RU2575925C1 - Tetravalent neoglycoconjugates with carbohydrate branching core and method for obtaining thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to obtaining tetravalent neoglycoconjugates with carbonhydrate branching core and derivative of L-glutamic acid in hydrophobic block, suitable for preparation of liposomes, of formula

. Method includes conjugation of azidoethyl carbonhydrate derivative, selected from 1-O-(2-azidoethyl)-α-D-mannopyranozide or 1-O-(2-azidoethyl)lactoside, with bishexadecyl-N-({3-[N'-2,3,4,6-tetra-O-propynyl-β-D-glycopyranozyloxy]-carbamoyl}-propyonyl)-L-glutamate.

EFFECT: increase of affinity of carbohydrate residuals to receptors, effective method of thereof obtaining.

2 cl, 1 dwg, 8 ex

Description

The invention relates to the field of bioorganic chemistry, in particular to derivatives of carbohydrates and amino acids containing four terminal carbohydrate residues.

Inflammation is an immune response to tissue damage and pathogen invasion, in which the penetration of white blood cells from the blood into the tissues is a key process. However, an excessive influx of leukocytes in certain pathological conditions may be the result of undesirable reactions. For example, localization of leukocytes at the site of tissue damage can cause further damage, leading to deepening, expansion and worsening of tissue damage, as well as irreversible necrosis and permanent loss of tissue function [Kaila N. / N. Kaila, K. Janz, S. DeBernardo , PW Bedard, R.T. Camphausen, S. Tam, D. H. H. Tsao, J.C. Keith Jr., C. Nickerson-Nutter, A. Shilling, R. Young-Sciame, Q.J. Wang // Med. Chem. - 2007. - V. 50. - P. 21-39].

An important role in the strong attachment of leukocytes to the endothelium is played by CD11b / CD18 leukocyte integrins. Studies of the biological release mechanism using fluorescently labeled multivalent lactosides have shown that they are targets of CD11b on the surface of leukocytes. In addition, when studying the treatment of severe burn shock, the clinical syndrome of which is associated with insufficient blood flow to vital organs and tissues, it was demonstrated that multivalent lactosides can effectively inhibit the adhesion of leukocytes in endothelial cells [Watterson S.N. / S.N. Watterson, Z. Xiao, D.S. Dodd, D.R. Tortolani, W. Vaccaro, D. Potin, M. Launay, J. Stetsko // Med. Chem. - 2010. - V. 53. - P. 3814-3830].

The closest technical solution to the claimed invention is a glycoconjugate with two terminal lactose residues, which, having two hydrophobic chains, is able to integrate into bilayer liposomes and effectively bind to lectins.

The technical result of the invention is to increase the affinity of carbohydrate residues for receptors by creating new tetravalent neoglycoconjugates with D-glucose as a branching nucleus and a derivative of L-glutamic acid in a hydrophobic block specific for the corresponding receptors and containing residues of various carbohydrates (for example, lactose , D-mannose and others) having the following general formula:

To achieve the technical result, a method for producing neoglycoconjugates has been developed, including the synthesis of a tetrapropargyl ether derivative of D-glucose with L-glutamic acid dihexadecyl ether, the synthesis of an azidoethyl derivative of a carbohydrate and conjugation of these components to form target compounds.

The implementation of the present invention is confirmed by the following examples.

Example 1

Synthesis of N- (23,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy) succinimide. To a solution of 2.00 g (5.13 mmol) of 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranoside in 22 ml of ethyl acetate was added 0.450 ml (5.64 mmol) of boron trifluoride ether complex . After 15 minutes, 1.18 g (10.3 mmol) of N-hydroxysuccinimide was added to the reaction mixture and held at 40 ° C for 12 hours. Then the reaction mass was neutralized with 25% ammonia solution to pH = 7. The solution was washed with 5 × 100 ml of water, the organic layer was dried, and the solvent was removed in vacuo. The product was purified by column chromatography in a toluene-acetonitrile 5: 1 system. 1.03 g (45%) of N- (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl) succinimide was obtained as white crystals, R _f (toluene-acetonitrile 2: 1) 0 , 45. T _pl = 177-178 ° C (from isopropyl alcohol). IR spectrum (liquid paraffin, v _max , cm ^-1 ): 2915, 2850, 1410, (CH), 1750 (C = O), 1232 (CO), 1168-1048 (CO, 4 bands, carbohydrate skeleton).

