Method of producing salts of muramylpeptides

专利摘要:
The invention relates to substituted peptides, in particular the sodium salts of muramyl peptides: N-acetyl-mura-mil-b-alanyl-B-glutamyl (C 1) -L-ala-nyl-2- (1,2-dipalmitoyl-3n-glycerol- 3- -oxiphosphoryloxy) -ethylslmide (l); N- -acetyl-muramyl-L-alanyl-B-glutamyl- (Cot) -alkyl ester () - (CV) -L- -alanyl-2- (I, 2-dipalmito-1-5n-glycero-3- oxyphosphorylox-O-ethylamide (s), which have antiviral activity and can be used in medicine. To detect the activity; new salts I and II were obtained among the substituted peptides. Synthesis is carried out from N-acetyl-muramyl-L-alanyl-D -glutamic acid or its C (alkyl () ester, which in the medium of the solvent mixture (CH01 +) in the presence of N-oxysuccinimide and dicyclohexylcarbodiimide) is condensed with the sodium salt of L-alanyl- 2- (1,2-β-dipalmitoyl-8n-glycero-3-oxyphosphoryloxy) ethylamide at room temperature. Tests I and II show that they have better antiviral activity than the known H-acetyl-muramyl-b-alanyl -2- (1,2-dipalmitoyl-8n-glycero-3-hydroxyphosphoryl-oxy) -stenslamide (pyrogenicity in LT ° C, is 0.48-1.1 vs. 2). I and II can be used against viral and bacterial pathogens and for the prevention of disease. 4 tab. With oo with with SL O5 CM
公开号:SU1299516A3
申请号:SU833622707
申请日:1983-07-22
公开日:1987-03-23
发明作者:Хартманн Альберт；Вакер Оскар；Башанг Герхард；Торчай Лайош
申请人:Циба-Гейги Аг (Фирма)；
IPC主号:

专利说明:

This invention relates to a process for the preparation of salts of muramyl peptides, new biological active compounds that can be used in medicine.
The purpose of the invention is to obtain new derivatives in the range of muramyl peptides, which have a higher antiviral activity and low toxicity.
Example 1. 3.60 g (5.66 mol (Co1 -) - N-acetylmuramyl-L-alanyl-B-glutamic acid p-butyl ester, 0.6 mol of water and 0.88 g (7.54 mmol ) With continuous stirring, N-oxysuccinimide is dissolved in 20 ml of a mixture of chloroform and methanol at a volume ratio of 4: K. 1.6 g (7.54 mmol) of solid dicyclohexyl-carbodiimide are added to the mixture and dropwise within 4 h at room temperature, a solution of 3.7 (4.72 mmol) of sodium salt of L-alanyl-2- (1,2-dipalmitoyl-glycero-3-hydroxyphosphoryl-oxy) ethyl-amide in 40 ml of chloroform is added. The mixture you hold for 1 h, evaporated on a rotary impl rer, to the residue was added 200 ml of dioxane, a solution of lyophilized
The product is suspended in 100 ml of chloroform, 600 ml of acetic acid ester is added, the mixture is cooled in an ice bath with continuous stirring for 1 hour. The undissolved part of dicyclohexylcarbodiimide is filtered off and the filtrate is evaporated. The residue is dissolved in 14 ml of chloroform-methanol mixture and the mixture 70: 30: 5 (volume ratio) and purified on a column (diameter 4.5 cm, length 80 cm) filled with 700 g gel of silicic acid 60 (Merck, 70-230 Mesh, ASTM) in the indicated solvent mixture. Prog-; Ny 500ml of this mixture, then the same mixture elutes the product, collecting fractions of 10 ml No. 133-152 and 153- 236, they are separately subjected to diofiltration. The percentage of the main fraction is dissolved in 300 ml of bidistilled water, quickly heated to 40 ° C, after cooling it is centrifuged, removing the undissolved particles of dicyclohexylurea.
The supernatant is supplied to the dialyzer (Amikon Corporation, Dan-Ver, Mass., 01932 USA, Model Z02, RM ultrafilter, 10176 mm diameter, inert, neon polysulfone-based polymer, medium
0
five

the mask is 10 A) c, dialyzed at 4 atm tech. against, 5 liters of bidistilled water, 0.5 liters of sodium phosphate-chloride buffer solution (0.1 M, 1: 1, pH 7) and 1.5 liters of water for complete leaching of chlorides.
The resulting dialysis product (70 ml) is sterile filtered on a filter with milipores (0.2 μm) and lyophilized. ; The sodium salt of N-acepsht-Muramyl-L-alanyl-B-glutamyl- (Cd) -n-butyl ester- (cr) -b-alanine-2- (1,2-dipalmitoyl-3n-glycerol 3-hydroxy-phosphoryloxy) ethylamide (3.4 mol H O) in the form of a colorless powder in an amount of 4.13 g (67% of theory):
  + 14 ° C (s 0.44; water);
Rf 0.37 (chloroform: methanol: water 70: 30: 5);
Rf 0.64 (ethyl acetate: n-butanol-pyridine: acetic acid: water - 42: 21: 21: 6: 10)
Calculated,%: C 54.77; H 8.94; N 5.07; P 2.24; H, 0 4.71
SbN issOjoNsPIIa 0.41 H20 (1381.63)
Found,%: C 54.3; H 9.1; N 5.6; 2.3, H, jO 4.7.
Calculated: Na 1.75
.s (1316,59)
Found ;: Na 1, 57.
The original product is obtained as follows.
5.5 g (8.1 mmol) of (c) -p-butyl- (c) -benzylether-4, b-O-isopropylidene-N-acetyl-muramyl-b-alanyl-P-glutamic acid dissolved in 60 ml of 60% acetic acid and left overnight at room temperature. The yellowish solution is evaporated to about half on a rotary is, par with, lyophilized.
4.8 g (7.5 mmol) of the obtained product are dissolved in 100 ml of a mixture of dimethoxyethane and water (9: 1), mixed with 0.8 g of a catalyst consisting of palladium oxide on coal (10%) and treated with hydrogen for 4 hours, the catalyst is filtered off. The filtrate was evaporated, the residue was purified twice by silica gel 60 chromatography (Merck, 1: 200, fractions 10 and 5 ml) in chloroform: methanol: water 70: 30: 5. The fractions containing the product are combined, evaporated.
The resulting product is partially presented in B1ode sodium salt
five
0
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31
(from silica gel) was dissolved in AO ml of distillate and passed through a 50 ml well-cleaned ion exchanger (Dowex 50w x 8 50/100 mesh, Form H) column 100 ml of bidistillate, the eluate was filtered through a filter of 0.2 millipores RcM and lysophilized. 3.7 g (697, from theory) (Co1) -p-6-utilenether-N-acetyl-muramyl-b-alanyl-B-glutamic acid are obtained in the form of a colorless powder, containing 0.6 mol of water.
Cc C +45 j GS (c 1.142, methanol);
Rf 0.23 (chloroform: methanol: water 70: 30: 5);
Rf 0.40 (n-butanol: acetic acid: water 75: 7.5: 21),
The starting material is prepared as follows.
4.4 g (11 mmol) of sodium salt of K-acetyl-4,6-0-isopropylidene-muramly acid (2.5 mol, 1 g) are suspended in 60 ml of dimethylformamide. With vigorous stirring, 4.4 g (P mol) of hydrochloride (Co (.) - p-butyl ester- (Cc.) - benzyl ester of L-alanyl-B-glutamic acid, 2.53 g ( 22 mmol of N-oxysuccinimide and 2.5 g (12.1 mmol of dicyclohexylcarbodiimide), the mixture is stirred overnight at room temperature.
For processing the suspension, the latter is diluted with 100 ml of ethyl acetic acid and, after half an hour of stirring at 0 ° C, undissolved particles are filtered off (dicyclohexaurea, sodium chloride. The filtrate is evaporated in vacuo at 30 ° C, the residue is taken up in 400 ml of acetic acid and ethyl acetate with bidylated water (10x50 ml) o After drying the organic phase and evaporating the solvent, 5.6 g (Cot) -p -butylether (Col) -benzyl ether of N-ace-TSP1-4,6, O-isopropolido- B-alanyl-b-glutamic acid in the form of an amorphous powder (75% of theory).
/ ot / +30 + 1 ° С (c 0.732; methanol);
Rf 0.87 (chloroform: methanol: water 70: 30: 5);
Rf 0.83 (h-butanol: acetic acid: water / 5: 7.5: 21).
The original product is obtained as follows.
5 o
five
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0
five -

