of dichloromethane, and then added 1.1 g of NAC and 0.8 ml of triethylamine, and stirred at room temperature overnight.
  • the mPEG 4()K -O-(CH 2 )rCOOH prepared in the previous step was dissolved in 700 ml of toluene, and 250 ml of toluene was distilled off under a nitrogen atmosphere.
  • To the reflux liquid 6.5 mmol of anhydrous pyridine and 6.5 mmol of thionyl chloride were added, and the mixture was refluxed for 10 hours and then stirred overnight.
  • the reaction mixture was concentrated in vacuo, then EtOAc EtOAc EtOAc EtOAc. After the reaction mixture was filtered, the filtrate was concentrated to dry. Add 350 ml of isopropanol, heat until the solution is clear, pour into a beaker and stir to cool and crystallize. The crystals were filtered off, washed twice with isopropanol, and dried to give mPEG 40K -O-(CH 2 ) 3 -CO-NAC o
  • tert-butyl 6-hydroxycaproate was dissolved in 80 ml of toluene, heated under a nitrogen atmosphere, and heated to azeotropic distillation until the remaining volume was about 50 ml.
  • the solution was cooled to room temperature and poured into 20 ml of 0.25 g of sodium hydride. In a toluene solution, mix well. The solution was stirred for 5 hours in a 37 ° C water bath, filtered, and cooled to room temperature.
  • the solution was mixed with a solution of the compound mPEG 5K -O-SO 2 -CH 3 prepared in the above step in 80 ml of anhydrous toluene, and the reaction was stirred at 125 ° C for 25 hours.
  • the reaction solution was vacuumed under reduced pressure, and then concentrated, and then, then, 100 ml of cold diethyl ether was precipitated, and the precipitate was collected and dried to obtain mPEG 5K -O-(CH 2 ) r CO 2 -C 4 H 9 .
  • the mPEG 5K -O-(CH 2 ) 5 -COOH prepared in the above step was dissolved in 150 ml of toluene, and 70 ml of toluene was distilled off under a nitrogen atmosphere. 1.5 mmol of anhydrous pyridine and 1.5 mmol of thionyl chloride were added to the reflux, and the mixture was refluxed for 4 hours and then stirred overnight. The reaction solution was concentrated in vacuo and then evaporated to ethyl ether. The precipitate was collected, dissolved in 50 ml of dichloromethane, and then 0.33 g of NAC and 0.3 ml of triethylamine were added and stirred at room temperature overnight.
  • the insoluble salt was filtered off, concentrated under reduced pressure, and then diethyl ether (180 ml).
  • the precipitate was filtered, dissolved in 100 ml of deionized water, adjusted to pH 12 with sodium hydroxide, stirred at room temperature for 1 hour, adjusted to pH 3 by adding 1 N hydrochloric acid, and the solution was extracted three times with dichloromethane, 100 ml each time.
  • the extract was dried over sodium sulfate / magnesium sulfate and filtered. After concentration under reduced pressure, the precipitate was added 400ml cold ether, the precipitate was collected and dried in vacuo to save, to give mPEG 1QK -S- (CH 2) 2 -COOH.
  • the mPEG 1QK- S-(CH 2 ) 2 -COOH prepared in the above step was dissolved in 250 ml of toluene, and under a nitrogen atmosphere, 120 ml of toluene was distilled off. 1.5 mmol of anhydrous pyridine and 1.5 mmol of thionyl chloride were added to the reflux, and the mixture was refluxed for 4 hours and then stirred overnight. The reaction solution was evaporated to dryness. The precipitate was collected, dissolved in 100 ml of dichloromethane, and then added with 0.5 g of NAC and 0.45 ml of triethylamine, and stirred at room temperature overnight. After the reaction mixture was filtered, the filtrate was concentrated to dry.
  • R 2 are hydrogen atoms; Y is -NHCO-; m is 2; n is 454)
  • reaction solution was filtered, and the filtrate was concentrated, dissolved in 150 ml of diluted hydrochloric acid, and adjusted to pH 3.0 with NaOH, and then extracted with dichloromethane three times, 150 ml each time, and the extract was combined and concentrated to dryness under reduced pressure.
