GB2211503A

GB2211503A - New saccharides, their preparation and pharmaceutical compositions containing them

Info

Publication number: GB2211503A
Application number: GB8830017A
Authority: GB
Inventors: Ingolf Macher; Frank Michael Unger; Christian R H Raetz
Original assignee: Sandoz AG
Current assignee: Sandoz AG
Priority date: 1985-06-28
Filing date: 1988-12-22
Publication date: 1989-07-05
Anticipated expiration: 2008-12-22
Also published as: GB2211503B; GB8830017D0

Abstract

Compounds of formula III <IMAGE> where X and Y independently are oxygen or imino, but not both imino and R1 and R2 independently are acyl of 14 carbons, optionally monosubstituted by hydroxy or acyloxy of 14 carbon atoms except when X is imino and Y is oxygen, R1 and R2 are not both (R)-3-hydroxytetradecanoye are useful as modulators of antimicrobial resistance, for enhancing immune response and unspecific immunity, for preventing endotoxin shock, and for treating malignant tumors and inflammation.

Description

NEW SACCHARIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM The invention relates to saccharides, their preparation, pharmaceutical compositions containing them and their use as pharmaceuticals.

In particular the invention provides new compounds of formula III

wherein X and Y independently are oxygen or imino afld R1 and R2 independently are acyl of 14 carbon atoms ontinnaily .mono- substituted by hydroxy 3r acyloxy ur 14 carbon atoms with the provisos that 1) X and Y are not both imino and 2) when X is imino and Y is oxygen, then R1 and R2 are not simultaneously (R)-3-hydroxy-tetradecanoyl, in free form or in form of acid addition salts.

The compounds of formula III may exist in free form or where appropriate in the form of acid addition salts. A free form may be converted into a salt form in conventional manner and vice-versa.

R1 and R2 preferably are identical. The configuration at the carbon atom in the 3 position may be (R) or (S). Thus Rl and R2 may be present in a compound of formula III in achiral, (R), (S) or racemic form. This also applies to compounds of formula hereunder. The configuration remains unchanged during the process of the invention, i.e. when a compound in (R), (S) or racemic form is used as starting material a corresponding (R), (S) or respectively, racemic end product is obtained.

Compounds of formula III may be obtained by a process comprising a) for the production of compounds of formula lIIa

wherein R1 and R2 are as defined above and either X' and Y' are oxygen or Y' is imino and X' is oxygen, converting in compounds of formula I

wherein R1 , R2, X' and Y' are as defined above and R is a protecting group, the unprotected hydroxy group to a protected phosphate group or b) for the production oF compounds of formula Ilib

wherein R1 and R2 are as defined above, acylating corresponding compounds of formula IlIc

wherein R; and R are as defined above and R' is a protecting group, and thereafter splitting off the protecting groups.

Process variant a) may e.g. be effected by dissolving a compound of formula IV in an inert solvent, e.g. a cyclic ether such as tetrahydrofurane and reacting at lower temperature, e.g. at .700 C, with butyllithium in an aliphatic hydrocarbon such as hexane, and thereafter adding dibenzylphosphorochloridate. The product may be recovered from the reaction mixture and if desired purified in known manner. The free hydroxy groups of the phosphate residue are protected, e.g. by benzyl. The splitting off of the protecting groups may also be effected in known manner. For example, a protecting group may be cleaved in conventional manner under acidic conditions, e.g. with an aqueous acid (ion-exchanger), or hydrogenolytically.

Process variant b) may e.g. be effected by dissolving a compound of formula IIIc in an inert solvent, e.g. in a chlorinated hydrocarbon such as methylene chloride, together with an acylating agent, appropriately with addition of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and letting the reaction take place at lower temperatures, e.g. at about 40C.

The product may be isolated from the reaction mixture and if desired purified in known manner. Thereafter the protecting groups present are split off in known manner.

As protecting group any group commonly used for protection in saccharide chemistry may be used. For example both R substituents may together be the benzylidene or isopropylidene group. Also, as protecting groups for the phosphate moiety known protecting groups such as benzyl may be used.

The compounds of formula lIIc are new and may be obtained in accordance with the following reaction scheme:

TBDMS = tert.-butyldimethylsilyl The compounds of formula IV may be obtained according to the following reaction schemes, whereby the hydroxy groups not participating in the reaction depicted may appropriately be in protected form. The substituents have the following meanings: R, R' = protecting groups R5 = both independently have the significance indicated above for R1 or R2 Ac = acetyl TBDMS = tert.-butyldimethylsilyl 1. Preparation of compounds of formula IVa:

2. Preparation of compounds of formula IVb:

3.Preparation of compounds of formula IVc:

Insofar as the preparation of any particular starting material is not particularly described this is known or may be effected in conventional manner or in analogous manner to that described in the Examples.