0
164
5.6 g (12.1 mmol) of () -n-butyl ether- (cy) -benzyl ether N-tertiary. Butyloxycarbonyl-L-alanyl-B-glutamic acid is dissolved in 20 ml of abs, acetic acid ethyl ester, with vigorous stirring and excluding the ingress of moisture, 50 ml of 4.5 N are added dropwise. hydrochloric acid in abs. ether and the mixture is left to stand for one hour at 0 ° C. The yellowish solution is evaporated at room temperature to approximately 20 ml, mixed with 150 ml of dioxane and lyophilized .. 4.75 g (97% of theory) of (Cc) -n-butyl ether- (CJ) -benzylether-b are obtained. -alanyl-B- -glutamine acid as a colorless oil
  + 19 + (C 0.313; methanol);
Rf 0.58 (chloroform: methanol: water 70: 30: 5);
Rf 0.56 (ethyl acetate: p -butanol: pyridine: water 42: 21: 6: 10).
The original product is obtained as follows.
To 8.5 g (25.8 mmol) of hydrochloride- (Cct) -n-butylether- (C y) -benzylether-D-glutamic acid and 7.4 g (25.8 mmol) of N-t-butyloxycarbo- nyl-b-alanine-K-oxysuccinimide ester. dissolved in 150 ml of dimethylformamide, 2.6 g (25.7 mmol) of N-methylmorpholine are added and the mixture is left to stand for 20 hours at room temperature. The clear yellow solution was evaporated in vacuo at 30 ° C, the semi-solid residue was added to 500 ml of ethyl acetate and extracted seven times with anhydrous sodium sulfate. The residue after evaporation of the ethyl acetate is purified on 600 g of silica gel 60 (Merck, grain size 70-230, ASTM mesh) in chloroform: methanol 98: 2. The product in fractions 36-82 is collected and dried. Get (Col) - -butyl ether- (C 4) -benzylether-K-tert. - butylloxycarbonyl-b-alanine-B-glutamic acid as a colorless oil (6 g, 50% of theory).
oLl. - 1 ° С (c 0.315; dimethylformamide);
. .Rf 0.73 (chloroform: isopropanol:: acetic acid 70: 8: 2);
Rf 0.75 (p-butanol: acetic acid: water 75: 7.5: 21).
The original product is obtained as follows.
To 11.0 g (30 mmol) (Sy) -p-butyl ether- (C3) -benzyl ester of N-tert-buty-loxycarbonyl-B-glutamic acid, dissolved in 25 ml of abs, ethyl acetate, is added in the cold with constant stirring and exclusion of moisture 25 ml of 4 n hydrochloric acid in abs. ethyl acetate and leave it all to stand for one hour. The volatile constituents are evaporated at, the oily residue is introduced into 150 ml of abs. diethyl ether and the mixture was evaporated again (3 ml). The oily residue is dried over sodium asbestos at half vacuum. Get hydrochloride (Co.) - p-butyl ether- (soo) - -benzyl ester of D-glutamic acid (8.5 g; 92.4% of theory).
 9 + HS (with 0.646; methanol);
Rf 0.82 (chloroform: methanol: water 70: 30: 5);
Rf 0.68 (ethyl acetate: p-butanol: pyridine: acetic acid: water 42: 21: 21: 6: 10).
The original product is obtained. As follows.
20.0 g (64.6 mmol) of N-tert.-butyloxycarbonyl-D-glutamic acid benzyl ester (cU) is dissolved in 750 ml of abs. Tetrahydrofuran, 25.05 g (64.6 mmol) of cesium carbonate (Fluka , clean) dissolved in 80 ml of water are added dropwise and the whole mixture is distilled under vacuum. The residue was dissolved in 200 ml of abs. Dimethylformamide, the solvent% was evaporated and the whole operation was repeated. The crystalline residue obtained after drying under high vacuum is dissolved in 1 l abs. dimethylformamide and 13.3 g (97 mmol) of p-butyl bromide are added dropwise with constant stirring.
The suspension, obtained after 18 hours stirring at room temperature, is filtered, the filtrate is evaporated by half and after adding 1 liter of ethyl acetate, extract with water (10 x 100 ml). The organic phase is dried over sodium sulfate and evaporated. The oily residue is taken up in 100 ml of diethyl ether and crystallized by the addition of 1800 ml of petroleum ether and by holding at. Crystalline mass
five
0
five
0
five
0
five
0
five
sucked off, washed and dried. Receive (Sy) -p-butyl ether- (with dL) -6eH-zylether N-tert.-butyloxycarbonyl-D- -glutamic acid as colorless needles (21 g; 89% of theory). That 70-71 ° C;
 20 ° C (with 1.149, meta- .0 -
Rf 0.77 (chloroform-isopropanol: acetic acid 30: 8: 2);
Rf 0.92 (ethyl acetate: 1-butanol: pyridine: acetic acid: water 42: 21: 21: 6: 10) o
L-alanine-2- (1,2-dipalmitoyl-3n-β-glycero-3-hydroxyphosphoryloxy) ethyl amide, which serves as the starting product, is prepared as follows.
13.25 g (14.9 mmol) N-tert.-butyrotoxycarbonyl-b-alanine-2- (1,2-dipalmitoyl-Bp-glycero-3-hydroxyphosphoryl-oxy) ethyl, amide feed with a constant stirring, t are introduced into 170 ml of a mixture of trifluoroacetic acid and methylene chloride cooled to 0 ° C at a ratio of 1: 3 (V: V), and a clear solution is obtained. After soaking for 2 1/2 hours at room temperature, the solution is evaporated on a rotary evaporator at. The semi-solid residue to remove excess trifluoroacetic acid is mixed repeatedly each time with 100 ml of methylene chloride and then evaporated.
After five times rubbing the residue each time with 100 ml abs. diethyl ether and standing the resulting product to give a well-filtered suspension. It is filtered on a suction filter, washed with a solid product with diethyl ether, then twice with 100 ml of hot acetone and dried in vacuum at 60-70 ° C. 10.9 g of L-alanine-2- - (1,2-dipalmitoyl-Bp-glycero-3-hydroxyphosphoryloxy) ethylamide are obtained in the form of colorless crystals with Tn 138-147 ° C (95.6% of theory) .
 +30 + I C (with 1, chloro. Form: methanol: water 70: 30: 5);
Rf 0.14 (chloroform: methanol 7: 3);
Rf 0.40 (chloroform: methanol: water 70: 30: 5).
The product thus obtained is converted into the sodium salt as follows.
3.82 g (5 mmol) of b-alanine-2- (1,2-β-dipalmitoyl-3p-glycero-3-hydroxyphosphoryloxy) ethylamide are dissolved in 100 ml of a mixture of chloroform: methanol 7: 3 with brief heating to 35 C. Cool and carefully add 5 ml of IN sodium hydroxide solution dropwise. The clear solution is evaporated to approximately 1/3. the original volume and after adding 200 ml of dioxane lyophilized. The sodium salt of L-alanine-2- (1,2-dipoly to-to-Sn-hlsh epo-3-oxy-phylophyloxy) - -ethyl amide is obtained in the form of a friable powder (0.41 g).
Example 2 3.5 gSb, 9 mmol of (N-acetyl-muramyl-L-ethenyl-B-glutamic acid, methyl ester of N-acetyl-muramyl-L-ethenyl-B-glutamic acid and 1.2 g of N-oxysuccinimide are dissolved in 50 ml of a mixture consisting of from dimethylformamide, isopropanol and chloroform (-1: 3: 6; V: V: V), 2.10 g (10.35 mmol) of dicyclohexyl-carbodiimide was added and, with the exclusion of lagium, I kept it for 1 hour at room temperature temperature The suspension is mixed with 50 ml of ethyl acetate and the mixture is stirred for 30 minutes in an ice bath and then the insoluble particles of dicyclohexyl urea are filtered out. The filtrate is evaporated on a rotary evaporator to approximately 40 ml, as a result of the addition of 150 abs. ethyl ester falls out (twice), is filtered and dried, and ether is obtained with N-hydroxy-succinimide with Rf 0.43 (chloroform: methanol: water 70: 30: 5) —the raw material in the amount of 5.1g
3.43 g (4.4 mmol) of b-alanine-2- (1,2-dipalmitoyl-3p-glycero-3-hydroxyphosphoryloxy) ethylamide are suspended in 45 ml of chloroform and at 40 ° C for 0.86 ml (6.1 mmol) of triethylamine dissolved in 5 ml of chloroform was added dropwise to the mixture for 5 minutes, thus obtaining a clear solution. A solution of the indicated active complex in 100 Ml of a mixture consisting of dimethyl fbrmamide, chloroform and dioxane (1: 14: 6; V: V: V) is added dropwise to it while vigorously throwing, with excluding the ingress of moisture for 5 minutes. .