  • the CH 3 O-PEG 4 o K -NHCO 2 - (CH 2 ) 4 -COOH prepared in the above step was dissolved in 650 ml of toluene, and 200 ml of toluene was distilled off under a nitrogen atmosphere. 6.0 mmol of anhydrous pyridine and 6.0 mmol of thionyl chloride were added to the reflux, and the mixture was refluxed for 10 hours and then stirred overnight. The reaction mixture was concentrated in vacuo, and then evaporated, evaporated, evaporated, evaporated. After the reaction mixture was filtered, the filtrate was concentrated to dry.
  • the CH 3 O-PEG 1()K -(CH 2 )2-COOH prepared in the above step was dissolved in 250 ml of toluene, and under a nitrogen atmosphere, 120 ml of toluene was distilled off. 1.5 mmol of anhydrous pyridine and 1.5 mmol of thionyl chloride were added to the reflux, and the mixture was refluxed for 4 hours and then stirred overnight. The reaction solution was evaporated to dryness. The precipitate was collected, dissolved in 100 ml of dichloromethane, and then 3.0 mmol of 3-aminopropionic acid and 0.6 mmol of triethylamine were added and stirred at room temperature overnight. The reaction solution was filtered, and the filtrate was concentrated, poured into 250 ml of ethyl acetate to precipitate, and the precipitate was collected and stored in vacuo to give
  • the CH 3 O-PEG 1QK -(CH 2 ) 2 -CONH-(CH 2 ) 2 -COOH prepared in the above step was dissolved in 250 ml of toluene, and under a nitrogen atmosphere, 130 ml of toluene was distilled off. 1.5 mmol of anhydrous pyridine and 1.5 mmol of thionyl chloride were added to the reflux, and the mixture was refluxed for 4 hours and then stirred overnight. The reaction solution was evacuated, concentrated to dryness and then evaporated and evaporated.
  • Example 3 mPEG 2() K -propionic acid, i.e., CH 3 O-PEG 2GK -(CH 2 ) 5 -COOH, was prepared. b) CH 3 O-PEG 2 . Preparation of K -(CH 2 ) 2 -COO-(C3 ⁇ 4) 5 -COOH
  • the CH 3 O-PEG 2QK -(CH 2 ) 5 -COOH prepared in the above step was dissolved in 360 ml of toluene, and under a nitrogen atmosphere, 140 ml of toluene was distilled off. 1.5 mmol of anhydrous pyridine and 1.5 mmol of thionyl chloride were added to the reflux, and the mixture was refluxed for 4 hours and then stirred overnight. The reaction solution was concentrated to dryness in vacuo. EtOAc was evaporated. Et. The reaction solution was filtered, and the filtrate was concentrated and poured into cold diethyl ether. The precipitate was collected and dried to give CH 3 O- PEG-(C3 ⁇ 4) 2 -COO-(CH 2 ) 5 -COOH.
  • the CH 3 O-PEG 20K -(CH 2 )2-COO-(CH 2 ) 5 -COOH prepared in the above step was dissolved in 380 ml of toluene, and 160 ml of toluene was distilled off under a nitrogen atmosphere. To the reflux liquid were added 3.0 mmol of anhydrous pyridine and 3.0 mmol of thionyl chloride, and the mixture was refluxed for 6 hours and then stirred overnight. The reaction solution was vacuumed under reduced pressure, and then concentrated, and then, 380 ml of cold diethyl ether.
  • the residue was dissolved in a mixture of 200 ml of toluene and 160 ml of 1,4-dioxane, and 5.0 mmol of ⁇ -aminopropionic acid was added thereto, and 0.7 ml of triethylamine was added dropwise thereto, and the mixture was stirred at room temperature for 2 hours.
  • the reaction solution was filtered, and the filtrate was concentrated and then precipitated with 600 ml of cold diethyl ether. The precipitate was collected and dried.
