RU2359975C2 - Method for production of modified arabinogalactan - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to new derivants of arabinogalactan. The method for production of modified arabinogalactan by oxygenising its primary hydroxy groups with system of 2,2,6,6,-tetramethylpiperydine-1-oxyl radical NaBr-NaClO-H₂O with pH 10.2 to carboxyl groups and further conjugation of carboxy groups of oxidised arabinogalactan with amino containing compounds in the presence of condensing agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in aqueous medium at the ambient temperature and pH 4.7-4.8 is described. A number of pharmaceutically important amines are used as amino containing compounds: n- and o-aminophenols , 5- and 4-aminosalicylic, anthroic and n-aminobenzoic acids, ethyl ester of n-aminobenzoinc acid (benzocaine), (3-diethylaminoethyl ester of n-n-aminobenzoinc acid (ethocaine hydrochloride), 1-phenyl-2,3-dimethul-4-amonipyrazolon-5(4-aminoantipyrine), isonicotinic acid hydrazide (isoniaside), sulfanamides, such as n-sulphanilamide (streptocide), n-aminobenzolsulfacetamide-sodium (sulfacyl sodium), 2-aminoethanesulfonic acid (taurine), natural α-amino acids (amino acetic acid, diamino-hexanoic acod, etc), diamines (hydrazine, ethylene diamine) and dihydrazides (dehydrazide of hexandioic acid). The derivants of arabinogalactan which can be used in medicine and pharmacology are derived. Conversion of carboxygroups to amide groups of oxidised arabinogalactan achieves 90-95%.

EFFECT: invention can be applied in medicine, pharmacology

1 cl, 2 dwg, 1 tbl, 15 ex

Description

The invention relates to the field of chemistry, in particular to a method for producing new substances - derivatives of arabinogalactan, which can be used in medicine, pharmacology.

Arabinogalactans - a class of polysaccharides, the building material of the cell wall of many trees and plants [Medvedeva E.N., Babkin V.A., Ostroukhova L.A. // Chemistry of plant materials. 2003. No. 1. S. 27-37]. Western larch (Larix occidentalis) and Siberian (Larix sibirica) are commercially significant sources of arabinogalactan, which is used as an emulsifier or stabilizer in the manufacture of certain food products. High molecular weight arabinogalactan is recommended for use as an immunostimulating agent that activates macrophages [Application WO 2006082707], and in combination with iron salts for iron deficiency anemia [US Pat. RU 2208440].

The arabinogalactan molecules consist of the main chain (1 → 3) - linked D-galactose residues with L-arabinose and D-galactose (1 → 6) residues - branches at almost every D-galactose residue in the main chain [Methods of carbohydrate chemistry. Edited by N.K. Kochetkov Moscow: 1967. 317 S.]. In larch arabinogalactans, the ratio of galactose: arabinose is from 5: 1 to 10: 1. Like many polysaccharides, the molecular weight of arabinogalactans varies from 10 ⁴ to 2 · 10 ⁶ Yes.

Not so long ago it was found that L-arabinose and D-galactose, which are part of arabinogalactan, unlike, for example, monosaccharides such as glucose or mannose, show affinity for asialoglycoprotein receptors of liver cells, 4-OH- plays an especially important role in this galactose group [Groman EV, Enriquez PM, Jung C, Josephson L. // Bioconjug. Chem. 1994. V. 5 (6). P.547-556]. This property stimulated work on the synthesis and study of the properties of modified arabinogalactans as drug delivery systems to liver cells. For example, in the presence of NaBH ₃ CN with poly-L-lysine, arabinogalactan graft copolymer was prepared by the reductive amination reaction between the reducing sugar residue at the end of the arabinogalactan chain and the ε-amino group of poly-L-lysine. A dendritic copolymer as a system for delivering DNA to liver cells showed high specificity for asialoglycoprotein receptors, which is promising for the use of the copolymer in gene therapy [Park JU, Ishihara T., Kano A., Akaike T., Maruyama A. // Prep. Biochem. Biotechnol. 1999. V.29 (4). P.353-370].