After 2 1/2 hours stirring at room temperature, the somewhat turbid solution is evaporated on a rotary evaporator until dry (30 ° C. The resulting product is purified on silica gel 60 (Merck, grain size 70-230 Mesh, ASTM) in chloroform: methanol: water 70: 30: 5 (5 ml fractions). Fractions containing product are collected (DS). The residue after solvent evaporation is dissolved in 250 ml of double-distilled water and purified in the same way as described in example 1, by means of dip-filtration ( AMICON agitator, type 402 (0.30 / 76 mm ultrafilter). Solution remaining in I Alka, filtered through a filter with millipores (0.2 MKm) and lyophilized, the sodium salt of C-acetyl is obtained. Muramyl-b-alanyl-B- -glutamide- (Co.) -methylether- (C 5:) -1 -alanine-2- (1,2-dipalmitoyl-5p-glycero-3-hydroxyphosphoryloxy) ethyl amide, 2.96 mol But in the form of a colorless powder.
s. +11 j Gs (with 0.285; 10% acetic acid);
Rf 0.25 (chloroform: methanol: water 70: 30: 5);
Rf 0.39 (ethyl acetate: p -butanol-pyridine: acetic acid: water 42: 21: 21: 6: 10) (in the amount of 3.1 g; 55% of theory).
The starting compounds are prepared as follows.
7.7 g (21.2 mmol) of hydrochloride (CoL} -methylether- (C V) -benzyl ester of b-alanyl-B-glutamic acid and 8.96 g (23.3 mmol) of K-acetyl-4 sodium salt , 6-0-isopropylidene-muraminic acid are combined analogously to example 1 according to the method of dicyclohexycarbodiimide): N-hydroxysuximide. Without further purification, the obtained product was split into 100 ml of 60% acetic acid. After 5 1/2 hours of stirring at room temperature, the solution is evaporated, mixed with water, again evaporated and lyophilized after addition of 100 ml of dioxane. The residue is cleaned with a 60 ml gel of silicic acid in chloroform: methanol:. : water 70: 30: 5 (7 ml fractions), the process is carried out twice.
The fractions, the content of the desired product, are combined, the solvent is evaporated. The residue (Co (.) Is methyl ester- (C) -benzyl ester of N-acetyl-myramyl-L-alanyl-B-glutamic acid as a colorless foam with Rf 0.63 of chloroform: methanol: water 70:30: 5) in the amount of 6.9 g.
6.0 g (10 mmol) (Co () - methyl ether - - (SU) -benzyl ether N-acetyl-muramyl chromatograph on 550 g gel of silicic acid 60 „
The product is collected, mixed with 200 ml of double distilled
fO
conditions (o, 45 µm) and lyophilized. The sodium salt of N-acetyl-muramyl-b-alanil-B-glutamyl- (Cot) -t-butyl ester- (sous) -b-alanine-2- (1, 2-di-palmitoyl-8n-glycero-3-hydroxyphosphoryl oxy) ethyl amide as a colorless powder containing 3.4 mol of water.
 j ° -1-14 1 ° C (C 0.817; 10% acetic acid);
 f5
-B alanyl-B-glutamic acid, ravod (), filtered in sterile mixture of 1,2 dimethoxyethane and water 95.5 (V: V), hydrogenated in the presence of 0.6 g of palladium-on-carbon catalyst ( 10%) for 1 1/2 hours at normal pressure. The catalyst is filtered off and the filtrate is evaporated at room temperature under elevated pressure. The residue is purified on 400 g of silica gel 60 in chloroform: methanol: water 70: 30: 5 (10 ml fractions), as indicated above.
The fractions containing the product are combined, the solvent is evaporated. The residue in the form of sodium salt is purified from salts, as described in example 1, in 50 ml of Dowex 50 WX 8 (50/100 Mesh, form H, strongly acidic cation exchanger). The filtrate is passed through a filter with millipores (0.45 microns) and. lyophilized. Get (Cot) -methyl ester of H-acetyl-muramyl-b-alanyl-B-glutamic acid (1.27 mol
20
25
Rf 0.29 (chloroform: methanol: water 70: 30: 5);
Rf 0.53 (ethyl acetate: P -butanol: pyridine: acetic acid: water 42: 21: 21: 6: 10). The output of the sodium salt of 8.39 g of 78% of theory Peptide derivative, used as the original product, was prepared as follows.
38.3 g (63.5 mmol) (Co () - tert.-β-butyl ester- (CU) -benzyl ether (4-biphenylyl) -propyloxycarbonyl -b- -alanyl-B-glutamic acid is dissolved in 700 m a mixture of trifluoroethane 30 and water 9: 1 (V / V) and as a result of adding dropwise 1.23 n hydrochloric acid in the same solvent mixture (1 part by volume of concentrated hydrochloric acid and 9 volume h (trifluoroethano-35), the pH value decreases to 1.5 (2 1/2 hours, consumption 51.66 ml, i.e. 81% of theory).
H „0) in the form of a colorless powder with +47 - 1 ° С (С 1.249; meta -
(58%
from tenol) in the amount of 3.1 g of Oriya).
Rf - 0.80 (chloroform: methanol: water 70: 30: 5);
Rf 0.30 (acetonnitrile: water 3: 1);
Rf rr 0.43 (ethyl acetate: acetic acid: water: methanol 67: 10: 23: 12) o
38.3 g (63.5 mmol) (Co () - tert.-β-butyl ester- (CU) -benzyl ether (4-biphenylyl) -propyloxycarbonyl -b- -alanyl-B-glutamic acid is dissolved in 700 m a mixture of trifluoroethane 30 and water 9: 1 (V / V) and as a result of adding dropwise 1.23 n hydrochloric acid in the same solvent mixture (1 part by volume of concentrated hydrochloric acid and 9 volume h (trifluoroethano-35), the pH value decreases to 1.5 (2 1/2 hours, consumption 51.66 ml, i.e. 81% of theory).
Light yellowish solution on the rotor
Nominal evaporator in a vacuum, creating a PREAMER 3. 3. 9.4 g (11.1 mmol) with a 0 m water jet pump, with C (C) —T-butyl ether N-acetyl-mura — evaporated to almost 100 ml then the dol-b-alanyl-b-glutamic acid is translated as in example 2 into the active ester and then as described. combined with 6.48 g (8.2 mmol) of L- -alanine-2- (1,2-dipalmitoyl-3p-glycerol-3-hydroxyphosphoryloxy) ethylamide in the presence of triethylamine. The resulting product is suspended in 250 ml of bidisbavl 200 ml of dioxane, the mixture is dried in the cold.
The semi-solid residue is dissolved in 30 ml of abs. diethyl ether and at 0-5 ° C with constant stirring, and the product is precipitated with 150 ml of petroleum ether. Determine in the cold (-2b ° C), decant and repeat this.
(procedure twice. The oily residue is then introduced into 100 ml of tert.-butanoethyl water and heated quickly to 37 ° C, stirred for 15 minutes in an ice bath, filtered insoluble dicyclohexylurea particles and the filtrate is subjected to diafiltration.
(procedure twice. The oily residue is then introduced into 100 ml of tert.-butanol, lyophilized and the residue is dried over sodium asbestos under high vacuum. Hydrochloride is obtained
as described in ripHMepe 1 (AMICON - IU - 5-5 (CoO-tert-butyl ester (SU) -benzyl-shalka 402, ultrafilter PM And 30/76 mm of ether b-alanyl-B-glutamic acid remaining in the mixer The solution (120 ml), in the form of a highly hygroscopic south-west, is lyophilized and the product is cleaned with a powder.
a 550 g chromatograph of 60 „silica gel
The final product is collected, mixed with 200 ml of double distilled
conditions (o, 45 microns) and lyophilized. The sodium salt of N-acetyl-muramyl-b-alanil-B-glutamyl- (Coth) -t -butyl ester (s) -b-alanine-2- (1,2-dipalmitoyl-8n- glycero-3-hydroxyphosphoryl-oxy) ethyl amide as a colorless powder containing 3.4 mol of water.
 j ° -1-14 1 ° C (C 0.817; 10% acetic acid);
water (), filtered in sterile
f5