The following Examples illustrate the invention. All temperatures are in degrees Centigrade. EDTA means ethylenediaminetetraacetic acid, DTA is 1,4-dithioerythrite, UDP stands for uridine phosphate and tris is tris(hydroxymethyl)aminomethane.

Example l: 3-deoxy-2-O[(R)-3-hydroxytetradecanoyl]-3-4(R)-3-hydroxyte.

tradecanowlamido]-l-O-Phosphono-a-D-qlucopyranose a) 2-04(R)-3-benzy loxytetradecanoy l)-3 (R)-3-benzy loxy te tradecanoyl amido]-3-deoxy-1-O-dibenzylphosphono-4,6-O-isopropylidene-α-D-glucopy- ranose To a solution of 4.85 g 2-O-[(R)-3-benzyloxytetradecanoyl)-3-(R)-3-benzyl- oxytetradecanoylamido]-3-deoxy-4,6-O-isopropylidene-D-glucopyranose in 40 ml anhydrous tetrahydrofurane cooled to -700 are added 8 ml of 1.6 M butyllithium in hexane. After 5 minutes a solution of 3.8 g dibenzylphosphorochloridate in 10 ml benzene is added at the same temperature. Agitation is pursued at -700 for 10 minutes, 0.3 ml of acetic acid are added and the solution concentrated to 1/4 the initial volume.The solution is diluted with 200 ml of methylene chloride, extracted with 50 ml of water, 50 ml of dilute sodium hydrogen carbonate solution and 50 ml of sodium chloride solution, dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (toluene/ethyl acetate 7/3).

Rf (toluene/ethyl acetate 2/1) = 0.58.

b) 3-deoxy-2-O4(R)-3-hydroxytetradecanoyl]-3-[(R)-3-hydroxytetradeca- noylamido)-1-O-ohoshono-a -D-olucoyranose A solution of 980 mg 2.O{(R)-3-benzyloxytetradecanoyl)].3(R).3.benzyl- oxytetradecanoylamido]-3-deoxy-1 -O-dibenzy lphosphono-4,6.O-isopropyli- dene--D-glucopyranose in 100 ml tetrahydrofurane is hydrogenated about 1 hour with 300 mg 10 % palladium on charcoal under atmospheric pressure. Then the catalyst is filtered off, water (10 ml) and Dowex AG50WX8 H+ are added and the mixture is stirred at room temperature until the isopropylidene group is completely cleaved. The ion exchanger is filtered off, the solution neutralised with tris and tetrahydrofurane and water evaporated under reduced pressure. The residue is dissolved in methanol and chromatographed over Sephadex LH 20.

Rf (chloroform/methanol/acetic acid/water 125/75/10/20) = 0.65.

Example 2.: 2 3-di-O-{(R)-3-hydroxytetradecanoyl]-1-O-phosphono-α-D- 2,3-di-O-[(R)-3-hydroxytetradscanoyl]-1 glucopyranose a) 4,6-O-benzylidene-2,3-di-O(R)-3.benzyloxytetradecanoyl]-1-O-diben- zylDhosphonc-t-D-qlucopyranose To a solution of 1 g 4,6-O-benzylidene-2,3-di-O{(R)-3-benzyloxytetradeca- noyl]-D-glucopyranose in 10 ml of dry tetrahydrofurane cooled to .700 are added dropwise 0.9 ml of 1.6 M butyllithium in hexane. After 5 minutes at that temperature a solution of 420 mg dibenzylphosphorochloridate in 4 ml benzene is added dropwise. Stirring is continued for 10 minutes at -700, the solution is neutralised with acetic acid and evaporated to dryness. The residue is chromatographed (silicagel, hexane/toluene/ethyl acetate 4/4/1).

Rf (toluene/ethyl acetate 6/1) = 0.65.

b) 2 3.di-O4(R)-3-hydroxytetradecanoyl)-1-O-phosphono.a -D-plucopyra- nose 230 mg 4,6-O-benzy lidene-2,3-di-O{(R)-3-benzy loxytetradecanoy 1]-1-O-di benzylphosphono-α-D-glucopyranose are dissolved in tetrahydrofurane/wa ter (9/1) and hydrogenated for about 5 hours under atmospheric pressure with 80 mg of 10% palladium over charcoal. The catalyst is filtered off, the solution neutralised with tris and evaporated to dryness. The residue is chromatographed with methanol over Sephadex LH 20.

Rf (chloroform/methanol/acetic acid/water 125/75/10/20) = 0.65.

Example 3 : 2-deoxy-3-O-[(S)-3-hydroxytetradecanoyl]-2-[(S)-3-hydroxyte- tradecanowlamido]-1-O-phosphono-α-D-glucopyranose a) 4,6-O-benzylidene-3-O(S)-3-benzyloxytetradecanoyl]-2(S)-3-benzyl- oxytetradecanoylamido].2-deoxy.1-O-dibenzylphosphono-α-D-glucopyranose Rf (tohene/ethyl acetate 4/1) = 0.44.

