SU1652319A1 - Chitooligosaccharide derivatides possessing immunomodulating and antitumoral activity - Google Patents

Abstract

The invention relates to sugars, in particular chitooligosaccharides, 4-oligosaccharides 2-amino-2-deoxy-O-glucose of the general formula 8e 1 j, sp0 l-e (o) - (si, -c1o) it "H-ctol-CHi -c- j-lCH n-en, where a) to 1,1-0. m 1, n 6, Rt-H; 6) k 1. l.m-l.n-10. Ri-H; e) k-1. M-1, n-14, Ri- H; r) k 1, I-O, m - 1. n 10, Ri-OH; fl) k 1.1 0, m-1.n-10, Ri-0-C 0 - CH3; e) k - 0.1 - 1, m - 1. n - 10. Ri - OH; g) k - 2. I - 0, m - 1, n - 10, Ri - H; 3) k 2.l 0, m 1, n 10, Ri-OH; n) k 1, I 0, m 2. n 10, Ri-OH; K) k 3, 1 0. m 1. n 10, Ri - H; l) k 3, I 0, m 1. n 10, Ri-OH; M) k 1, I 0, m 1, n 2, Ri - H, n) k 1, l 0, m 1. n 10, Ri-0-C 0 -CH3, possessing immunomodulating and anti-tumorous activity, which can be used in medicine and biology. The purpose of the invention is the creation of new less toxic compounds of the specified class. Synthesis is carried out by selective acylation of amino groups contained in the chitooligosaccharide molecule, interaction of equimolecular amounts of amino sugar and acylating agents, which use fatty acid chlorides or fatty acids mixed with condensing agents, or active fatty acid esters in a mixture of water and an organic solvent. . Yield,%, gross formula: a) 22.4; С22Н42ОЮ№С1; b) 38; C26HsoOioN2CI; c) 14; JUNE C; d) 16.3; S2bN5oOtsM2S1: e) 15.5; C4oH76Oi2N2CI; e) 49.8; g) 37.4; not; h) 26.1; Sz2Nbz015№S12; i) 6.8; SyuNadO zS: k) 26.5: Sz8N7b01dM4S1z; l) 37.8; SzvN7b02oM4S1z; m) .11: CieH340ioN2CI: n) 37.2; C2pH520i2N2CI. The new compounds have an LDz -92 toxicity of 110 mg / kg (versus 0.1-0.5 mg / kg). They stimulate the production of the cells of the immune system of interleukin-1 and the necrosa-tumor factor, increase: the tumor-static activity of macrophages, exhibit an antitumor effect in vivo. 7 tab. Mr. Fe O El Yu OJ Yu

Description

This invention relates to the chemistry of sugars specifically to novel chitool state saccharides (fi 1,4-oligosaccharide and 2-amino-2-deoxy-O glucose) of the general formula

im-cloJ-CKj-enR., Q

JlL

and -FNRCH-SNO D

where 1aH 1.I-0, m-1, n-6, Ri H; 16} k-1.1-0. m - 1, n 40, Ri - H; 1e) k- 1. 1-0, m - 1, n «14, Ri - H, 1r) k 1, I 0 rr. - 1. n 10. R-OH; 1 / vlk 1,, m t, t, - 50, Ri - CCA (CH2) 12CH3: 1e) k - 0. I 1. m 1, n - 10, Ri - OH; 1g) V 2, i 0, m 1, n 10, Ri-H; I3), 1 0, m 1, n 10. Ri-OH; 1i) k - 1,1 0, m 2, n 10, Ri - OH; IK) k 3. I 0.01 1, l 10, Ri-H; 1n) k 3, IO, m-1, n - 10, Ri - OH; 1m) k - 1, I - 0, m 1, n 2, Rf- H; 1n) k - 1, I - 0. m 1. n «10, Ri AHCOs with immunomodulating and antitumor activity that can be found. application in medicine and medicine

The purpose of izbrete1 and - lowering toxicity in a number of DERIVATIVES of carbohydrates possessing an immune module and antitumor activity

PRI me R 1. Mono-N K. eirichia chitobiose (1a). (2-Deoxy-2-decanoyl & g, ino-4-0- (2-amino-2-deoxy / 3-O-gluconoenosyl} -G glucose hydrochloride).