 water (), filtered in sterile
0
five
Rf 0.29 (chloroform: methanol: water 70: 30: 5);
Rf 0.53 (ethyl acetate: P -butanol: pyridine: acetic acid: water 42: 21: 21: 6: 10). The output of the sodium salt of 8.39 g 78% of theory. The peptide derivative used as starting material is prepared as follows.
38.3 g (63.5 mmol) (Co () - tert.-β-butyl ester- (CU) -benzyl ether (4-biphenylyl) -propyloxycarbonyl -b- -alanyl-B-glutamic acid is dissolved in 700 m mixtures of trifluoroethane 0 and water 9: 1 (V / V) and as a result of adding dropwise 1.23 n hydrochloric acid in the same solvent mixture (1 part by volume of concentrated hydrochloric acid and 9 volume h. trifluoroethano-5 la) the pH value decreases to 1.5 (2 1/2 hours, consumption 51.66 ml, i.e. 81% of theory).
A light-yellowish solution on the rotor with a 0 m water jet pump, with ZO s, is evaporated to almost 100 ml, then
Bavl 200 ml of dioxane, the mixture is dried in the cold.
The semi-solid residue is dissolved in 30 ml of abs. diethyl ether and at 0-5 ° C with constant stirring, and the product is precipitated with 150 ml of petroleum ether. Determine in the cold (-2b ° C), decant and repeat this.
(procedure twice. The oily residue is then introduced into 100 ml. tert-butano. 11
20
Cdl-j j: 1 ° C (C 1.630; chloroform);
Rf 0.60 (chloroform: methanol: water 70: 30: 5);
Rf 0.45 (pb, tanol: acetic acid: water 10: 1: 2: 8) 19.25 g (76.5% of theory).
fO
To a solution cooled to 0 ° C, consisting of 65.96 g (0.2 mol) of hydrochloride (Sy) -t-butyl ester– (SU) benzyl ester of D-glutamic acid and 65.47 g (0.2 mol) (4-biphenylyl) -propyl-rxi-car-6-C3-3-S-L-alanine in 650 ml of abs. 22 ml (0.24 mol) of I-methylformalin and then 59.11 g (0.24 mol) of 2-ethoxy-H-ethoxy-carbonyl-1,2-dihyd are added with constant stirring -, roquinoline. After 10 hours of stirring at room temperature, the yellow solution is evaporated on a rotary evaporator at 30 ° C. The residue is introduced into 1 liter of ethyl acetate, extracted with water (5x200 ml), the aqueous phase is extracted with 0.5 l of acetic acid ester and the collected organic phases are dried.
The product obtained after evaporation of the solvent (product 150 ml) was repeatedly purified on a silica gel 60 chromatograph (1:20) with ethyl acetate. Get (Coi) - tert. -butylether- (with If -benzylether-N Example 4. To a solution of 4.62 g (6.05 mmol) of 1, -alanine-2- (1, 2-dipalmi-thoyl-Sn-glycero-3-oxy-propyloxy) - - ethyl amide in 100 m chloroform:: isopropanol: water 70: 30: 2 while stirring at 15 ° C for 10 minutes, 14.52 g (7.26 mmol) of a 0.5 M solution of triethyl amine in a mixture are added dropwise chloroform: isopropanol: water 70: 30: -2 Then 4 portions of the mixture are added over a period of 20 minutes. 1.5 g in solid form of 6.0 g (7.26 mmol) of (Co1) methyl ester - (C) -C- -Hydroxysuccinimide ester of N-propionyl-desmethylmuramyl-b-alanyl-B-glutaminic acid, containing more dicyclohexyl urea, than the solution becomes cloudy eyes.
Then, after stirring for 15 minutes at 15 ° C, the cooling is stopped and stirring is continued for another 2 1/2 hours at room temperature. Then the turbid solution thus obtained is evaporated in vacuo at ZO C. The residue (10.75 g) is suspended in 170 ml of bidistilled water (pH 5.5), the suspension is adjusted to pH 6.0 by addition of 0.3 ml of triethylamine; insoluble particles (dicyclohexyl ureas) are filtered off and
25
The (4-biphenylyl) -propsh 10xycarbonyl-5 filtrate is lyophilized in high va-b-alanyl-B-glutamic acid as a colorless oil, oi. -14 + 1 ° С (С 1.625; ethyl acetate ethyl acetate) (100 g, 83% of theory);
Rf 0.73 (chloroform: isopropanol:: acetic acid 70: 8: 2);
Rf 0.51 (toluene: ethyl acetate 1: 1).
The hydrochloride used as the starting material (Cc /.) - tert.-butyl ester- (SU) -benzine 1-ester of D-glutamic acid is obtained by conducting a reaction between the (su) -benzyl ester of D-glutamic acid and isobutanol in a mixture, consisting of 1,4-dioxane and sulfuric acid, the product is isolated as colorless needles with T ;, 108-109 ° C.
ten
 -16 + (with 1.235;
ethanol);
Rf 0.84 (chloroform-methanol: water 70: 30: 5);
- five
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- 29951612
Rf 0.64 (ethyl acetate: h-butanol: pyridine: acetic acid: water 42: 21: 21: 6: 10) in the amount of 61% of theory).
Example 4. To a solution of 4.62 g (6.05 mmol) of 1, -alanine-2- (1, 2-dipalmi-thoyl-Sn-glycero-3-oxy-phosphorycloxy) -ethyl amide in 100 m of a mixture of chloroform:: isopropanol : water 70: 30: 2 while stirring at 15 ° C for 10 minutes, 14.52 g (7.26 mmol) of a 0.5 M solution of triethyl amine in chloroform: isopropanol: water 70:30 : -2. Then, over 4 portions, 1.5 g are added in solid form, 6.0 g (7.26 mmol) of a (Co1) methyl ester- (C) -K-hydroxysuximine ether nyl-desmethylmuramyl-b-alanyl-B-glutamic acid, containing more dicyclohexyl urea, and the solution becomes cloudy before the eyes.
Then, after stirring for 15 minutes at 15 ° C, the cooling is stopped and stirring is continued for another 2 1/2 hours at room temperature. Then the turbid solution thus obtained is evaporated in vacuo at ZO C. The residue (10.75 g) is suspended in 170 ml of bidistilled water (pH 5.5), the suspension is adjusted to pH 6.0 by addition of 0.3 ml of triethylamine; insoluble particles (dicyclohexyl ureas) are filtered off and
25
0
five
cuome.
The lyophilisate thus obtained is dissolved in 300 ml, then lyophilized under high vacuum.
The sodium salt of N-propionyl-desmethylmuramyl-b-alanyl-B-glutamyl- (Co1) -methyl ester- (c) -b-alanine--2- (1,2-dipalmitoyl-8n-glycero-3 is obtained -oxaciphosphoryloxy) ethylamide as a colorless powder containing 1.90 mol of water.
  3.7 + O, GS (C 0.672; chloroform);
0 ° +1.7 T Oh, GS- (C 1.044; water);
Rf 0.42 (chloroform: methanol: water 70: 30: 5);
Rf 0.62 (ethyl acetate: p-butanol: pyridine: acetic acid: water 42: 21: 21: 6: 10) in the amount of 5.64 g (71.2% of theory).
The original product is obtained as follows.
five
5.12 g (10.1 mmol) of dL-methyl ester of M-propionyl demethylmuramyl-β-alanyl-B-glutamic acid are completely dissolved in that ml of chloroform: isopropanol 7: 3 and as a result of the addition of 10 ml of dimethyl formamide.
2.68 g (13 mmol) of dicyclohexylcarbodiimide and 1.50 g (13 mmol) of N-oxysuccinimide were added to this solution and the resulting clear colorless solution was stirred for 1 1/2 hours at room temperature and then held 17 h at 4 ° C ..
The suspension thus obtained is mixed with 400 ml of diethyl ether and then stirred for an additional hour at room temperature.