The title compound is obtained in a manner analogous to the following which yields 4,6-O-benzylidene-3-O[(R)-3-benzyloxytetradecanoyl]-2[(R)-3-benzyl- oxytetradecanoylamido]-2-deoxy-l -O-dibenz ylphosphono- ar-Dlqlucopyranose : To a solution of 3.9 g 4,6.O.benzylidene.2(R).3.benzyloxyte tradecanoyl- amido].2-deoxy-1-O-dibenzylphosphono-α-D.glucopyranose, 2 g (R)-3-ben- zyloxytetradecanoic acid and 50 mg 4-dimethylaminopyridine in 20 ml of methylene chloride cooled to -100 are added 2 9 of dicyclohexylcarbodi.

imide and the reaction mixture held at 40 overnight. The mixture is then filtered, the filtrate evaporated to dryness, the residue taken up in a small amount of toluene/ethyl acetate (8/2) and chromatographed with the same solvent. M.P. 96-980.

[α]D20 = +33.0 = +33.3 (c = 1, chloroform) Rf (toluene/ethyl acetate 2/1) = 0.5.

b) 2-deoxy-3-0{(5)-3-hedroxY tetradecanoy l]-2i(S)-3-hydroxy totradeca- noylamido]-1-O-phoshono-α-D-glucopyranose M.P. 150-2000 (decomp.) Rf (methylene chloride/methanol/ammonia 1/1/1; lower phase) = 0.5.

The title compound is obtained in a manner analogous to the following, which yields 2-deoxy-3-O-[(R)-3-hedroxetetradecanoyl]-2{(R)-3-hydroxytetradeca- noylamido}1-O-phosphono-α-D-qlucopyranose: 4.2 g 4,6-O-benzylidene-3-O{(R)-3-benzyloxytetradecanoyl]3-2{(R)-3-ben- zyloxytetradecanoylamido3-2-deoxy-1-O-dibenzylphosphono--D-glucopyra- nose are dissolved in 900 ml of tetrahydrofurane/water (85/15) and hydrogenated with 1.5 g 10 % palladium on charcoal for 2 hours at 10 bar and 400. The catalyst is then filtered off, the tetrahydrofurane evaporated and the aqueous suspension lyophilized.

20 La]0 = +28.5 (c = 0.2, chloroform + 1 drop methanol) Rf (chloroform/methanol/acetic acid/water 125/75/10/20) = 0.56.

Example 4 : 2-deoxy-3-O{(R)-3-hedroxetetradecanoYI]-1-O-phosphono-2- [(R)-3-tetradecanoy loxytetradecanoylamido].a.D.qlucopyrano5e a) 4,6-0-benzyIidene-3-OS(R)-3-benzy loxytetradecanoy 1]-2-deoxY-1-O-di- benzylphosphono.24(R).3.tetradecanoyloxytetradecanoylamido].α.D-gluco- pyranos.

The title compound is obtained in a manner analogous to Example 3a).

M.P. 82-950 L]D = +24.10 (c = 1, chloroform) Rf (toluene/ethyl acetate 2/1) = 0.7.

b) 2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-1-O-phosphono-2{(R)-3-te- tradecanoyloxytetradecanoylamido]α-D-glucopyranose [α]D20 = +14.3 (c = 1, tetrahydrofurane/pyridine) Rf (chloroform/methanol/acetic acid/water 80/25/5/5) = 0.35.

The title compound is obtained in-a manner analogous to the following, which yields 2.acetamido-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl-]1 -0-phosohono.

α-D-glucopyranose: 2.08 g 2-acetamido-4,6-O-benzylidene-3-O{(R)-3-benzyloxytetradecanoyl]- 2.deoxy-1-O-dibenzylphosphono-α-D-glucopyranose are dissolved in tetra hydrofurane/water (9/1) and hydrogenated for 3 hours with 1 g 10 % palladium on charcoal under atmospheric pressure. The catalyst is filtered off and the filtrate first evaporated and then lyophilised. The lyophilisate is dissolved in methanol, neutralised with tris(hydroxymethyl)aminome thane and chromatographed with methanol over Sephadex LH 20.

20 [α]D20 = + 43.3 (c = 1, methanol) Rf (chloroform/methanol/acetic acid/water 125/75/10/20) = 0.4.

Example 5: 2-deoxv-1-O-phosphono-3-O-tetradecanoyl-2-tetradecanoyl- amido-a-D-qlucopyranose a) 4,6-O-benzylidene-2-deoxy-1-O-dibenzylphosphono-3-O-tetradecanoyl-2- tetradecanowlamido-α ido-a-D-glucopyranose The title compound is obtained in a manner analogous to Example 3a) using tetradecanoic acid and with toluene/ethyl acetate (4/1) as eluent.