Other graft copolymers of arabinogalactan are obtained by graft copolymerization with acrylamide and subsequent treatment with dimethylamine and formaldehyde according to the Mannich reaction. Such copolymers are used to improve dehydration and increase paper strength [Auth. St. No. 633965].

Esters of arabinogalactans with cinnamic acid or its derivatives containing alkyl, allyl, CN, Hal or NH ₂ groups are known; in many cases, at least 50% of the hydroxy groups of arabinogalactan have been modified [Application JP 60219202]. Conjugates with thiophenecarboxylic acids (they can be useful as carriers, adsorbents or resins [Application JP 60219201]) and some fatty acids (palmitic, stearic and decanoic) [Application US 2001036933] were obtained from the hydroxyl groups of arabinogalactans. Water-soluble biocompatible arabinogalactans with lipophilic substituents can be used as adjuvants, inhibitors of cell adhesion, infection or inflammation.

A number of arabinogalactan derivatives exhibiting affinity for asialoglycoprotein receptors have been synthesized in order to obtain compounds with antiviral, anti-inflammatory, radioprotective properties [Pat. WO 9325239]. Arabinogalactan was first modified with hydrazine or ethylenediamine or conjugated to poly-L-lysine. The antiviral agents adenosine-5'-monophosphate and adenine arabinoside-5'-monophosphate, as well as the anti-inflammatory agent pepstatin, were added to the free amino groups obtained using 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (CDI). Phosphate groups in phosphorylated arabinogalactan were esterified in the presence of CDI S-2- (3-aminopropylamino) ethylthiophosphoric acid having radioprotective properties [US Pat. WO 9325239].

Acidic forms of arabinogalactans are found in nature, containing a small percentage of uronic acids in their composition. Carboxy-functional arabinogalactans are also obtained synthetically - by esterification of hydroxy groups with succinic or glutaric anhydride or by treatment of arabinogalactan with diethylene triaminopentaacetic acid (DTPA) dianhydride. The content of carboxy groups in the arabinogalactan hemisuccinate was 1.96 mEq / g of the product; in the arabinogalactan ether with DTPA, it was 0.117 mEq / g. Carboxymethyl and carboxyethyl arabinogalactans obtained by reaction with bromoacetic and 2-bromopropionic acids contained 5.2 and 1.3 mEq carboxy groups per gram of the final product [US Pat. WO 9325239].

Another way to introduce carboxy groups into the arabinogalactan molecule is to oxidize its primary alcohol group. When oxidized with hydrogen peroxide in an aqueous medium at an elevated temperature (90 ° C) with bubbling through a reaction mixture of air, the oxidation products contained up to 10-12% of uronic acids [Borisov IM, Shirokova EN, Mudarisova R.Kh. and others // Proceedings of the Academy of Sciences. Series chem. 2004. No. 2. S.305-311]. The destruction of macromolecules also occurs, accompanied by the oxidation of the anomeric center to a carboxyl group.

The selective way to oxidize the primary hydroxy group in polysaccharides (hyaluronic acid, cellulose, chitin) is to oxidize the 2,2,6,6-tetramethylpiperidinium-1-oxyl radical (TEMPO) -NaBr-NaClO-H ₂ O (pH 10.2) system [Jiang B ., Drouet E., Milas M., Rinaudo M. // Carbohydr. Res. 2000. V.327. P.455-461; Isogai A., Kato Y. // Cellulose. 1998. V.5. P.153-164]. However, this method was not used for the oxidation of arabinogalactan.

Closest to the proposed invention is a method for producing conjugates of alginic acid with compounds containing an amino group, in the presence of CDI at room temperature in an aqueous medium (pH 4.7-4.8) [US Pat. RF number 2283848]. In this way, conjugates with conversion of carboxy groups to amide up to 100% are obtained.

The task of the invention is to obtain modified arabinogalactans.