Then the crystallized acid is stripped off, washed with diethyl ether and dried in vacuum over phosphorus pentoxide.
Cgry (CdO-methylether-. -4c y) -K-hydroxysuccinimide ester-M-propionate-desmethylmuramyl-b-alanyl-O-glutamic acid is obtained as colorless crystals, containing dicyclohexylurea in the form of impurity and used without further purification. .
Rf 0.46 (chloroform: methanol 5: 1);
Rf 0.72 (chloroform: methanol 7: 3). The yield of ester 8.34 g,
The original product is obtained as follows.
A solution of 13.0 g (21.75 mmol) (Cot) -methyl ester- (C) -benzyl ester of N-propionyl-d zmethylmuramyl-b-alanyl-B-glutamic acid in 250 ml of a mixture of dimethoxyethane: water 20 : 1, 2.5 g of the catalyst are hydrogenated with palladium on carbon (10%) for 1 h at room temperature and normal pressure.
The catalyst is then filtered and the filtrate is evaporated to dryness in vacuo and the resulting residue is mixed three times with 50 ml of water and then evaporated again.
The precipitate is dissolved in approximately 100 ml of bidistilled water, filtered through a millipore filter (NAL GENES: 0.2 µm) and lyophilized under high vacuum. N-propionyl-desmethylmuramyl-b-alanyl-B-glu ot-methyl ester is obtained.
0
five
0
five
0
five
0
five
0
five
tamic acid (0.68 mol of water) in the form of colorless lio-lilisate +14.9 4. 0, ° С (s 1.067; methanol), in the amount of 11.0 g (96% of theory).
Rf 0.78 (chloroform: methanol: water 7,: 30: 5);
Rf 0.57 (ethyl acetate: p-butanol: pyridine: acetic acid: water 42: 21: .21: 6: 10).
The original product is obtained as follows.
A solution of 20.7 g (32/46 mmol) (CN) - methyl ester- (C) -benzyl ester of 4.6-0-isopropylidene-I-propionyl-desmethylmuramyl-b-alanyl-B-glutamic acid in 400 ml of 60% acetic acid is stirred for 21 hours at room temperature, then evaporated in vacuo at 30 ° C and the resulting residue is sequentially mixed three times each time with .100 ml of water and again evaporated. The resulting crude product is purified by chromatography on a column of 1000 g of silica gel 60 (Merck, 0.063-0.200 mm) in methylene chloride: methanol 85:: 15 (15 ml fraction) Fractions 207-290 are collected and evaporated in vacuo at 30 ° C . The (Cc1) methyl ester- - (c) -benzylether of N-propionyl-desmethylmuramyl-b-alanyl-B-glutamic acid containing 0.24 mol of water is obtained.
  +16 + 0.1 С (С 1.463; methanol);
Rf 0.24 (chloroform: methanol 9: 1);
Rf 0.53 (chloroform: methanol 5: 1);
Rf 0.94 (chloroform: methanol: water 70: 30: 5) in the amount of 13.7 g (70.1% of theory).
The original product is obtained as follows
To a suspension of 13.2 g (35.77 mmol) of sodium 4,6-0 isopropylidene-H-propionium, p-desmethylmuraminic acid in 200 ml of dimethylformamide was added at room temperature with stirring 9.6 g (46.5 mmol) of dicyclohexylcarbodiimide, 5.35 g (46.5 mmol) of N-oxysuccinimide and 12.8 g (35.77 mmol) of hydrochloride (CcA) methyl ester- (C11) -be: nzylether of L-alanine-B-glutamic acid and all stir for 21.h at room temperature,
The white suspension thus obtained is mixed with 200 ml of vinegar151.
ether, stirred for 1 h at, separated from the crystallizer (dicyclohexyl urea), on a suction filter, rinsed with ice-cold acetic ether and the filtrate evaporated in vacuo at 30 ° C. The residue thus obtained was introduced into 300 ml of ethyl acetate and 50 ml of 2N are sequentially washed twice. acid, water, 10% sodium hydro carbonate solution and water again.
The ether layers are combined, dried over sodium sulfate, filtered and evaporated in vacuo.
The (Cc) 1) -methylether- (C} 1) -benzylether of 4,6-0-isopropylidene-M-propionyl-desmethylmuramyl-b-alanyl-B-glutamic acid is obtained.
Rf 0.65 (chloroform: methanol 9: 1) ;.
Rf 0.86 (chloroform: methanol 5: 1) in the amount of 21.3 g (93.3% of theory).
Both initial produsts are obtained as follows.
To a solution of 20.3 g (48.0 mmol) of (Cc) -methyl ester (s) -benz1-ester of N-tert-butyloxycarbonyl-b-alanyl-D-glutamic acid in 100 ml of absolute acetic ester is added at 0 ° C 100 ml of approximately 5 N hydrochloric acid in absolute ether and stirred for 2 hours at 0 ° C.
The yellowing solution is evaporated in vacuo, the residue obtained is mixed twice each time with 100 ml of absolute ether and again evaporated. After digesting with absolute diethyl
ether (2x100 ml) the residue is dried
in vacuum and get hydrochloride
(Cc1) -methyl ester- (C}) -benzyl ester of L- -alanyl-B-glutamic acid, soda p ”gaşe1y 0.31 mol of water.
     (from 0.043
methanol);
Rf 0.72 (chloroform: methanol: water 70: 30: 5);
Rf 0.44 (chloroform: methanol 5: 1);
Rf 0.31 (chloroform: methanol 9: 1) in the amount of 1.7 g (94.8% of theory).
A solution of 34.4 g (73.8 mmol, 2.14 mmol) containing sodium chloride 1oC-O-benzyl-4,6- -0-isopropylidene N-propionyl-desmethylmuraminic acid sodium salt in 340 ml of water is hydrogenated at constant value
1616
pH 7.1 6.0 g of a 10% catalyst palladium-carbon for 23 hours at room temperature and normal pressure. Then the catalyst is filtered off, the filtrate is evaporated in vacuo at 30 ° C and at a pH of 7.1; and the residue thus obtained is dried under high vacuum over phosphorus pentoxide. Sodium salt of 4,6-0-isopropylidene-L-propionyl-desmethylmuraminic acid is obtained (16.6 g, 94.8% of theory)
Rf 0.50 (chloroform: methanol: water 70: 30: 5) ;.
Rf 0.66 (acetonitrile: water 3: 1).
The original product was prepared as follows.
A solution of 14.3 g (32.7 mmol) of methyl ester of 1 s.-0-benzyl-4,6-0-isopropylidene-M-propionyl-desmethylmuraminic acid in 130 ml of methanol is mixed with 24.4 ml ( 48.7 mmol) of 2N sodium hydroxide solution and stirred for 1 1/2 hours at room temperature.
Then, the clear yellowing solution thus obtained is obtained using 1N. hydrochloric acid is adjusted to pH 7.0 and evaporated in vacuo at 30 ° C.
After drying over phosphorus pentoxide, the sodium salt of I-0-g-benzyl-4,6-0-isopropyliden-N-propio-nyl-desmethylmuraminic acid is obtained in the form of colorless crystals in quantities of 27.2 g.
Rf 0.67 (chloroform: methanol: water 70: 30: 5);
Rf "0,74 (acetonitrile: water 3: 1
The original product is obtained as follows.
To a suspension of 6.75 g (224.7 mmol sodium hydride in 120 ml of absolute tetrahydrofuran is added dropwise at 5 ° C in a nitrogen atmosphere with constant stirring for 5 minutes a solution of 31.3 g (85.65 mmol) of 1Y .-0-β-benzyl-2-deoxy-4,6-0-isopropylidene-2-propionamide-L-B-glucopyranoside in 200 ml of absolute) tetrahydrofuran, and the temperature rises to 20 ° C.