M.P. 123-129 Rf (toluene/ethyl acetate 2/1) = 0.6.

b) 2-deoxy-1.O-hosphono-3-O-tetradecanoyl-2-tetradecanoylamido-α-D- glucopy ranose The titel compound is obtained in a manner analogous to Example 4b Rf (ch loroform/methanol/acetic acid/water 125/75/10/20) = 0.65.

Example 6 : 2-deoxy-2{(R)-3-hYdroxY tetradecanoylamido]-1-O-phosphono- 3-O4(R)-3-tetradecanoy loxy tetradecanoyl]- a-D-q lucopy ranose a) 4,6-O-benzylidene-2[(R)-3-benzyloxetetradecanoylamido]-2-deoxy-1-O- dibenzy lphosphono-3-O{(R)-3-tetradecanoy loxyte tradecanoy 1]- a-D-q lucopyranose The title compound is obtained in a manner analogous to Example 3a), using (R)-3-tetradecanoyloxytetradecanoic acid and with toluene/acetic ester (4/1) as solvent mixture.

M.P. 89-900.

20 [α]D20 = +30.9O (c = 1, chloroform/methanol 1/1).

Rf (toluene/ethyl acetate 4/1) = 0.5.

b) 2-deoxy-2-[(R)--hydroxytetradecanoylamido]-1-O-phosphono-3-O-[(R)- 3-tetradecanoy loxytetradecanoy 1]- a-D-plucopyranose The title compound is obtained in a manner analogous to Example .4b).

The lyophilisate is dissolved in methanol and chromatographed over Sephadex LH 20 using the same solvent.

Rf (chloroform/methanol/acetic acid/water 80/25/5/5) = 0.32.

Example 3:: 2-deoxy-2{(R)-3-hvdroxytetradecanoylamido]-1-O-phosphono- 3-O-tetradecanoyl-α-O-glucopyranose a) 4,6-O-benzylidene-2-[(R)-3-benzyloxytetradecanoylamido]-2-deoxy-1-O dibenzylphosphono-3-O-tetradecanoyl- a-D-olucopyranose The title compound is obtained analogous to Example 3s),using tetradeca noic acid and toluene/ethyl acetate (3/1) as solvent mixture.

M.P. 125.5-126.5 .

Rf (toluene/ethyl acetate 3/2) = 0.6.

b) 2-deoxy-2(R)-3-hydroxytetradecanoylamido]-1-O-hosphono-3-O-tetra- decanoyl-a-D-qlucopYranose 354 mg 4,6-O-benzylidene-2(R)-3-benzyloxytetradecanoylamido]-2-deoxy- 1-O-dibenzy lphosphono.3-O-tetradecanoyl- a-D-glucopyranose are dissolved in 30 ml tetrahydrofurane/water (9/1) and hydrogenated with 200 mg palladium on charcoal for 10 hours under atmospheric pressure. The catalyst is then filtered off and the solution adjusted to pH 7.5 with tris(hy droxyinethyl)aminomethane. The tetrahydrofurane is evaporated under reduced pressure and the aqueous solution lyophilised. The lyophilisate is triturated twice with ether and dried.

Rf (chloroform/methanol/acetic acid/water 125/75/10/20) = 0.6.

Example 8 -: 1-0 .phosphono-2,3di-O-(R)-3-tetradecanoy loxytetradeca- noyl]-α-D-glucoperanose a) 4,6-O-benzYìidene-1-O-dibenzelphosphono-2,3-di-O{(R)-3-tetradeca- noyloxytetradecanoyl]α-D-glucopyranose The title compound is obtained analogous to Example - 2a),under chromatographic purification over silicagel with ether/hexane (1/1) as eluent.

Rf (ether/hexane 1/1) = 0.5.

b) 1-O-phosphono-2,3-di-O-[(R)-3-tetradecanoy loxytetradecanoylEa-D-qlu- copy ranose 470 mg 4,6-O-benzylidene-1-O-dibenzy Iphosphono-2,3-di-O{(R)-3-tetra- decanoyloxytetradecanoyl]-a-D-glucopyranose are dissolved in 47 ml tetra hydrofurane and hydrogenated 1 hour with 230 mg palladium on charcoal under atmospheric pressure. The catalyst is filtered off, the filtrate concentrated to dryness and the residue chromatographed over silicagel (chloroform/methanol/water/triethylamine 15/10/2/0.2). Fractions containing product are pooled and evaporated to dryness. The title compound is obtained as di-triethylamine salt.

Rf (chloroform/methanol/acetic acid/ water 80/25/5/5) = 0.5.