Example 2

Synthesis of N- (β-D-glucopyranosyl) -3- (carboxymethyl) propanamide. To a solution of 100 mg (0.224 mmol) of N- (2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside) -succinimide in 5 ml of methanol was added dropwise a 0.1 M solution of sodium methylate in methanol to pH = 8. The reaction mixture was left for 24 hours. Then the reaction mixture was treated with KU-2 (H ⁺ ) ion exchange resin to pH = 7. The resin was filtered off, the solvent residue was removed in vacuo. IR spectrum (in the film, v _max , cm ^-1 ): 3370 (OH), 2895, 1470, 1354 (С-Н), 1750 (С = О), 1671 (С = O, I amide band), 1632 (NH, II amide band), 1225 (С-О), 1110-1044 (С-О, 4 bands, carbohydrate skeleton).

Example 3

The synthesis of N- (β-D-glucopyranosyl) -3- (carboxy) propanamide. 62.0 mg of N- (β-D-glucopyranoside) -3- (carboxymethyl) propanamide was stirred in a 0.05 M solution of NaOH in methanol at room temperature for 24 hours. The reaction mixture was neutralized to pH = 7 with KU-2 ion exchange resin ( H ⁺ ), the resin was filtered. The remaining solvent was removed in vacuo and dried over P ₂ O ₅ in a vacuum desiccator. 50.0 g (67%) of N- (β-D-glucopyranosyl) -3- (carboxy) propanamide were obtained as an amorphous substance, R _f (chloroform-methanol-water 18: 6: 1) 0.67. IR spectrum (v _max , cm ^-1 ): 3332 (OH), 2890, 1476, 1350 (С-Н), 1715 (С = O), 1671 (С = O, I amide band), 1632 (NH, II amide band), 1280 (С-О), 1118-1041 (С-О, 4 bands, carbohydrate skeleton).

Example 4

Synthesis of bishexadecyl-N - ({3- [N′-β-D-glucopyranosyloxy] carbamoyl} propionyl) -L-glutamate. To a solution of 50 mg (0.169 mmol) of N- (β-D-glucopyranosyl) -3- (carboxy) propanamide in DMF was added DCC and DMAP as a catalyst. The mixture was cooled to 0 ° in an ice bath for 15 minutes. 100 mg (0.169 mmol) of L-glutamic acid dihexadecyl ester in DMF was added. The reaction was carried out in an inert atmosphere for 3 days. The precipitate was filtered off, the solvent residue was removed in vacuo. The product was isolated by preparative chromatography in chloroform-methanol 5: 1, toluene-acetonitrile 1: 1 systems. Received 33 mg (21%) of the product, R _f (1: 1 chloroform-methanol) 0.4. IR spectrum (in the film, v _max , cm ^-1 ): 3322 (OH), 2917, 2848 (С-Н), 1731 (С = O), 1695 (I amide band, С = O), 1650 (II amide strip, NH), 1467 (C-O), 1197-1035 (C-O, 4 lanes, carbohydrate skeleton). Mass spectrum, m / z (I _rel ,%): 895.6 [M + Na] ⁺ (100), 429.2 (18.9), 256.7 (25), 163.4 (21.8), 147.2 (36), 71.7 (34).

Example 5

Synthesis of bishexadecyl-N - ({3- [N′-2,3,4,6-tetra-O-propynyl-β-D-glucopyranosyloxy] carbamoyl} propionyl) -L-glutamate. To 33 mg (0.0368 mmol) of bishexadecyl-N - ({3- [N′-β-D-glucopyranosyloxy] -carbamoyl} propionyl) -L-glutamate in 3 ml of DMF, 88 mg (0.368 mmol) of sodium hydride was added in portions. ) at a temperature of 0 ° C. After 20 minutes, propargyl bromide was added dropwise. Left on a magnetic stirrer at 0 ° C for 12 hours. The solvent was removed in vacuo. The residue was dissolved in ethyl acetate and washed with water. The organic layer was dried over anhydrous Na ₂ SO ₄ . The remaining solvent was removed in vacuo. Purification was performed using preparative chromatography in a 1: 1 hexane-ethyl acetate system. 20 mg (53%) of the compound were obtained as an amorphous substance, R _f (2: 1 hexane-ethyl acetate) 0.8. IR spectrum (liquid paraffin, cm ^-1 ): 3324 (О-Н), 2920, 2850 (С-Н), 2130 (С≡С), 1700 (С = O), 1619 (C = O, I amide lane), 1580 (NH, II amide band), 1420 (С-О), 1189-1070 (С-О, carbohydrate skeleton). Mass spectrum, m / z (I _Rel ,%): 1025.497 (100) [M] ⁺ , 989.933 (11), 978.933 (9), 816.801 (10), 716.341 (12).