The suspension thus obtained is stirred for another 2 hours at 40 ° C, then cooled to 0 ° and added dropwise under a nitrogen atmosphere and continuous stirring for 30 minutes.
171
to cooled to -15 ° C solution of 20.7 g (135.77 mmol) of methyl bromoacetic acid ester in 95 ml of abs. tetrahydrofuran. The suspension thus obtained is stirred for an additional 3 hours at 0-5 ° C, then 20 ml of methanol and 20 ml of tetrahydrofuran are added. The pH is adjusted to 6 using 4.5 ml of glacial acetic acid and the solution is evaporated in vacuo at.
The residue is dissolved with 200 ml of methylene chloride and the resulting solution is washed with water (3x125 ml). The collected methylene chloride fractions are dried over sodium sulfate, filtered and evaporated in vacuo at 30 C.
The product is obtained in the form of yellowish crystals, which are then purified by chromatography on a column of 500 g of neutral alumina (Yoelm N, super 1) using ether as. eluent (Yu ml fractions). Fractions 14-36 are combined and evaporated in vacuo.
1ot-0-β-benzyl-4, b-O-isopropylidene-N-n-pionyl-desmethylmuraminic acid methyl ester is obtained in the form of colorless crystals with Tp 121-123 ° (diethyl ether: oil ester 1: 2).
  +147.9 + 0.1 ° С (С 0.849; chloroform) - 32.2 g (86.2% of theory).
Rf 0.67 (chloroform: methanol 9: 1).
Example 5. Analogously to Example 4, it is obtained from 5.525 g (6.43 mmol) of crude (it also contains insignificant amount of dicyclohec of urea) (SY.) - tert-butyl ether - (SU) -I-oxy-succinimide ester of Nn-pionyl -desmethylmuramyl-b-alanyl-B-glutamic acid and 3.78 g (4.95 mmol) of L-alanine-2- (1,2-dipalmitoyl-5p-glycerr-3-hydroxyphosphoryloxy) ethylamide sodium salt of N-n-popionyl-desmethylmuramyl-b-alanyl-B- -glutamyl- (Cd) -tert.-butylether- (C y) - -b-alanine-2- (1, 2-dipalmitoyl-5p- - -glycero-3-hydroxyphosphoryloxy) ethyl amide as a colorless powder containing 2.64 mol of water.
LuLl ° +2.5 + 0.1 ° C (with 0.649; water);
  +5.8; + 0.1 ° С (С 0.694; chloroform);
1618
Rf 0.60 (chloroform: methanol: water 70: 30: 5), in the amount of 4.32 g (64.0% of theory).
A source product is obtained as follows.
Analogously to Example 3 of 3.44 g (6.15 mmol) of cat-tert.-butyl ester of H-propionyl-desmethylmuram il-b-alanyl-D-glutamic acid, 1.68 g (8.14 mmol) of dicyclohexylcarbodimide and 0.94 g (8.14 mmol) of N-hydroxysuccinimide are obtained as crude () -t-β-butyl ester- (s) -K-hydroxysuccinimide ester of N-propionyl-desmethyl-mymyl-L-alanyl-D-glutamine acid in an amount of 5 , 38 g in the form of colorless crystals containing dicyclohexyl urea, and used without further purification.
Rf 0.61 (chloroform: methanol: water 70: 30: 5).
The original product was prepared as follows.
A solution of 5.1 g (7 mmol) (CcA) -t-butyl ester- (Su) -benzyl ether of d-0-benzyl-K-propionyl-desmethylmuramsh-1-l-alanyl-B-glutamic acid in 100 ml of dimethoxyethane mixture: water 9: 1 is hydrogenated with 1.5 g of a 5% catalyst palladium on carbon (E 101N, Degussa) for 20 hours at room temperature and normal pressure. After 20 hours, the catalyst is filtered off and hydrogenated again with 1.5%. g fresh catalyst for 26 hours as indicated. Then the catalyst is filtered off and the filtrate is evaporated to dryness in vacuo at 30 ° C.
The residue thus obtained is dissolved in a mixture consisting of 100 ml of methylene chloride and 5 ml of isopropanol, the solution is mixed with 500 ml of a mixture of 3: 2 distillate n petroleum ether and stirred for another 1 hour at room temperature. Then remove the precipitated product and wash it with diethyl ether. Prepared with N-n-popyl-de-methylmeramyl-1-alkanyl-D-β-glutamic acid α-tert-butyl ether as a colorless powder containing 0.52 mol of water (3.7 g, 94.7, from theory).
 +20.2 + 0.1 ° С (С 0.902; water);
Rf 0.32 (chloroform: methanol: water 70: 30: 5);
Rf 0.54 (acetonitrile: water 3: 1).
191
The initial product is obtained in the following manner.
In analogy to Example 4, 7.3 g (9.5 mmol) of (Cdl) -t-butyl ether- (C y) -benzyl ether-1 oL-0-benzyl-4,6-0-isoproxy-1 propionyl-desmethylmuramyl-1-alanyl-B-glutamic acid and 150 ml of 60% acetic acid (Cot.) -term. -butylether p- f C y) - -benzylether 1 ° C-O-benzyl-K-propnyl-desmethylmuramyl-b-alanyl-B-glutamic acid as colorless crystals with T 152-153 C (methanol:: water 1: 5).
With. +68.9 J- 0, HS (with 0.991; methanol);
Rf 0.40 (chloroform: methanol 9: 1);
Rf 0.70 (anethylamine: water 3: 1), the amount of acid 5.9 g (85.3% of theory).
The original product is obtained as follows.
Analogously to example 4 of 4.65 g (9.62 mmol, 2.066 mmol / g, contains sodium chloride) sodium salt of 1c. -0-benzyl-4, b-O-isopropylidene-N- -propionyl-de serpentylmuramines oh acids, 2.38 g (11.54 mmol) of dicyclohexylcarbodiimide, 1.33 g (11.54 mmol) of N-hydroxysuccinimide, and 3.86, g (9.62 mmol) of hydrochloride (Cot) -tert. -butylether- (C%) -benzyl ester of b-alanyl-B-glutamic acid get (Cct) -tert.-butylether- (CV) - -benzylether 1c ".- 0-benzyl-4,6-0-from - propilidan-N-propionyl-desmethyl-mipa-mil-ala-n-D-glutamine acid containing O, 31 moles of water.
  +38.4 + 0.1 ° С (С 1.086 methylene chloride); , 86 (acetonitrile: water 3: 1)
Rf 0.83 (chloroform: methanol 9: 1), acid yield 7.3 g (97.9% of theory).
Example 6. Analogously to Example 4 from 3.07 g (4 mmol) of crude (still contains dicyclohexylurea) (Cot) -p-butylether - () - M-hydroxysuc11inimide-ester W-propionyl-desmethylmuramyl-l-apanil-B- of glutamic acid and 2.37 g (3.1 mmol) of L-alanyl-2- (1,2-β-dipalmitoyl-3p-glycero-3-hydroxyphosphoryloxy) ethylamide, the sodium salt of N-propionyl-desmethylmuramyl is obtained.
-B-alanyl-b-glutamyl- (col) -h -butyl-ether- (c4) -b-alanine-2- (1,2-dipalmitoyl-bp-glycero-3 - oxyphosphoryloxy) 1620
-ethylamide in the form of a colorless powder containing 2.93 moles of water.
20- + 6.9 -f (c 0.504; methylene chloride);
Rf 0.65 (chloroform: methanol: water 70: 30: 5);
Rf 0.63 (chloroform: methanol 7: 3), powder yield 2.93 g (70.9%)
The original product is obtained as follows.
Analogously to Example 4 of 2.2 g (3.9 mmol) dt of N-propane n-butyl ester of N-p-pionyl-de-emmethylmyl amyl-L-alanyl-D-β-glutamic acid, 1.2 g ( 5.8 mmol) of dicyclohexylcarbodiimide and 0.69 g (5.8 mmol) of N-oxysuccinimide are crude (СЫ) -n-butyl-ether- (сУ) -И-oxisuccinimidzir N- -propionyl-desmethylmuramyl-b alanyl-D-glutamic acid in the form of colorless crystals containing dicyclohexyl urea and used without further purification (number of crystals 3.07 g).
Rf 0.66 (chloroform: methanol: Db-70: 30: 5).
The original product is obtained as follows.
