Method for preparing modified glycosaminoglycans possessing analgesic property

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to a method for preparing modified glycosaminoglycans possessing analgesic properties. Method involves interaction of glycosaminoglycans with 1-phenyl-2,3-dimethyl-4-aminopyrazolone-5-(4-aminoantipyrine) in aqueous medium at pH = 4.7-4.8 in the presence water-soluble 1-ethyl-3-[3-(dimethlamino)propyl]carbodiimide as a condensing agent at room temperature followed by purification from low-molecular reagents. Method involves a single step that simplifies technology in preparing modified glycosaminoglycans.

EFFECT: improved preparing method.

3 ex

The invention relates to the field of chemistry and biochemistry, in particular to a method for producing new substances modified glycosaminoglycans, which can be used in medicine, pharmacology.

Acidic glycosaminoglycans (GAGS) from connective tissues of animals and humans are heteropolysaccharides a linear structure with different molecular weight, degree of sulfation, and include D-glucuronic or L-Euronova acid. The most representative of the class of GAGS are hyaluronic acid (desulfuromonas GAGS), chondroitin sulfates and heparin (most sulfated GAGS).

On the basis of GAG produced the following preparations.

High molecular weight hyaluronic acid (M=10⁶Yes) is part of a viscoelastic gels "Provisk" (Sweden) and "Healon" Canada), used in ophthalmic surgery operations cataract extraction, is the active substance in the films "Seprafilm" to prevent adhesive disease of the peritoneum. Low molecular weight hyaluronic acid impregnated wipes "Gaplus", intended for the treatment of burn disease. Drug "Curiosin" (in the form of a solution or gel) for the treatment of venous ulcers and acne represents Incoloy salt of hyaluronic acid, and the drug Cystistat" is intended to repair a damaged mucosa of the urinary bladder in interstitial cystitis. Hyaluronic acid is a component of cosmetic creams and tonics to stimulate hair growth. Patented the use of hyaluronic acid in the treatment of herpes, psoriasis, and also for the manufacture of suppositories for various purposes.

The chondroitin sulfates from the cartilage of cattle included in the ointment "handaxe" (for local treatment of osteoarthritis), are the active ingredient of drugs "Honored" (for acceleration of reparative processes) and "Romulan" (for treatment of joints).

The known heparin as anticoagulant and antithrombotic agents and is used intravenously for the operations of the vessels. In addition to the anticoagulant and antithrombotic agents, heparin revealed antibacterial, antiviral, anti-inflammatory [Bychkov S.M. New data on heparin. // Questions of medical chemistry. 1981. No. 6. S-735], and antitumor properties [Lapierre F., Holme, K., Lam, L. Chemical modification of heparin that diminish anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties. // Glycobiology. 1996. V.6(3). P.355-366]. Also found an inhibitory effect of heparin on the growth and replication of human immunodeficiency virus [C.C. Rider The potential for heparin and its derivatives in therpy and prevention of HIV-1 infection. // Glycoconj. J. 1997. V.14(5). P.639-642]. Heparin ointment, in which, in addition to heparin, is benzocaine (a local anesthetic agent), apply externally with superficial thrombophlebitis and ulcers of the extremities, thrombosis hemorrhoid veins, thus diminishing the inflammatory processes [Mashkovsky PPM Medicines. - Kharkov: Torsing, 1997. 1, 2. - 590 S.].

Covalent binding of low molecular weight biologically active compounds with polymer matrix contributes to reducing drug toxicity, increase the length of their actions, achieves a more effective delivery of drugs to organs-targets. For example, in combination with hyaluronic acid, some of the drug substance form in the epidermis like depo these drugs and are released from the composition slower than in the absence of hyaluronic acid [Vercruysse K.R., G.D. Prestwich // Therapeutic Drug Carrier Systems. 1998. V.15(5). P.513-555].

Known to produce the conjugate of hyaluronic acid (ha-COOH) with ibuprofen, or brovana (d,1-2-(4-isobutylphenyl)-propionic acid), a drug with anti-inflammatory, analgesic and cough activity. Synthesis of conjugates is carried out in three stages:

(1) using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) first get conjugates (IIa-b) hyaluronic acid with Digita Itami amber (Ia), adipic (IB) and saburovoi acids (IB) [conversion of carboxyl groups is 30%(IIa), 56 (IIB) and 46 (IIB)];

(2) from ibuprofen and N-hydroxysuccinimide (NHS) in the presence of dicyclohexylcarbodiimide (DCC) synthesize N-hydroxysuccinimidyl ester of ibuprofen (NHS-ester of ibuprofen);

(3) derivatives of hyaluronic acid (IIa-b) in a mixture of 0.1 N. NaHCO₃(pH 8.5) and DMF (1:2) react with the NHS-ester of ibuprofen with the formation of conjugates (IIIa-C). Thus it is possible to introduce 24 mol%. ibuprofen (relative to the carboxyl groups of hyaluronic acid).