To a solution of 200 mg (0.48 mmol) of chitobiose hydrochloride in a mixture of 2 ml of water and 18 ml of ethanol, 0.175 ml of triethylamine is added, followed by stirring 95.3 mg (0.5 mmol of hydrochloride, stirred for 18 hours at 20 ° C. acidified with hydrochloric acid, evaporated off, the residue is extracted with acetone, dissolved in 10 ml of water.CMF-CJ is filtered, the filtrate is extracted with p-butanol (2x10 ml). Butanol extract is evaporated, the residue is dissolved in 5 ml of ethanol, the product is precipitated the precipitate is filtered off by the addition of 50 ml of acetone. The yield of compound 1 is 57.5 mg (22.4%). White powder with a) 5 + 15.9 ° (with 0.35; water).

Found,%: C 48.66; H 7.93; N 5.32.

С22К42010№С1.

Calculated% C 49.85; H 7.99; N 5.29.

EXAMPLE 2. Mono-M-myristization hitoi-pei (1b).

To a solution of 750 mg (1.81 mmol) of hydrochloride chitobiose in a mixture of 5 ml of water and 4B ml of ellole, 0.66 ml of triztilamine and

Q

15 0 25

 i

40 45 50

410 g (9 mmol) of myristoyl chloride. Further processing is carried out according to Example 1. Exit. And „„ d1 nzchi 16404 mg (38 0%) White powder g / J5rj 14.7 ° (s 0.3; water).

Naively. % C 52.86; H 8.64; N 4 89

With bNch OyG With

Calculated,% C 53.28; H 8.60; N 4.78.

PRI m o about 3. Mono-M-stearate hitobiozy Po).

To a solution of 200 mg (0.48 mmol) of chitozoic hydrochloride in a mixture of 1 ml of water and 4 ml of DMF, 0.15 mp of triethylamine and 235 mg {0.54 mmol) of m-hydroxysuccinimide ester of stanoic acid in 10 ml of ethanol are added. Further processing is carried out according to the reference code RU 1 of the SLEEP of compound IB 43.8 mg (14%),

Wimpy 1. with. a + 29 0 ° (s 0.1, hearth - ethnnol 1: 1)

Found,% C 54 B 5 H 9 57; V 4 64.

C: / iH - QicN2Cl

You incnei o,%; C, 56.10; H 9,10, N 4.36.

Pr i wi ep 4 Mono-fi-R S-3-oxymorbate chitobiasis. (1 g).

A solution of 290 g / g (0.7 mmol) of hydrochloride vi-tg iozch in 5 ml of water is passed through i-p, OHKV r 2 mp ion exchange hmopa Dowex p, g, m ring tsom ayut 3 mp water, “ : Oe and ripoiaiBHbie of water are added to a volume of 2 fn, to the ssm solution, pcC-aors of 1CO mg (0.7 mmop) of RS-3-oxy-hydrochloride tinic acid, 10 pyrigna per 290 (1.4 mmol A) K1M-diiikpohexylcarbodiimira in 10 ml of f iridine. The mixture is stirred for 24 hours at 20 ° C, the precipitate is filtered, the filtrate is evaporated, and the mixture is extracted with acetone. Further treatment of the solution of residue 1 was carried out as in Example 1. Compound yield 1 g, 82.4 mg (19.5%). For purification, the product is dissolved in water, applied to a polychrome-1 sorbent gum (5 mlX elution is carried out with water (10 ml), and then increasing gld1 erynol R water. Concentration TLC in the system n-ViCM - EtON - H20 20% 40 40.155 (iio oemu). Compound 1 g is dispensed from the column in 20-30% ethanol. After precipitating the alcohol with acetone, outputting the compound is 1 g 68.9 mg (16.3%). White powder

from af5 to 6.0 ° (from 0.3, water)

1%: C 53.02, H 8.67; G 4.53.

, НОоои№С1

E o. %; C 55.12; H 8.83, N 4.95.

P i and me 5, Mono-N-R, S-3-miriyuykimiristiat chitsbiosis (1e).

To a solution of 200 mg (0.48 mmol) of chitobiose hydrochloride and a mixture of 1 ml of water and 4 ml of DIPL, 0.15 ml of triethylamine are boiled. 1 Rustor 230 mg (51 mmol) of N-oxysuccinimide ester of R, Svf-myristoxychristino acid in 1 ml of ethanol. A further sample is carried out in Example 1. After additional purification on the C / i with Polybrom-1 eorbent, the output of Compound 1d is eluted, - Cggos in 40-50% iic-M etamol, 51 mg (15.5%). %).