Example - 9 : 2-deoxy-3-O-](R)-3-hydroxytetradecanoyl]-2-[(S)-3-hydroxyte- tradecanoyiamido]-1-O-phoshono-α-D-glucopyranose a) 4,S-O-benzylidene-P;O-C(R)-3-benzy loxy tetradecanoy ll-2CS)-3-benzyl- oxytetradecanoylamido)-2-deoxy-1-O-dibenzylphosphono-α-D-glucopyranose The compound is obtained in a manner analogous to Example. 3a).

Rf (toluene/ethyl acetate 4/1) = 0.44.

b) 2-deoxy-3-O-L(R)-3-hydroxytetradecanoyl]-2-[(S)-3-hydroxytetradecanoyl- amido]-1-O-phosphono-α-D-glucopyranose The compound is obtained in a manner analogous to Example. 3b).

Rf (methylene chloride/methanol/ammonia 1/1/1, lower phase) = 0.5.

Example No: NMR-Spectra 1 5.77 (dd, 3,5 a. 7,5 Hz, lH); 4.77 (ddd, 2,5, 3,5 a. 10,5 Hz, (CDC1 / 1H); 4.38 (dd, 9,5 a. 10,5 Hz); 3.8 to 4.1 (m, 4H); 3.73 (dd, CD3O) 5,5 a. 12 Hz, lH); 3.53 (t, 10 Hz, 1H).

2 2 5.71 (dd, 3,5 a. 7,5 Hz, 1H); 5.48 (t, 10 Hz, lH); 4.77 (ddd, 2, (CO30D) 4 a. 10 Hz, 1H); 3.9 to 4.05 (m, 3H); 3.86 (dd, 2 a. 12 Hz, 1H); 3.7 (dd, 5,5 a. 12 Hz, 1H); 3.67 (s, Tris); 3.58 (t, 10 Hz, 1H).

3 5.58 (dd, 3 a. 7 Hz, lH); 5.22 (t, 10 Hz, 1H)q 4.19 (dt, 3 a.

(CDC1 / 10 Hz, lH); 3.8 to 4.1 (m, 4H); 3.7 (s, Tris a. dd, 1H); 3.46 (t, CD3OD) 10 Hz, 1H).

4 5.46 (dd, 3 a. 7 Hz, 1H); 5.22 (t, 10 Hz, 1H); 5.17 (m, 1H); (CDCl3/ 4.19 (dt, 3 a. 10 Hz, 1H); 3.9 to 4.1 (m, 3H), 3.7 (s, Tris); CD3OD) 3.65 (dd, 1H); 3.51 (t, 10 Hz, 1H).

5/step a) 7.38 (m, 15H); 5.70 (dd, 3,5 a. 6 Hz, 1H); 5.63 (d, 9,5 Hz, (protected) 1H); 5.50 (s, 1H); 5.26 (t, 10 Hz, 1H); 5.08 (m, 4H); 4.38 (m, (CDCl3) 1H); 4.08 (dd, 5 a. 10 Hz, 1H); 3.95 (dt, 5 a. 10 Hz, 1H); 3.73 (t, 10 Hz, 1H); 3.69 (t, 10 Hz, 1H); 2.29 (m, 2H); 1.89 (m, 2H).

6 /step a) 7.2 to 7.45 (m, 20H); 6.34 (d, 9 Hz, 1H); 5.77 (dd, 3,5 a. 6 Hz, (protected) 1H); 5.49 (s, 1H); 5.29 (t, 10 Hz, 1H); 5.14 (quintet, 6 Hz, (CDCl3) 1H); 5.0 (m, 4H); 4.51 a. 4.41 (AB, 12 Hz, 2H); 4.41 (m, 1H); 4.08 (dd, 5 a. 10 Hz, 1H); 3.95 (dt, 5 a. 10 Hz, 1H); 3.7 (m, 3H); 2.58 a. 2.47 (ABX, 5,5, 7,5 a. 15,5 Hz, 2H).

7/step a) 7.2 to 7.45 (m, 20H); 6.29 (d, 9 Hz, 1H); 5.74 (dd, 3,5 a. 6 Hz, (protected) 1H); 5.49 (s, 1H); 5.30 (t, 10 Hz, 1H); 5.0 (m, 4H); 4.52 a.

(CDCl3) 4.42 (AB, 11 Hz, 2H); 4.44 (m, 1H); 4.03 (dd, 5 a. 10 Hz, 1H); 3.94 (dt, 5 a. 10 Hz, 1H); 3.7 (m, 3H); 2.15 to 2.35 (m, 4H).

8/step a) 7.3 to 7.45 (m, 15H); 5.92 Cdd, 3,5 a. 6,7 Hz, 1H); 5.60 (t,9,5 (protected) a. 10 Hz, 1H); 5.48 Cs, 1H); 5.02 to 5.22(m, 6H); 4.95 Cddd, (i3) 2,5, 3,5 a. 9,5 Hz, 1H); 4.10 (dd, 4,5 a. 10 Hz, 1H); 3.96 (ddd, 5, 9 a. 10 Hz,1H);3.67 (t,9,5 Hz, 2H); 2.60 (m, 2H); 2.38 Cd, 6,5 Hz, 2H); 2.24 (t,7,5 Hz,2H); 2.12 (t,7,5 Hz, 2H).