This is achieved as follows. The primary hydroxy groups of arabinogalactan from larch are selectively oxidized to carboxyl in the 2,2,6,6-tetramethylpiperidinium-1-oxyl radical (TEMPO) -NaBr-NaClO-H ₂ O (pH 10.2) system. In this way, arabinogalactan containing carboxyl groups (AG-COOH) is obtained, which is then reacted with compounds containing a primary amino group (RNH ₂ ) in the presence of a condensing reagent CDI. The reaction is carried out in an aqueous medium (pH 4.7-4.8) at room temperature for 20-30 minutes at a reagent ratio of AG-COOH: RNH ₂ : KDI = 1: 2: 1.5 or AG-COOH: RNH ₂ : KDI = 1: 5 : 1.5 if α-amino acids and their derivatives are used as RNH ₂ . Pharmacologically significant n- (1) and o-aminophenols (2), n-aminobenzoic (3) anthranilic (4), 4- (5) and 5-aminosalicylic (6) acids, n-aminobenzoic acid ethyl ester are used as amine-containing compounds. (anestezin) (7), n-aminobenzoic acid β-diethylaminoethyl ester (novocaine) (8), isonicotinic acid hydrazide (isoniazid) (9), 2-aminoethanesulfonic acid (taurine) (10), 1-phenyl-2,3 -dimethyl-4-aminopyrazolone-5 (4-aminoantipyrine) (11), as well as sulfonamide preparations containing a free amino group, such as n-aminobenzenesulfamide (streptocide) ( 12), n-aminobenzenesulfaacetamide sodium (sulfacyl sodium) (13). Other amines can be used: α-amino acids (glycine (14), lysine (15), etc.) or their esters, various diamines (hydrazine, ethylenediamine, hexamethylenediamine) and dihydrazides, for example adipic acid dihydrazide.

It should be noted that according to the method proposed in the work of Jiang B. et al., Hyaluronic acid was oxidized by primary hydroxy groups by no more than ~ 80%. Arabinogalactan was 100% oxidized at the primary hydroxy groups, which is 5.1 mEq carboxy groups per 1 g of the product AG-COOH (in the form of sodium salt). Other methods described in the description of the prior art, such a result is not obtained. The high content of uronic acids in the structure of the modified arabinogalactan allows increasing the pharmacological load on the macromolecule, which is achieved by effective carbodiimide conjugation of carboxy groups with amines.

The invention is illustrated by the following examples.

Example 1

a) Arabinogalactan (AG) is oxidized according to the method described in [Jiang B., Drouet E., Milas M., Rinaudo M. Study on TEMPO-mediated selective oxidation of hyaluronan and the effects of salt on the reaction kinetics // Carbohydr . Res. 2000. V.327. P.455-461]. Ag oxidation reaction conditions: pH 10.2, 0 ° С, TEMPO = 0.027 mmol, NaBr = 0.5 mmol, AG = 2.5 mmol in 200 ml of water, and 4 ml of a 13% aqueous solution of sodium hypochlorite (NaClO), reaction time - 1 hour. The oxidized product is isolated from the aqueous solution by precipitation into methanol. The precipitate was separated by centrifugation, washed with methanol, diethyl ether and dried under reduced pressure and a temperature not exceeding 60 ° C. A yellowish water-soluble powder is obtained. The selectivity of oxidation of CH ₂ OH groups to carboxyl groups is confirmed by IR data (by the appearance of an intense peak at 1600 cm ^-1 for the carboxylate anion) and ¹³ C NMR spectroscopy. In the ¹³ C NMR spectrum of oxidized arabinogalactan (AG-COOH) in the region of 176-178 ppm. the appearance of a group of signals corresponding to carbon atoms of the carboxyl groups of the oxidized residues of arabinose and galactose (arabinuronic and galacturonic acids, respectively). In this case, the signals of carbon atoms of CH ₂ OH groups in the region of 62-63 ppm absent, which also indicates the complete transformation of the primary hydroxy groups of arabinogalactan into carboxyl (Fig. 1).