White powder with. - 16.3 ° (with 0.3: water). Found,% C 57.61; H 9.38; N 4.00.

C40H76012N2CI

Calculated. %: C 59.13; H 9.43; N 3.45.

PRI me R 6. N-R, Z-Z-Oksirisisat-N -succinate chitobiose (1e).

To a solution of 390 mg (0.65 mmol) of compound 1 g in 20 ml of methanol was added 0.08 mg of triethylamine. Mixed portions with stirring - 150 mg succinic anhydride. After 3 h with the mixture being evaporated, the residue is dissolved in 4 ml of w / 7b. basified to pH 9.0 with tripgiltamine, the solution is acidified to pH 3.0 with acid, the precipitate is separated by centrifugation. The yield of sss- dinene 1e 215 mg (49.8%). 5th powder

s, 2 ° (with 0.2: water).

Found %: C, 53.77; H 8.59; N 5.00.

C30H54014N2

Calculated,% C 54.04; H 8.76; N 4.20.

PRI me R 7. MOHO-N-M chronic of chitotriose (1).

To a solution of 500 mg (0 C2) of chitotriose hydrochloride in 5 ml of choda and 45 ml of ethanol, 0.56 ml of triethylamine and 212 mg (0.86 mmM) of myristoyl chloride were added. Further processing is carried out according to Example 1. The yield of the compound is 1 g, 240 mg (37.4%). White powder with a, 5 + 7.2 ° (with 0.3; zod).

. PRI me R 8. Mono-N-R, S-3-oximirystat chitotriose (1 h) and di-N, N-R, S-3-okimimyotiat chitotrisza (1 and).

To a solution of 2CC mg (0.33 mmol) of chitotriose hydrochloride in 2 ml of water is added 0.116 ml of triethylamine and a solution of 117 mg (0.34 mmol) of the N-hydroxysuccinimide ester of R. S-3-oximeristoic acid in 20 ml of ethanol. Further processing was carried out as described in Example 1. The mixture of sediments 1z and 1 and 110 mg was removed, the product was dissolved in water and applied to the column with the hydrophobic sorbent Ocfadecyl SI 60 Polyol (Sevva, HGF), the elution was carried out on a make-up artist 4, the separation of substances is controlled by TLC. After precipitation from ethanol with acetone, the yield of the compound

1 68.5 mg (26.1%). White powder with + 6.3 ° {0.3; water).

Found,%: C 48.05; h 8.40. N 5.07.

SzzNvzOschMzSts

Calculated,%: C 48.0; H 7.93; N 5.25,

0

0

five

0

WITH

about

0

five

0

five

Upon precipitation from ethanol with dcetone, the yield of compound 1 and 22 mg (6.8%). White powder from 13.5 ° (from 0.25: water).

Found,%; C 54.38; H 9.26; N 4.72.

C46HtyOl7N3CI

Calculated,%: C 55.77; H 8.95; N 4.24.

PRI me R 9. Mono-M-myristate chitotraose (1 k).

0.34 ml of triethylamine and 1GO mg (0.65 mmol) of myristoyl chloride are added to a solution of 500 mg (0.62 mmol) of hitrograzy hydrochloride in 5 ml of water and 45 ml of ethanol. After 24 h at 20 ° C, the precipitate formed is filtered off, further processing of the filtrate is carried out as in Example 1. The yield of the compound is 1k 160 mg (26.5%). White powder with atf + 1.5 (with 0.3; water).

Found %: C 45.27: H 8.03; N 5.24.

SzeN7b019M, 1S1z

Calculated,%: C 45.67; H 7.67; N5,61.

PRI me R 10. Mono-N-R, S-3-oximeristate chitotetraose (1 l).

To a solution of 200 mg (0.25 mmol) of chitotetraose hydrochloride in 1 ml of water and 4 ml of DMF, 0.112 ml of triethylamine and a solution of 88.6 mg (0.26 mmol) of R, S-3-oximyristinic acid ester in 5 ml are added ethanol. Further processing is carried out according to Example 1. After purification on a column with a Polychrome-1 sorbent, the yield of compound 1l, zlyuyushchegos in 15-25% ethanol,

95 mg (37.8%). White powder with + 2.6 ° (with 0.3; water).

Found %: C, 44.91; H8.11: N5.56.