Example 6

Synthesis of 2,3,4,6-tetra-O-acetyl-1-O- (2-bromoethyl) -α-D-mannopyranoside. To a solution of 3 g (7.69 mmol) of 1,2,3,4,6-penta-O-acetyl-α-D-mannopyranoside in 22 ml of anhydrous methylene chloride was added 0.710 ml (8.84 mmol) of boron trifluoride ether complex . After 15 minutes, 0.985 ml (8.84 mmol) of 2-bromoethanol was added to the reaction mixture and held for 12 hours at 20 ° C. The reaction mass was stirred while heating to 40 ° C. Then the reaction mass was neutralized with 25% ammonia solution to pH = 7. The solution was washed with 3 × 100 ml of water, the organic layer was dried through a pleated filter moistened with methylene chloride, and the solvent was removed in vacuo.

The oil formed after processing the reaction mass was chromatographed using column chromatography, eluting with a 9: 1 toluene-acetonitrile solvent system. 2.2 g (59%) of pure compound are obtained. R _f (chloroform-methanol 9: 1) 0.45, T _mp = 56-58 ° C.

IR spectrum (v _max , cm ^-1 ): 2836 (С-Н), 1755 (С = O), 1460, 1380 (С-Н), 1218 (С-O), 1158-1035 (С-О, 4 stripes, carbohydrate skeleton).

¹ H-NMR spectrum (CDCl ₃ , δ, ppm): 1.99, 2.05, 2.10, 2.16 (s, 12H, COCH ₃ ), 3.50-3.53 (t, 2H, OCH ₂ CH ₂ ), 3.86- 4.0 (m, 2H, OCH ₂ CH ₂ ), 4.10-4.16 (m, 2H, H-6), 4.24-4.30 (dd, 1H, H-5), 4.87 (d, 1H, H-1, J ₁₂ 1.88), 5.26-5.31 (m, 2H, H-2, H-4), 5.34-5.37 (dd, 1H, H-3).

Example 7

Synthesis of 1-O- (2-azidoethyl) -α-D-mannopyranoside. To a solution of 200 mg (0.40 mmol) of 2,3,4,6-tetra-O-acetyl-1-O- (2-bromoethyl) -α-D-mannopyranoside in 5 ml of anhydrous dimethylformamide was added 80 mg (1, 20 mmol) of sodium azide and stirred at room temperature for 20 hours. An excess of anhydrous diethyl ether was added to the solution. The precipitate formed was filtered off, and the solvent was removed in vacuo.

Removal of the solvent gave 157 mg (85.5%) of (2,3,4,6-tetra-O-acetyl-1-O- (2-azidoethyl) -α-D-mannopyranoside as an amorphous substance, R _f ( toluene-acetonitrile 2: 1) 0.65.

IR spectrum (v _max , cm ^-1 ): 2831 (С-Н), 2099 (N = N), 1742 (С = O), 1460, 1380 (С-Н), 1250 (С-N), 1210 (C-O), 1217-1040 (C-O, 4 bands, carbohydrate skeleton).

¹ H-NMR spectrum (CDCl ₃ , δ, ppm): 1.99, 2.05, 2.10, 2.16 (s, 12H, COCH ₃ ), 3.40-3.52 (m, 2H, OCH ₂ CH ₂ ), 3.62- 3.9 (m, 2H, OCH ₂ CH ₂ ) 4.0-4.15 (m, 2H, H-6), 4.24-4.32 (dd, 1H, H-5), 4.85-4.87 (d, 1H, H-1, J ₁₂ 1.88), 5.26-5.30 (m, 2H, H-2, H-4), 5.34-5.38 (dd, 1H, H-3).

To a solution of 200 mg of 2,3,4,6-tetra-O-acetyl-1-O- (2-azidoethyl) -β-D-mannopyranoside in 5 ml of anhydrous methanol, 0.1 ml of freshly prepared was added with stirring at room temperature. 0 , 1 M solution of sodium methylate in methanol until a pH of 8 is reached and the mixture is heated in a water bath. The solution was desalted by KU-2 ion exchange resin (H ⁺ form), filtered and the solvent removed in vacuo.