9/step a) 7.2 to 7.42 (m, 25H); 6.49 Cd, 9 Hz, 1H); 5.77 Cdd, 3,5 a. 6 Hz, (protected) 1H); 5.45 (s,1H); 5.35 (t, 10 Hz, 1H); 5.0 (m, 4H); 4.54 a.

(CDCl3) 4.40 CAB, 11 Hz, 2H); 4.47 a. 4.36 CAB, 11 Hz, 2H); 4.45 (m, 1H); 3.9 to 4.1 (m, 2H); 3.6 to 3.8 (m, 4H); 2.58 (dd, 6,5 a.

15,5 Hz, 1H); 2.34 Cdd, 6 a. 15,5 Hz, 1H); 2.2 (m, 2H).

The compounds used as starting materials may be obtained as follows: A) 4,6-O-benzylidene-2[(R)-3-benzyloxytetradecanoylamido]-2-deoxy-1-O- dibenzylphosphono-α-D-glucopyranose (for Examples 6 and 7) a) 2{(R)-3-benzyhxytetradecanoylamido > 2-deoxy-D-qiucopyranose A mixture of 5.4 g D-glucosamine-hydrochloride, 10.8 g N-[(R)-3-benzyl- oxytetradecanoyloxy]succinimide and 5 ml diisopropylethylamine in 25 ml dry dimethylformamide is stirred 24 hours at room temperature. The bulk of the solvent is distilled off and the residue dissolved in a mixture of 150 ml chloroform, 300 ml methanol and 120 ml water. After addition of a further portion of 150 ml chloroform and 150 ml water the lower phase is separated and washed twice with tha upper phase of 100 ml chloroform, 100 ml methanol and 90 ml water. The solution is evaporated to dryness and dried under high vacuum. The resultant product ist used in the next step without further purification.

For analysis a small portion of crude product is chromatographically purified (toluene/ethanol 9/1).

M.P. 150-1580.

[α]D20 = +53 (c = 1, dimethylformamide) Rf (chloroform/methanol 9/l) = 0.3.

b) 4,6-O-benzYlidene-2CR).3-benzyloxytetradocanoylamido].2.deoxy.D.

glucopyranose A solution of 5.83 g 24(R)-3-benzyloxytetradecanoylamido]-2-deoxy-D-glu- copyranose, 3 g benzaldehydedimethylacetal and 500 mg p-toluene-sulfonic acid-monohydrate in 200 ml of anhydrous dimethylformamide is kept for about 3 hours at 55-600 and 30-40 mbar on a rotary evaporator. Thereafter no starting material is remaining. The major part of the dimethylformamide is distilled off, 500 ml of methylene chloride are added and the solution is extracted twice with 200 ml of diluted sodium hydrogen carbonate solution and 200 ml of water. The solution is dried with sodium sulfate, evaporated to dryness and the residue chromatographed (toluene/ethyl acetate 6/4).

M.P. 162-1650.

20 La] = + 5.5 Cc = 1, chloroform) Rf (toluene/ethyl acetate 1/1) = 0.2.

c) 4,6-O-benzylidene-2-[(R)-3-benzyloxytetradecanoylamido]-2-deoxy-1-O- dibenzylphosphono-α-D-glucopyranose To a solution of 4.86 g 4,6-O-benzylidene-2{(R)-3-benzyloxytetradecanoyl- amido]-2-deoxy-D-glucopyranose in 40 ml of anhydrous tetrahydrofurane cooled to -700 are added dropwise 8 ml of 1.6 M butyllithium in hexane.

After 5 minutes at that temperature a solution of 3.8 g dibenzylphosphorochloridate in 10 ml of benzene ist added dropwise. After further stirring for 10 minutes at -70 0.3 ml of acetic acid are added and the solution concentrated to 1/4 of its initial volume. The solution is diluted with 200 ml of methylene chloride, extracted with 50 ml of water, 50 ml of diluted sodium hydrogen carbonate solution and 50 ml of sodium chloride solution, dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (toluene/ethyl acetate 7/3).

M.P. 102-1050.

[α]D20 = + 31.7 (c = 1, chloroform) Rf (toluene/ethyl acetate 1/1)= 0.32.