The authors propose a one-step method for production of modified GAG^*(^*Hyaluronic acid and a mixture of chondroitin sulfates were obtained from the umbilical cord of newborns, heparin - of pharmacopoeial preparation for intravenous injection.), analgesic properties by interaction of GAG with 1-phenyl-2,3-dimethyl-4-aminopyrazole-5(4-aminoantipyrine) in aqueous medium at pH 4.7-4.8 in the presence of a condensing reagent is a water - soluble 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The reaction proceeds at room temperature for 60 minutes with a high conversion of the carboxyl groups of the GAG (83-100%). Cleaning products from low molecular weight reagents is achieved by two - or threefold deposition conjugates ethanol is the alcohol from the aqueous solution.

Spectra¹H-NMR were recorded for solutions in D₂O spectrometer Bruker AMX-300 (operating frequency for¹H-NMR 300.13 MHz), as internal standard used the sodium salt of 3-(trimethylsilyl)-1-propanesulfonic acid. Control the pH of the solutions was performed using a pH meter pH-340".

Water-soluble conjugates GAG from 4-aminoantipyrine was prepared as follows (scheme 2, examples 1-3).

Example 1. To a mixture of 0.15 mmol of hyaluronic acid (from the umbilical cord) and 91.4 mg (0.45 mmol) of 4-aminoantipyrine in 15 ml of N₂About add 0.1 N. NaOH (or, if you want, 0.1 N. HCl) to pH 4.7-4.8, then under vigorous stirring and the temperature of 20-22°make 86.4 mg (0.45 mmol) carbodiimide, maintaining pH 4.7-4.8 titration with 0.1 N. HCl. After 1 h to a cooled to 0°the reaction mixture is successively added 0.1 N. NaOH (to pH 7), 2-3 ml of a saturated solution of NaCl and 60-65 ml of chilled ethanol. Dropped the precipitate was separated by centrifugation, dissolved in 10 ml of 6% NaCl, add 30-40 ml of ethanol. Again precipitated precipitate is centrifuged, washed with ethanol (10×3 ml), then ether (10×3 ml) and dried at a temperature of ≤60°and reduced pressure. Get ˜60 mg conjugate of hyaluronic acid with 4-aminoantipyrine, soluble in water. Conversion of carboxyl groups is 83 mol%. (determined from the intensity ratio C the signals of the phenyl protons (δ 7.5 and 7.7 ppm) and the signal metal protons NHCOMe-group (δ 2.1 ppm) in N-acetylglucosaminidase levels of hyaluronic acid).

Example 2. The reaction of chondroitin-4-sulfate and chondroitin-6-sulfate (mixture) with a 4-aminoantipyrine carried out analogously to example 1. Get ˜80 mg of water-soluble product in the form of a white powder. Conversion of carboxyl groups was 87 mol%. (determined from the ratio of intensities of the signals of the phenyl protons (δ 7.5 and 7.7 ppm) and the signal metal protons NHCOMe-group (δ 2.1 ppm) in the levels of N-acetyl-D-glucosamine chondroitin sulfates).

Example 3. The reaction of heparin with 4-aminoantipyrine carried out analogously to example 1. Get ˜90 mg conjugate of heparin, a soluble in water white powder. Conversion of carboxyl groups is 100% mole. (determined from the ratio of intensities of the signals of the phenyl protons (δ 7.5 and 7.7 ppm) and two signals of anomeric protons of heparin in the field of 5.3-5.5 ppm).

Conjugates GAG from 4-aminoantipyrine are structural analogues of antipyrine, analgin, amidopirina, butadiona and have analgesic properties.

Analgesic effect of conjugates was studied on the model of acetic cramps caused by intraperitoneal rat 1 ml ˜1% solution of acetic acid [Trinus FP,Klebanov BM, Mokhort N.A. screening Methods and pharmacological study of anti-inflammatory, analgesic and antipyretic substances (HOWTO). Kiev, 1974. - 27 S.]. Conjugates of hyaluronic acid (1) and chondroitin sulfates (2) with 4-aminoantipyrine was administered at a dose of 50 mg/kg, the conjugate of heparin (3) at the dose of 40 mg/kg as the comparison drug used analgin. Prophylactic administration of the conjugate (1) reduced the frequency of painful manifestations 1.9 times compared to the untreated group of animals, conjugate (2) - 2.7 times, conjugate (3) - 1.6 times. Comparison drug analgin reduced the frequency of painful manifestations in 2.1-2.5 times. Analgesic properties of the conjugate of chondroitin sulfates with 4-aminoantipyrine dose of 50 mg/kg is comparable to aspirin.

A method of obtaining modified glycosaminoglycans, analgesic properties, wherein the glycosaminoglycan is subjected to interaction with 1-phenyl-2,3-dimethyl-4-aminopyrazole-5(4-aminoantipyrine) in aqueous medium at pH 4.7-4.8 in the presence of condensing agent is a water - soluble 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide at room temperature, followed by purification from low molecular weight reagents.