Analogously to Example 5 (duration of hydrogenation, 20 min) from 7.3 g (11.3 mmol) (Co (.) - p-butyl ether - (s)) benzyl ether TJ-propionyl-desmethylmuramyl-b-alanyl- L-glutamic acid get oL-n-butyl ester of N- -propionyl-desmethylmuramyl-L-alanyl-β-D-glutamic acid as a demon, a colored powder containing 0.88 mol of water, with an acid yield of 5.8 gS90.9 % of theory).
 +16.4 j 0, Gs (c 0.959; water);
Rf 0.61 (methylene chloride: methanol:: water 70: 30: 5);
Rf 0.52 (acetonitrile: water 3: 1).
The original product is obtained as follows.
Analogously to example 4 of 9.5 g ((3.97 mmol) (Co1) -n-butylether- (C) -benzylether 4, b-O-isopropylidene-N- -propionyl-desmethyl-muramyl-b-alanyl - D-glutamic acid and 120 ml of 60% acetic acid give (C.o (.) -P-butylether- (Cy) -benzg-ether C-propionyl-desmethylmuramyl-b-alanyl-B- glutamic acid, containing 0.41 mol of water, (7.5 g, 83%).
  +15.7 + n, l “C (C 1.019; methanol) ;.
Rf 0.23 (methylene chloride: methanol 9: 1);
Rf 0, D5 (methylene chloride: methanol 5: 1);
R.f 0.82 (methylene chloride: methanol:: water 70: 30: 5).
The original product was prepared as follows.
Analogously to example 4 of 3.81 g (10.76 mmol V 2.72 mmol / g, contains sodium chloride) sodium salt 4, b-0-isopropylidene-K-propionyl-α-desmethylmuramic acid, 2.44 g (11 , 83 mmol) of dicyclocarbodiimide, 1.36 g (11.83 mmol) of N-hydroxysuciniim and (10.76 mmol) of hydrochloride (S) - -p-butyl ether- (Co (,) - benzyl-b-alannl-D -glutamic acid get (CoL) -p-butylsulfur- (C V) -benzylether, 4,6-0-isopropylidene-K-propionyl-des-methylmuramyl-b-alanyl-B-glutamic acid (4.1 g, 56 , 2%).
Coi. ° + 10,, 1 C (C 0.928; methanol) .;
Rf 0.81 (methylene chloride: methanol:: water 70: 30: 5);
Rf 0.57 (methylene chloride: methanol 5: 1).
Example 7. To a solution consisting of 2.85 g (2.09 mmol) of the sodium salt of N-propionyl-desmethylmuramyl-L-alanyl-B-glutamyl (Col.) - tert.-butyl ether obtained according to Example 5 - (C y) -b-alanine-2- (1,2-dipalmitoyl-3p-glycero-3-hydroxyphosphoryloxy) ethylamide and 112 ml of abs, methylene chloride, trifluoroacetic acid is added and the whole is stirred for 3 hours at room temperature. Then the colorless clear solution is evaporated in vacuum at 30 ° C and the residue thus obtained is introduced several times into methylene chloride and again evaporated.
3.0 g of a colorless oil dissolved in 350 ml of a mixture of phosphate-buffered sodium chloride solution are obtained.
(at 0.1 M, 1: 1, pH 7), filtered in an AMICON dialyzer (model 402, ultrafilter PM 0 30/76 mm) at 3 atm. It is then filtered to remove chlorides in countercurrent with a total complexity of 2.1 liters of bidistilled BODA. The solution remaining in the dialyzer (approximately 50 ml) is lyophilized under high vacuum. Get colorless
0
five
,
P
d g
l
g
New lyophilisate, which is purified by chromatography on a column of 260 g of silica gel (60, 0.063-0.200 mm) in the system chloroform:: methanol: water 70: 30: 5 (10 ml fractions).
Fractions 90-260 are combined and evaporated under high vacuum without heating (cooling) until dry. The residue is introduced into 250 ml of bidistilled water and then in an AMICON dialyzer (model 402, PM 0 30/76 mm ultrafilter) is evaporated at 3 atm to approximately 50 ml, then filtered successively to remove chlorides in countercurrent 250 ml of phosphate buffer solution Sodium chloride (0.1 M 1: 1,) and 175 ml of bidistilled water. The solution remaining in the dialyzer is sterile, filtered, then passed through two filters with Millipore (NAL GENES, 0.45 µm, respectively 0.2 µm) and lyophilized under high vacuum.
The disodium salt of (Cii) -L-alanine-2- (1,2-dipalmitoyl-8n-glycerol-3-hydroxyphosphoryloxy) ethylamide H-propionyldesmethylmuramyl-b-alanyl-D-glutamic acid is obtained in the form of a colorless hydroscopic powder. It contains 4.8 mol of water.
y-
40
-6.1 + 1 ° С (with 0.489;
methylene chloride: ethanol 1: 1);
 Rf 0.28 (ethyl acetate: and-butanol: pyridine: acetic acid: water 41: 21: 21: 6: 10);
Rf 0.62 (acetonitrile: water 3: 1), salt yield 1.04 g (36.4%).
Example 8. 4.5 g (mmol) of sodium salt of N-acetyl-myramyl-L-ala-nyl-B-glutamyl- (CL) -trethan-butylsulfur- - (C1) -l-alanine-2- ( 1,2-Dipalmitoyl-Sn-glycero-3-hydroxyphosphoryloxy) ethyl amide, dried under high vacuum over phosphorus pentoxide, was dissolved in 75 ml of dry dichloromethane. It is cooled to 0 ° C and with stirring under absolute conditions, 25 ml of anhydrous trifluoroacetic acid are introduced and then heated to room temperature. After 2 1/2 hours, the clear, colorless solution is evaporated in a rotary evaporator at room temperature (up to about 10 ml), mixed several times with dichloromethane in an amount of 100 ml and finally evaporated again.
23
The crude product is purified twice by chromatography on 400 g of a gel of Koemevic acid 60 (for each time 400 g) in the system chloroform: methanol: water 70: 30: 5. The product contained in fractions 9-172, is dissolved in 100 ml of double-distilled water and is purified by filtration (AMICON - mixer 402, ultrafilter PM 10/76 mm) as in Example 1. The solution is filtered using a filter with millipora (0 , 2 μm) and lyophilized. Get .. disodium salt of (-b-alanine-2- (1,2-dipalmitoyl-Bp-glycero-3-hydroxyphosphoryloxy) -ethylamide K-acetyl-muramyl-b-alanyl-B-glutamic acid, containing 4.89 moles of water, in the form of a colorless friable powder.
 2. About
Ccilj, 10 + 1 С ° (С 0.675; methanol), salt yield 1.1 g (25%);
Rf 0.08 (chloroform: methanol: water 70: 30: 5);
Rf 0.15 (ethyl acetate: h-butanol: pyridine: acetic acid: water 42: 21: 21: 6: 10);
Rf 0.30 (chloroform: methanol: water: acetic acid 70: 40: 9: 1).
Biological tests of the salts of muramyl peptides obtained by the proposed method were carried out.
Rat alveolar macrophages obtained by washing the lungs are incubated 24 hours either with liposomes containing the active substance or in physiological saline with the active substance. Tumor cells are inserted, marbed I. The cells are incubated for 72 hours. The dead tumor cells are washed, the number of living cells is determined by their radioactivity. Macrophage activity is assessed on the basis of cell toxicity., I.e. according to the number of animals killed during the experiments. The specific cell toxicity (%) was calculated as follows:
100
cpm in tumor cells, embedded macrophages by active principle
cpm in tumor cells, inducible macrophages and PBS The experiments were carried out with the following compounds:
I - sodium salt of N-acetyl-mu ramyl-L-alanyl-B-glutamyl - (SU) -b-alanyl-2- (1,2-di-palmitoyl-Bp-gliyero-3-hydroxyphosphoryloxy) -ethylamide ;