  • the precipitate was dissolved in 600 ml of toluene, and 190 ml of toluene was distilled off under a nitrogen atmosphere. 6.0 mmol of anhydrous pyridine and 6.0 mmol of thionyl chloride were added to the reflux liquid, and the mixture was refluxed for 10 hours and then stirred overnight. The reaction mixture was concentrated in vacuo, then EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc EtOAc After the reaction solution was filtered, the filtrate was concentrated under reduced pressure, and then 350 ml of isopropyl alcohol was added thereto, and the mixture was heated until the solution was transparent, poured into a beaker, and cooled to crystallize. The crystals were filtered off, washed twice with isopropyl alcohol, and dried.
  • R 2 is a hydrogen atom; Y is -S-; m is 2; n is 113)
  • the C 6 H 5 -CH 2 -O-PEG 5K -S- (CH 2 ) 2 -CO-NAC prepared in the above step was dissolved in 50 ml of dioxane, and 2 g of 10% palladium carbide was added. The hydrogen gas was oscillated overnight. The solution was filtered, concentrated under reduced pressure, and then 50 ml of isopropanol was added, and the mixture was heated until the solution was transparent, poured into a beaker, stirred and cooled to crystallize. The crystals were filtered off, washed twice with isopropyl alcohol and dried to give HO-PEG 5K -S- (CH 2 ) 2 -CO-NAC.
  • C 6 H 5 -CH 2 -O- PEG 2 OK-OH was prepared starting from C 6 H 5 -CH 2 -0- PEG -rich -S- (CH 2) R COOH.
  • the sample prepared in the previous step was dissolved in 95 ml of C 6 H 5 -CH 2 -O-PEG 2QK -S- (CH 2 ) 2 -CO-NAC
  • To the oxetane 2.5 g of 10% palladium carbide was added and vortexed overnight with hydrogen.
  • the solution was filtered, concentrated under reduced pressure and then purified with diethyl ether.
  • the precipitate was collected and dried to give HO-PEG 2QK -S - (CH 2 ) 2 -COOH.
  • the sample prepared in the previous step HO-PEG 2()K -S-(CH 2 ) 2 -COOH, was dissolved in 350 ml of toluene, and under a nitrogen atmosphere, 110 ml of toluene was distilled off.
  • To the solution was added 3 ml of an ethanol solution containing 2.1 mmol of sodium ethoxide, and the reaction was heated to reflux until about 6 ml of a solution was distilled off.
  • the reaction solution was cooled to room temperature, and 2.0 mmol of allyl chloride was added, and the mixture was reacted under nitrogen overnight.
  • the CH 2 CH-CH 2 -O-PEG 2()K -S-(CH 2 )2-COOH prepared in the above step was dissolved in 360 ml of toluene, and under a nitrogen atmosphere, 120 ml of toluene was distilled off. To the reflux liquid were added 3.0 mmol of anhydrous pyridine and 3.0 mmol of thionyl chloride, and the mixture was refluxed for 6 hours and then stirred overnight. The reaction solution was vacuumed under reduced pressure, and then concentrated, and then evaporated. The precipitate was collected, dissolved in 180 ml of dichloromethane, and then added 1.1 g of NAC and 0.8 ml of triethylamine, and stirred at room temperature overnight.
  • CH 2 CH-CH 2 -O-PEG dish -S-(CH 2 ) 2 -CO-NAC.
  • the C 6 H 5 -CH 2 -O-PEG 1GK -O-SO R CH 3 prepared in the above step was dissolved in 60 ml of distilled water, and then poured into 200 mr of concentrated aqueous ammonia containing 20 g of ammonium chloride, and stirred at room temperature overnight. The solution was extracted three times with dichloromethane, 220 ml each time. The combined extracts were dried over sodium sulfate and filtered. The solution was concentrated under reduced pressure, and then a mixture of 300 ml of cold diethyl ether was added, and the precipitate was collected to obtain C 6 H 5 -CH 2 -O- PEG 1QK -NH 2 and stored in vacuo.