S.Ch N76020M4S 3

Calculated,%; C, 44.95; H 7.54; N 5.52.

PRI I, m 11. 11. Mono-NR, S-3-acetoxymyris of chitobiose (1 n).

To a solution of 200 mg (0.48 mmol) of chitobiose hydrochloride in 1 ml of water and 4 ml of DMF were added 0.15 ml of triethylamine and a solution of 193 mg (0.5 mmol) of the R, S-3-acetox N-ether. myristic acid in 5 ml of ethanol. Further processing was carried out according to Example 1. The yield of the compound was 1 n 116 mg (37.2%).

White powder with + 15.4 ° (with 0.3; water). Found,%: C 50.94; H 7.68; N4.11.

C28H52012N2CI

Calculated,%: C, 52.21; H 8.14; N 4.35.

PRI me R 12. MoHO-N-caproniate chitobiose (1 m).

To a solution of 300 mg (0.73 mmol) chitobiose hydrochloride in a mixture of 3 ml of water and 200 ml of ethanol was added 0.256 ml of triethylamine and 103 mg (0.75 mmol) of caproyl chloride.1 Further processing was carried out as in Example 1. The output of compound 1m 37 , 3 mg (11%). White

powder with + 18.3 ° (with 0.3; water).

Found,%: C 44 68; H 7.32; N 5, L.

CieH340ioN2CI

Calculated,% C 4G, 62; H 7.3, M 5 91

In the IR spectra of compounds 1j - n, there are bands - stubs at 1523, 1644, 2856,2922, characteristic of the amide bond bound fatty acid residue in the spectrum of the compounds (1 in, 1 and). In addition, there is an absorption band at 1716 cm 1, characteristic of the residue of fatty cells associated with an ester bond.

The structure of compound 1g as an MnO-M-3-oxymoristoyl derivative of chitobiose was proved using a spectroscope and 13C-NMR.

In tab. 1 shows the data of the 13C-NMR spectrum of chitobiose and compound 1g.

The study of the biological properties of: compounds was carried out in mice in experiments in vivo and in vitro. Toxic drugs are investigated beforehand in mice of the CBA line (18-20 g) according to the method of Kerbeoa. The observation time for animals after administration of the drugs is 15 days. It was established that the LRD of the studied compounds is, respectively, mg / kg: 1: 102; 16 92; 1 to 110; 1y98; 1d98; 1zh9b, 1z9 °: 1m 106, 1k97; 1l 100; 1m 108; 1n 95.

Thus, all researched preparations of chito-igosaccharide derivatives have a dose of 92–110 mg / kg and can be attributed to the discharge of low-current compounds.

The lethal dose for lipopolis; achary and lipmda A for mice is 0.1–0.5 mg / kg.

Among the various methods used for screening immunomodulators, tests were chosen that characterize the degree of monokin production by macrophages and spleen cells for the following reasons:

an increase in the production of interleukins-1 (IL-1) and tumor necrosis factor (PMF) is directly corrected by retardation, of primary growth n vivo;

These metsdikm are widely used to evaluate the immune modulating action of lipopolysaccharides (LPS), lipid A and its analogues. The results of the experiments are given in table 2-6

As can be seen from the data table. 3 compounds 1g, 1d, 1e, 1z, 1l according to the potential of stimulating production. IL-1 macrophages, increasing with increasing dose of the drug, and compound 1d at a concentration of 10 mHg / ml is a more active stimulant than lipid A. In the experiment with low doses of compounds, equimol is 100 pg / ml lmpolysaccharide, compound 1a is more active , -m LPS (CT / MV index

ption for compound 1a is 2.27, dl / PS-1.6)

B. "and the scientific compounds are HINP1, TNFα, macrophages or spleen cells of mice in in vitia (Table L), and in the first case they are more active, and in the second they are similar in activity to LPS and lipid L

Table 2 shows the production of IL-1 by peritonezal macrophages of mice during the extraction of chitooligosaccharide derivatives in v.tto.

Table 3 shows the production of TNF by peritone-epic macrophages., When the inactivation of prostate chitol oligosaccharides is in vitro.