15
25


9951624
11 - sodium salt of N-propionyl-normuramyl-11-alanyl-B-. -glutamyl (Cc) 1) -p-butyl ether-SU (SU) -l-alanyl-2- (1, 2-5-dipalmitoyl-3n-glycero-3-hydroxyphosphoryloxy) ethyl amide, (Example 5); III - sodium salt of N-propionyl-normuramyl-b-alanyl-B-glutamyl (COL) -p-methyl ether- (C) -b-alanyl-2- (1, 2-diplatmitoyl-Bp-glycero- H-oxyphosphoryloxy) ethyl amide (Example 4); IV - Sodium salt of N-acetyl- -muramyl-L-alanyl-B-glutamyl- (Cd) -tert.-butylether - (soo) -b-alanine-2- (1,2-diplatmoyl-3n -glycero-Z-20 -oxyphosphoryloxy) ethyl amide (Example 3); V is the sodium salt of N-acetyl-g-Muramyl-b-alanyl-p-gluta-mil- (Co1.) Methyl ester (SU) -b-alanine-2- (1, 2- -dipulmitoyl-Sn-glycero - -3-oxyphosphocyloxy) -ethyl-amide (example 2); VI - sodium salt of N-propionyl-normuramyl-b-alanyl-B- -glutamyl- (Col) is a complex tert.-butyl ester- (sous) - -alanine-2- (I, 2-dipalmittoyl-8n- glycero-3-oxyphos-35 foryloxy) - ethylamide (Example 6).
The results are summarized in table. one.
40
Comparison of derivatives
muramylpeptides of general formula I with a known compound.
The pyrogenicity test was carried out on rabbits and showed that with high doses of 30 mg / kg of K-acetyl-muramyl-L-alanyl-D-glutamyl (Cd.) -M-Putil ether (C , V} -L-alanine-2- (1,2-dipalmitoyl-5n-gly-Q-cerr-3-hydroxyphosphoryloxy) -ethylamide-sodium salt (compound II) does not show a pyrogenic effect.
In contrast, with the same doses of sodium salt, N-acetyl-j-Muramyl-b-alanyl-B-isoglutaminyl-b- -alanine-2- (1,2-dipalmitoyl-3n-glycerol-3-oxyphosphoryloxy) - ethylamide (VIl) there is a strong increase in temperature.
thirty
25
The results of the pyrogenicity test for intravenous administration of the tested compounds at a dose of 1 mg / kg are shown in Table. 2
The results show that the temperature variation (LT C) in the case of the proposed compounds is about 2-4 times lower than in the case of the known compound yNO. In addition, mura ilpeptides of formula I can be successfully used for the prevention and treatment of diseases caused by DNA viruses. with cubic symmetry and non-enveloped nucleocapsid, DNA - viruses with a virion in the shell, and also RNA - viruses with cubic symmetry of capsid and RNA - viruses with spiral symmetry of capsid.
Compounds of formula I are preferably used in the case of DNA viruses with virion in the shell and cubic symmetry of the capsid, in the case of RNA viruses with cubic symmetry of the capsid and varion without the shell, as well as in the case of RNA viruses with helical symmetry of the capsid in which the nucleocapsid shell is located at the outer surface of the membrane, and, moreover, in the case of Adenoviridae, Poxvirides, Coronaviridae, in particular, in human Coronaviren.
Primarily, the compounds of Cyporfly I are used in the case of Herpesvir dae, picornaviridae, Myxoviren, a
also in the cl. learn masfadenviren per hour





of human diseases, with a strong effect not only in the case of bacterial pathogens, but also against viral pathogens. These compounds exhibit a long-term prophylactic or therapeutic effect, lasting from many days to several weeks after a single dose of the compounds of formula I obtained by the proposed method, and also have an effective effect on a wide range of viral pathogens.
The antiviral activity of the proposed compounds was tested.
And the sodium salt of N-acetylmuramyl-b-alanyl-C-glutamic acid - (SU) -b-alanyl-2- (1,2-dipalmitoyl- -sn glycero-3-hydroxyphosphoric 1) - ethylamide.
In the sodium salt of N-acetylmuramyl-b-alanyl-B-glutamyl- (Su) -butyl ovine ester- (Cc1 -) - b-alanine



26
0
15
five
35 fdr
-2- (1,2-dipalmitoyl-sn-glycero-3-oxyphosphoryloxy) ethylamide.
With the sodium salt of N-acetylmuramyl-l-alanyl-B-glutamyl- (G Y) -methyl ester- (Cc1) -b-alanine-2- (1,2-dipalmitosh1-cs-glycero-3-oxyphosphoryloxy ) -Ethylamido Each of the 20 and 30 female mice — MF-2f SPP weighing 1A-16 g each, is infected through the nose, using light anesthesia and using a mixture of equal parts of diethyl ether, ethanol and chloroform, forming plates with units of virus strains Influenza A (Texas) 1/77 (adaptable) in the amount of 1 in the form of 0.05 ml suspension of viruses.
At the indicated time, each mouse is administered once in a single dose (single dose) according to the table. 1 quantity of the active ingredient in 0.2 ml of 0.005% by weight sodium carboxymethylcellulose B solution In twice distilled water through the nose or through the mouth.
The rest of the indicated mice, i.e. 10 or 20 animals serve as controls: they are given a placebo (a 0.005% by weight sodium carboxymethylcellulose solution)
Through the nose, the active ingredient is administered using light anesthesia using a mixture of equal parts of diethyl ether, ethanol and chlorine.
thirty
0
five
0
five
The percentage of mice remaining in 24 days. after infection, are given in table. 3
A comparison with a known antiviral agent (valid for Influenza A viruses) - amantadine. The effect of amantadine begins only at a dose of several mg / kg, the LDDd value for magica is 700 mg / kg.
The proposed compounds act already at a dose of 0.1 mg / kg, in certain cases already at 0.01 mg / kg, while values for mice are more than 3000 mg / kg. This means that the range of therapeutic effects of the proposed compounds compared with amantadine is much wider. In addition, the proposed compounds show an antiviral effect after their single use no more than 7 days before the appointment.
or after it, while amantadipip must be administered before infection, for example 24 hours before infection, and continue daily administration for at least 5 days, i.e. The proposed compounds exhibit both therapeutic and prophylactic effects, while amantadine exhibits only a prophylactic effect.
Ш20Н
29951628
In addition, derivatives of muramyl-peptides obpey (Formulas I have low toxicity. Table. A shows the acute toxicity determined in mice.

权利要求:
Claims (1)
[1]
Invention Formula
Method for preparing muramyl peptide salts of general formula I
)
DS-W (DI) TQH-ef-Bi
/ (D)
but)
O-TSJ-CH-C-IH-CH-CH CH-C-NH-II I 1 and c. about
/
about R
pp
about N SN.Z about
where R is —C — C — alkyl;
 E is hydrogen or C, -C-alkyl, characterized in that. compound of general formula L
sngön
(HiOH)
/
NzS-Sh Ш) -NH-C-Ri
a) (d) he
O- -N-CH-C-TQH-CH-CHr-CHz-C
f IT rvr
0 H CHa 0
/ H 0 orj
about
he
-CH-C-Ml-CH -CHj-Q-p-OCH
snsQ
HM
: -o-CH
0
n I
Hsi-Cjs-C-O-CHj l (Stv)
where R, - has the specified value; RJ.- With, -C, -alkyl or protective
carboxylic acid group, is introduced into interaction with L-alanyl- -2- (1,2-dipalmitoyl-3-glycero-3-oxyphosphoryloxy) ethylamide of formula
(L) H and H-N-CH-C-IN-SNg-ShgO-P-O-SNg
n snz oh oh I
4iiCi5-l -0-CH
V i.
H3iCe-C-0-CH2 llSn)
or its salt, having protective groups in the form of a salt.
groups split off and target product
40
29
Active substance
Specific cellular toxicity,%
0.2 ml RV of the active substance, mg / culture
0.021 0, 2
3 23
39 21 81 18 5 29
Table2 Compound M ° C
II + 1.1
III + 1.0
IV + 0.69
V, +0.65
VI + 0.48
Famous VII - + 2.0
Table 3
Active Input Type Time-Percentage of the amount remaining in the Ginging-Medium (the number of post-infection and treated days) in the active ingredient, mg / kg
before infection (n.t - not tested - tests were not conducted + after infection) 100 10 I 1 0.1 0.01. 0 control
Blowjobs blowjobs
+7
+7
129951630
Table i
The active substance in 100 mg of liposomes at 0.2 ml, mg / culture
20
0.02 0.2 1 2 J
20
74
64 64
56 10 76 68 60 73
51 23 79 52
44 11
80 70 80 n.t n.t 15 n.t n.t 58 30 30 О

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

---

CH452782|1982-07-23|

---