  • the C 6 H 5 -C 3 ⁇ 4-O-PEG 1()K -NH 2 prepared in the above step was dissolved in 90 ml of dichloromethane, and adipic acid chloride 10 mmol and 1,4 ml of triethylamine were added, and the mixture was stirred at room temperature for 2 hours.
  • the reaction solution was filtered, concentrated in vacuo and then evaporated with diethyl ether.
  • the precipitate was collected, dissolved in 100 ml of dichloromethane, and then 2.0 g of NAC and 1.5 ml of triethylamine were added and stirred at room temperature overnight.
  • the reaction solution was filtered, concentrated, and then evaporated with diethyl ether.
  • the precipitate was collected to give C 6 H 5 -CH 2 -O-PEG 1QK -NHCO-(CH 2 )4-CO-NAC, which was stored in vacuo.
  • R is (CH 3 ) 3 CO-CONH-CH 2 -CH 2 ;
  • R 2 is a hydrogen atom;
  • Y is -S-;
  • m is 2;
  • n is 454)
  • the precipitate was filtered, dissolved in 150 ml of distilled water, and then poured into 250 ml of concentrated aqueous ammonia containing 25 g of ammonium chloride, and stirred at room temperature overnight. The solution was extracted three times with dichloromethane, 200 ml each time. The combined extracts were dried over sodium sulfate. The solution was filtered, concentrated under reduced pressure after addition of 600ml of cold ethyl ether precipitation, the precipitate was collected to give H 2 -PEG 2QK -S- (CH 2 ) 2 -COOH, save and dried in vacuo.
  • the sample prepared in the previous step NH 2 -PEG 20K -S-(CH 2 ) 2 -COOH, was dissolved in 100 ml of dichloromethane, and then 3.0 mmol of anhydrous triethylamine and 1.2 mmol of di-tert-butyl carbonate were added and mixed uniformly. The reaction was allowed to proceed overnight at room temperature under a nitrogen atmosphere. The solution was concentrated under reduced pressure, dissolved in 200 ml of distilled water, and adjusted to pH 3 with hydrochloric acid. The solution was extracted three times with dichloromethane, 200 ml each time. The combined extracts were dried over sodium sulfate.
  • the extracts were combined and extracted with 30 ml of 1N hydrochloric acid and 30 ml of distilled water. The solution was dried over sodium sulfate. The solution was filtered, concentrated under reduced pressure, and then taken to a mixture of 600 ml of cold diethyl ether. The precipitate was collected and stored in vacuo.
  • the precipitate was dissolved in 180 ml of dichloromethane, and 6 g of mPEGsk-CO-NAC synthesized according to Example 1 was added successively, and 0.25 ml of triethylamine was uniformly mixed and allowed to react at room temperature overnight. The reaction was quenched by the addition of an excess of ethylamine. The mixture was concentrated under reduced pressure and dissolved with a small portion of distilled water.
  • the product was subjected to Sephadex-200 molecular sieve chromatography ( ⁇ 2.6 cm X 100 cm), a buffer solution of 20 mmol/L pH 7.0 sodium phosphate (containing 200 mmol/L NaCl), and 30 ml of a double-chain PEG aqueous solution was collected.
  • the pH of the aqueous solution of the double-chain PEG was adjusted to 3.0 with hydrochloric acid, and the solution was extracted three times with dichloromethane, and the combined extracts of 100 mL each were dried over sodium sulfate. Filter the solution, concentrate under reduced pressure and add 300 ml of cold ether. Precipitation. The collected precipitate was dissolved in 400 ml of toluene, and under a nitrogen atmosphere, 100 ml of toluene was distilled off. 6.0 mmol of anhydrous pyridine and 6.0 mmol of thionyl chloride were added to the reflux, and the mixture was refluxed for 10 hours and then stirred overnight.
  • reaction mixture was concentrated in vacuo, and then evaporated, evaporated, evaporated,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
  • Example 19 mPEG 20k -(CH 2 ) 2 -CO-NAC modified interferon ct -2a.
  • the mPEG 20k -(CH 2 ) 2 -CO-NAC was mixed, and 0.75 ml of sodium borate (pH 9.2) was added to start the reaction, and the reaction was carried out at 4 ° C for 2 hours.