b) To a mixture of 60 mg (~ 0.3 mmol) of AG-COOH and 66.5 mg (0.6 mmol) of n-aminophenol (1) in 10 ml of Н ₂ О add 0.1 N. NaOH (or, if required, 0.1 N HCl) to pH 4.7-4.8, then 86.3 mg (0.45 mmol) of carbodiimide (Ar-COOH: RNH ₂ : CDI = 1 reagent ratio) are added with vigorous stirring and a temperature of 20-22 ° C. : 2: 1.5), maintaining pH 4.7-4.8 by titration of 0.1 n. HC1. After 0.5 h, 0.1 N was successively added to the reaction mixture cooled to 0 ° C. NaOH (up to pH 7-8), 2-3 ml of a saturated solution of NaCl and 30 ml of chilled methanol. The precipitate was separated by centrifugation, dissolved in 10 ml of 6% NaCl, add 30 ml of methanol. The newly precipitated precipitate was centrifuged, washed with methanol (10 × 3 ml), then with ether (10 × 3 ml) and dried at a temperature of ≤60 ° С and reduced pressure. ~ 60 mg of conjugate is obtained in the form of a white powder. The conversion of carboxy groups to amide groups in the conjugate with n-aminophenol is ~ 100%.

The conversion of AG-COOH carboxyl groups to amide groups is determined by ¹ H-NMR spectroscopy using the integrated intensities of the characteristic proton signals of the residues of the corresponding amines with respect to the internal standard — the signal intensities of all protons of the test sample in the region of 3.25-4.50 ppm

¹ H-NMR spectra were recorded for solutions in D ₂ O on a Bruker AMX-300 spectrometer (operating frequency for ¹ H-NMR-300.13 MHz), 3- (trimethylsilyl) -1-propanesulfonic acid sodium salt was used as an internal standard. UV spectra were obtained on a Specord M-40 spectrophotometer. Control of the pH of the solutions was carried out using a pH meter "pH-340".

Examples 2-13. The oxidation of arabinogalactan and the reaction of the obtained AG-COOH with the corresponding amine (2-13) and purification of the products are carried out analogously to example 1. The conversion of carboxy groups to amides of oxidized arabinogalactan reaches 90-95%.

Examples 14, 15. The oxidation of arabinogalactan and the reaction of AG-COOH with glycine (14), D, L-lysine (15) are carried out analogously to example 1, but with the ratio of the reactants AG-COOH: RNH ₂ : CDI = 1: 5: 1.5; at this ratio, the conversion of carboxy groups to amides of oxidized arabinogalactan reaches 90-95%. Purification of the target conjugates is carried out analogously to example 1.

Some spectral characteristics of conjugates (in comparison with amines) are given in the table.

Claims (1)

A method of producing modified arabinogalactans, characterized in that arabinogalactan is oxidized in the system of 2,2,6,6-tetramethylpiperidinium-1-oxyl radical-NaBr-NaClO-H ₂ O, pH 10.2, to obtain arabinogalactan-containing carboxyl groups (AG-COOH) which, then in an aqueous medium at room temperature, pH 4.7-4.8, in the presence of a condensing agent 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (CDI) is reacted with compounds containing the primary amino group of the general formula RNH ₂ , where
RNH ₂ - n- or o-aminophenol, n-aminobenzoic or anthranilic acid, 4- or 5-aminosalicylic acid, n-aminobenzoic acid ethyl ester, n-aminobenzoic acid β-diethylaminoethyl, isonicotinic acid hydrazide, 1-phenyl-2 , 3-dimethyl-4-aminopyrazolone-5, as well as sulfonamide preparations containing a free amino group, such as n-aminobenzenesulfamide, n-aminobenzenesulfatamide sodium, with a molar ratio of AG-COOH: RNH ₂ : CDI = 1: 2: 1 , 5, and with amine-containing compounds of the general formula RNH ₂ , where RNH ₂ -2-aminoethanesulfonic acid, glycine, lysine, with a molar ratio of AG-COOH: RNH ₂ : CDI = 1: 5: 1.5.