B) 4,6-O-benzviidene-2,3-di-O4(R)-3-benzyloxytetradecanoyl]-D-glucopy- ranose (for ExeamDle 2) a) trichloroethyl 2,3,4,6-tetra-O-acetYI-ss-D-qlucoperanoside To an ice-cooled solution of 2.34 g penta-O-acstyl-íD-gIucopyranose in 9 ml trichioroethanol is added 0.9 ml of borontrifluoride etherate and the solution is maintained 4 hours at that temperature. Then the solution is poured onto 100 ml of ice-water, extracted with 100 ml of methylene chloride, the organic phase is separated, washed with 50 ml of sodium hydrogen carbonate solution and 50 ml of water, dried over sodium sulfate and evaporated to dryness.After chromatographic purification (silicagel, toluene/ethyl acetate 4/1) the product crystallises from ethyl acetate/petroleum ether M.P. 1381400.

Rf (toluene/ethyl acetate 1/1) = 0.67.

b) trichloroethyl 4,6-O-benzylidene-(3-D-qlucopyranoside 7.13 g trichloroethyl 2,3,4,6-tetra-O-acetyl-B-O-glucopyranoside are dissolved in a mixture of methanol and methylene chloride, the solution is cooled with ice and reacted with a solution of sodium methanolate (60 mg sodium in 26 ml of methanol). After 2 hours at 0 the solution is neutralized with Dowex AG 50 WX8 H+, the ion-exchanger is filtered off, and the solvent removed under reduced pressure. The residue is dissolved in 60 ml of benzaldehyde, 4.19 zinc chloride are added and the mixture is stirred for 10 hours at room temperature. The mixture is then poured onto icewater, extracted with ether, the ether solution is dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (silicagel, toluene/ethyl acetate 4/1).

Rf (chloroform/methanol 9/1) = 0.60.

c) trichioroethyl 4,6-O-benzylidene-2q3 ,-di-O4CR)-3-benzyloxytetradeca- noyli0-D-qlucopYranoside To a solution of 800 mg trichloroethyl 4,6-O-benzylidene-ss-D-glucopyrano- side, 1.4 g (R)-3-benzyloxytetradecanoic acid and 20 mg dimethylaminopyridine in 10 ml of methylene chloride cooled to 0 are added 930 mg dicyclohexylcarbodiimide and the reaction mixture kept overnight at 40.

The reaction mixture is then filtered, the filtrate evaporated to dryness, the residue dissolved in hexane/toluene/ethyl acetate (12/5/1) and chromatographed with the same solvent mixture.

Rf (hexane/ethyl acetate 5/1) = 0.52.

d) 4,6-O-benzylidene-213-di-O[(R)-3-benzyloxytetradecanoyl]-D-glucopy- ranose 480 mg trichloroethy 1 4,6-O-benzylidene-2,3-di-O4CR)-3 -benzyloxytetrade canoyl]-ss-D-glucopyranoside are dissolved in 50 ml of a mixture of dioxane and acetic acid (1/1) and reacted in portions at room temperature with zinc powder until all the starting material has been used up. The reaction mixture is filtered, evaporated to dryness and the residue chromatographed (silicagel, hexane/toluene/ethyl acetate 2/5/1).

Rf (toluene/ethyl acetate 9/1) = 0.32.

C) 2-O-[(R)-3-benzyloxytetradecanoyl]3-[(R)-3-benzyloxytetradecanoyl - amldoJ-3-deoxY-4,6-O-isoproDylidene-D-qlucopyranose (for Example 1) a) a-azido-3-deoxe-4,6-O-isopropYlidene-D-qlucopYranose 1.02 g 3-azido-3-deoxy-D-glucopyranose are dissolved in 10 ml of dry dimethylformamide, 940 jil of 2-methoxypropene and a catalytic amount of p-toluenesulfonic acid-monohydrate are added and the solution is kept at room temperature for about 2 hours. The p.toluenesulfonic acid is neutralised with sodium hydrogen carbonate, the solution evaporated to dryness and the residue chromatographed (silicagel, chloroform/methanol 9/1).

Rf (chloroform/methanol 9/1) = 0.64.

b) tert.-butyldimethy Isi lyl 3-azido --deoxy-4,6-O-isopropy li den e-O-D-qlu- copyranoside To a solution of 560 mg 3-azido-3-deoxy-4,6-O-isopropylidene-D-glucopy- ranose and 310 mg imidazole in 25 ml of dry methylene chloride are added 345 mg tert.-butyldimethylsilyl chloride and the mixture is stirred for 2 hours at room temperature. The excess imidazole chloride is filtered off and the filtrate is shaken twice with 10 ml of water, dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (silicagel, toluene/ehtyl acetate 12/1).