0.125 g (82%) of 1-O- (2-azidoethyl) -α-D-mannopyranoside was obtained as an amorphous substance, R _f (chloroform-methanol 2: 1) 0.4.

IR spectrum: (v _max , cm ^-1 ): 3325 (О-Н), 2900, 1435, 1340 (С-Н), 1215 (С-О), 1140-1030 (С-О, 4 lanes, carbohydrate skeleton).

Example 8

Obtaining a glycoconjugate with four terminal lactose residues. To 20 mg (0.323 mmol) of 1-O- (2-azidoethyl) -lactoside in acetonitrile was added 80 mg (0.094 mmol) of the bishexadecyl-N derivative - ({3- [N′-2,3,4,6-tetra- O-propynyl-β-D-glucopyranosyloxy] -carbamoyl} -propionyl) -L-glutamate dissolved in a mixture of acetonitrile-DMF (2: 1), a catalytic amount of CuI, DIPEA and stirred at room temperature for 7 days under argon atmosphere . The reaction mass was filtered off from the precipitate. The solvent was removed in vacuo. Purification was carried out using preparative chromatography in a chloroform-methanol 9: 1 system. 30 mg (35%) of the product were obtained as an amorphous substance, R _f (B) 0.8. IR spectrum (in the film, v _max , cm ^-1 ): 3374 (OH), 2929, 2854, 1477, 1444 (С-Н), 2700 (C = N), 1749 (С = O), 1683 (C = O, I amide band), 1627 (NH, II amide band), 1400 (N = N), 1280 (C-O), 1255 (CN), 1249-1074 (C-O, 4 lanes, carbohydrate skeleton) . Mass spectrum (I _rel ,%) m / z: 2670.550 (29) [M] ⁺ , 531.63 (39), 530.21 (100), 259.74 (56.1), 199.01 (10), 149.1 (51.9).

Similarly, from 50 mg (0.43 mmol) of 2,3,4,6-tetra-O-acetyl-1-O- (2-azidoethyl) -α-D-mannopyranoside and 90 mg (0.11 mmol) of the bishexadecyl-N derivative - ({3- [N′-2,3,4,6-tetra-O-propynyl-β-D-glucopyranosyloxy] carbamoyl} propionyl) -L-glutamate received 48 mg (39%) of a tetravalent glycoconjugate with terminal residues of D-mannose. Mass spectrum (I _rel ,%) m / z: 1949.950 (100) [M] ⁺ , 480.63 (39), 351.70 (32.9).

To study the possibility of using the synthesized compounds as marker molecules in liposomes, we studied the interaction of tetravalent neoglycoconjugate modified vesicle lactose residues with galactose-binding lectin of castor bean plant Ricinus communis (RCA ₁ ) using a spectrophotometric method of analysis.

It is known that glycoproteins with terminal galactosyl residues, giving a positive precipitation reaction with RCA ₁ lectin, also have an affinity for other galactose-specific lectins, in particular asialoglycoprotein receptors of human and animal hepatocytes. In this regard, it is logical to assume that glycosylated liposomes precipitating in the presence of RCA ₁ will also be characterized by increased affinity for hepatocytes.

The interaction of liposomes modified with bi- and tetravalent glycoconjugates containing lactose derivatives as terminal residues with castor bean lectin RCA _{1 was} investigated. The tetravalent compound has shown greater binding efficiency than the bivalent compound. This confirms the literature on an increase in receptor binding with an increase in the number of carbohydrate residues. The addition of D-galactose led to a decrease in optical density, which indicates a specific interaction of conjugates with RCA ₁ lectin.

Thus, the obtained data indicate the presence of carbohydrate specificity of RCA ₁ lectin to terminal galactose of the formed model systems. An important factor affecting the efficiency of marker-receptor binding is the amount of terminal carbohydrate residues. For example, with an increase in the number of galactose residues, the affinity of the modified liposome for RCA ₁ lectin increased.

Claims (2)

1. Tetravalent neoglycoconjugates with a carbohydrate branching core and a derivative of L-glutamic acid in a hydrophobic block:

2. A method of producing tetravalent neoglycoconjugates described in paragraph 1, comprising conjugating an azidoethyl derivative of a carbohydrate selected from 1-O- (2-azidoethyl) -α-D-mannopyranoside or 1-O- (2-azidoethyl) lactoside, with bishexadecyl -N - ({3- [N'-2,3,4,6-tetra-O-propynyl-β-D-glucopyranosyloxy] carbamoyl} propionyl) -L-glutamate.

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