  • 0.1 ml of 2 M glycine was added, and after 10 minutes at room temperature, 0.5 ml of 1 M sodium acetate (pH 5.0) was added.
  • the sample was dialyzed, and the dialysate was 20 mM pH 5.0 sodium acetate.
  • the sample was directly applied to Sepharose-CM at a flow rate of 1.5 ml/min. After washing, it was eluted with 100 mM NaCl, 200 mM NaCl, 400 mM NaCl in 20 mM sodium acetate pH 4.5, respectively.
  • the modified and purified samples were collected and analyzed by SDS-PAGE. The results are shown in Figure 1.
  • the channel M is the protein standard
  • the channel 1 is the modified sample
  • the channel 2 is the ion-exchanged eluent.
  • 4 is a 100 mM elution sample
  • lane 5 is a 200 mM elution sample
  • lanes 6, 7 are 400 mM elution samples.
  • the apparent molecular weight of monoPEG interferon is about 60 kd
  • the molecular weight of interferon is 15 kd.
  • the results showed that PEG-NAC can be modified by interferon under mild conditions, and the product is stable and easy to separate and purify.
  • Example 20 mPEG 5k -(CH 2 ) 2 -CO-NAC modified bovine hemoglobin.
  • mPEG 5k -(CH 2 ) 2 -CO-NAC was prepared according to the procedure of Example 3.
  • the reaction was initiated by 0.75 ml of sodium borate (pH 9.0) and reacted at 10 ° C for 2 hours. 0.2 ml of 1 M glycine was added, and the reaction was terminated by a room temperature for 10 minutes. After the reaction, the sample was ultrafiltered with a 5kD membrane, and the ultrafiltered sample was collected for SDS-PAGE electrophoresis.
  • the electropherogram is shown in Figure 2.
  • channel 1 is hemoglobin
  • channel 2 is modified hemoglobin
  • channel 3 is protein standard.
  • the hemoglobin subunit has a molecular weight of about 15 kD
  • the PEG monomodified hemoglobin subunit has an apparent molecular weight of about 26 kD.
  • the results show that PEG-NAC can modify hemoglobin under mild conditions.
  • the N-acetyl cysteine derivative provided by the present invention is mainly used for modifying an amino group in a protein or a polypeptide, and the modified product is stable, and is advantageous for long-term preservation of a protein or a polypeptide product, and can be used as a modifier of a protein or a polypeptide.
  • the amino group in the protein or polypeptide is mainly modified, thereby having high selectivity and specificity.

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Abstract

Il est exposé des dérivés de la N-acétylcystéine et l'utilisation de ceux-ci. Les dérivés de la N-acétylcystéine selon la présente invention répondent à la formule I, dans laquelle POLY désigne des polyalkylèneglycols ayant un poids moléculaire allant de 1000 Da à 500 000 Da, des copolymères, terpolymères et copolymères mixtes de ceux-ci, dont le polyéthylèneglycol, le polypropylèneglycol et des copolymères de l'éthylèneglycol et du propylèneglycol. Les présents composés sont principalement utilisés pour modifier le groupe amino dans des protéines et des polypeptides. Ils ont une sélectivité, une spécificité et une réactivité modificatrice plus élevées. La N-acétylcystéine produite n'a pas d'effets secondaires. Il est facile d'effectuer le procédé de modification et les produits modifiés sont stables. En conséquence, les composés selon la présente invention peuvent être largement utilisés pour modifier des médicaments tels que des protéines et des polypeptides.
PCT/CN2006/000422 2005-03-23 2006-03-20 Dérivés de la n-acétylcystéine et utilisation de ceux-ci WO2006099794A1 (fr)

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CN103204789B (zh) * 2012-01-16 2014-09-24 中国科学院化学研究所 一种半胱氨酸衍生物、非离子型聚半胱氨酸及其制备方法
CN113527657B (zh) * 2021-07-16 2023-05-30 武汉纳乐吉生命科技有限公司 一种聚乙二醇修饰的胱氨酰胺衍生物、其制备及应用

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