 In addition, it conducts tests of JOB on i /. mommuno; I have a very active, criterion for the production of factor tumor necrosis (FIO) In vlv. - P experiment ° ha inbred

f. MO.V tzSl. )

Bm / s, line B 3 / s entered Dytvn l ribKK1 once a day by 5 Cmkg / kg / 1 tolio1OS1x ipujnoB i w LIR yes A, or 5 mg / Vr astasigi-dd (ester cx | and saturated fatty acids) which corresponds to one-dose / therapeutic dose of drugs for 1 time two hours, vm.yutnykh.by take blood and and s HPG KV test n. ichi. factor checkers, and tumors by cytocnecyrchid and otdizn1 and, Veritelnaya} and kg. tumor current 1 1.3. Og.nu the reaction is carried out by the standard method of dyeing aunts with crystal violet followed by OI 1 1 cytophotometry

From the presented data, it can be concluded that the studied hygooligosaccharides have the ability to gtmulate the production of tumor necrosis factor And in vivo, without significantly

 lipid A indicator In contrast to highs / sh. -opes. Harmides l, w w u A in this conject: we read p m i p ot the ichness of organisms. AP "pidche ppas inductsoy cht tg.iaa, which allows u to

 compared with the chitooligosaccharide-) io-c) low immunomodulating and antiglucose effect of this drug.

O. United 1g products

IT in the serum of normal mice, Tg with xe, both lipopolises read and lipid A (tab rz rz compound 1a 1g, 1o. 1l individual.) Increase the macrostagic activity of macros in relation to tumor

KLRGKE 1 (Table. P).

In tab 5 dsnch production of TNF in the sychochoma of nop mice in 2 hours after injection of compounds 1 g of lipid A and LPS

In tab. 6 d of the tumor-forming activity of makofoveg relative to tumor clerks R 815.

Thus, the studied compounds are immunomodulatory stimulants that stimulate key elements of antitumor immunity, and can be considered as potential antitumor drugs.

The antitumor activity of the compounds is studied in CBA mice weighing 18–20 g (males) with transplantable linear non-specific ascites Ehrlich carcinoma. The criterion of the antitumor effect of the drugs is a comparison of the average life expectancy (ALE) of animals in the tumor and control groups, the drugs are administered at a dose of 60 mg / kg for 5 days (Table 7).

Table 7 shows the antitumor effect of chitooligosaccharide derivatives.

From the results given in table. 7, it can be seen that all of the studied compounds exhibit antitumor properties, the most active is compound 16 (ILA 188%). Thus, the antitumor effect of chitooligosaccharide derivatives on the ascitic form of Ehrlich carcinoma has been established.

Thus, compounds 1-1n are much less toxic than structural analogs (lipid A and LPS), and at the same time these

The compounds possess a number of valuable biological properties: they stimulate the production of IL-1 cells and the full name by cells of the immune system, enhance the tumor-static activity of macrophages, exhibit an antitumor effect in vivo, and are also easy to obtain.

Claims (2)

Claims of the Invention Derivatives of chitooligosaccharides of the general formula I 0 15 where 1a), 1 0, m 1.n 6, Ri - H; 16), l 0, m 1, n 10, Ri-H; 1e) k 1,1 0, m - 1, n 14, Ri-H; 1r) k 1,1 0, m. 1.n 10. Ri - OH; 1e) k 1, I 0. m - i. n 10, Ri CCA (CH2) 12CH3; 1e) k-0. l-1.m-1.n- 10, Ri - OH; 1g) k 2.1 0. m 1, n 10. Ri - H 1s) k 2,1 0, m 1 ,. Ri-OH; 1n) k 1, i-o. m-2. n-IO. Ri.-OH; lK) k-3. I -0. m - 1. n 10, Ri - H; 1l) k - 3, I - 0. m - 1. n is 10. Ri-OH; lM) k- 1. 1-0. m 1. n - 2. Ri - H; 1h) k - 1. I - 0. m - 1. n - 10, RI - AOSOS, possessing immunomodulating and antitumor activity. Table 1  In addition, the spectrum contains signals of carbon atoms of the fatty acid residue at 14.4; 23.2; 26.4; 30.6; 32.6; 37.8; 8 44; 2.69; five; 173.3 ppm table 2  The dose of 10 μg / ml.  The dose of drugs equimolar 100 ug / ml lipopoliseharida.  The serum was tested in a titer of 1: 4.  Blood serum is tested in a titer of 1: 4.  Compounds are administered at doses of 100 ng / ml lipopolysaccharide. Table 3 Table 4 Table 5 Table 6  The increase in the life expectancy of mice compared with the control. Table 7