Rf (toluenelethyl acetate 1/1) = 0.6.

c) tert.-butyldimethy Isilyl 2-O~(R)-3-benzyloxytstradecanoyll-3(R) benzyloxytetradecanoylamido]-3-deoxy-4,6-O-isopropylidene.B-D-glucopy- ranoside A solution of 3.1 g tert.-butyldimethylsilyl 3-azido-3-deoxy-4,6-O-isopro pylidene-ss-D-glucopyranoside in 100 ml of methanol is hydrogenated for 2 hours with 300 mg 10 % palladium on charcoal. The catalyst is filtered off and the filtrate evaporated to dryness. The residue is dissolved with 5.35 g (R)-3-benzyloxytetradecanoic acid and 100 ml of dimethylaminopyri dine in 50 ml of dry methylene chloride, the solution is cooled with ice and 4.1 g dicyclohexylcarbodiimide are added. After about 3 hours at room temperature the solution is filtered, the solvent evaporated and the residue chromatographed (silicagel, toluene/ethyl acetate 9/1).

Rf (toluene/ethyl acetate 6/1) = 0.42.

d) 2-O-(R)-3-benzyloxytetradecanoyl]-3-(R)-3-benzyloxytetradecanoyl- amido j-5-deoxy-4,6-O-isopropylidene-D-qlucopyranose To a solution of 5 g tert.-butyldimethylsilyl 2-O{(R)-3-benzyloxytetrade- canoylJ-3-l(R)-5-benzy loxytetradecanoylamido]-3 deoxy-4,6-O-isopropyli- li- dene-6-D-glucopyranoside in 100 ml of anhydrous tetrahydrofurane cooled to -600 are added dropwise 5.2 ml of a solution of tetrabutylammonium fluoride in tetrahydrofurane. This solution is allowed to come to -40 and kept at this temperature until starting material is no longer present (about 30 minutes). 5 ml of methanol are then added, the temperature is brought to room temperature and the solvent evaporated.The residue is extracted with a mixture of methylene chloride and water, the organic phase dried over sodium sulfate and the solvent evaporated. After chromatographic purification (silicagel, toluene/ethyl acetate 3/1) the title compound is obtained as a mixture of anomeres.

Rf (toluene/ethyl acetate 1/1) = 0.48 and 0.52.

D) 4,6-O-benzy lidene-2(S)-3-benzy loxytetradecanoy 1]-2-deo xy-1-O-diben- zylphosphono-α-D-glucopyranose (for Example 3) a) 24'(S)-3-benzyloxy tetradecanoylamido]-2-deoxy-D-glucopyranose The title compound is obtained in a manner analogous to Example Aa).

Rf (chloroform/methanol 7/1) = 0.37.

b) 4,6-O-benzylidene-2[(S)-3-benzyloxytetradecanoylamido)-2-deoxy-D glucopyranose The title compound is obtained in a manner analogous to Example Ab).

[α]D20 = -3.5 (c = 1, chloroform) Rf (toluene/ethyl acetate 1/1) = 0.37.

c) 4,6-O-benzylidene-2-[(S)-3-bentyloxytetradecanoylamido]-2-deoxy-1-O- dibenzylphosphono-α-D-glucopyranose The title compound is obtained in a manner analogous to Example Ac).

(α]D20 = +39.20 (c = 1, chloroform) razz Rf (toluene/ethyl acetate 1/1) = 0.46.

E) 4,6-O-benzylidene-2-deoxy-1-O-dibenzylphosphono-2(R)-3-tetradeca- noyloxytetradecanoylamido]-α-D-glucopyranose (for Example 4.) a) 2-deoxy-2-[(R)-3-tetradecanoyloxy tetradecanoylamido]-D-glucose A mixture of 650 mg D-glucosamine-hydrochloride, 1,9 g N-t(R)-3-tetrade- canoyloxytetradecanoyloxy]succinimide and 0.5 ml diisopropylethylamine in 15 ml dimethylformamide is agitated for 20 hours at room temperature.

Then the solvent is distilled off and the residue chromatographed over silicagel using ethyl acetate/methanol (8/1) as an eluent.

[co3D = +250 Cc = 1, methanol) Rf (ethyl acetate/methanol 6/1) = 0.5.

b) 4,6-O-benzylidene-2-deoxy-2[R)-3-tetradecanoyloxytetradecanoyl- amido]-D-plucopyranose A solution of 5.83 g 2-deoxy-2[(R)-3-tetradecanoyloxytetradecanoylami- do)-D-glucose, 3 g benzaldehydedimethylacetal and 500 mg p-toluenesulfonic acid-monohydrate in 200 ml of dry dimethylformamide is kept on a rotary çvaporator for 3 hours at 55-60 and 30-40 mbar. After this time starting material is no longer present. Most of the dimethylformamide is then distilled off, 500 ml of methylene chloride ars added and the solution extracted twice with 200 ml of dilute sodium hydrogen carbonate solution and 200 ml of water. After drying over sodium sulfate and evaporation to dryness the residue is chromatographed (toluene/ethyl acetate 6/4).

M.P. 162-1650.

[a]D0 = 1.50 (c = 1, chloroform/methanol 1/1) Rf (toluene/ethyl acetate 1/1) = 0.32.

c) 4,6-O-benzylidene-2-deoxy-1-O-dibenzylphosphono-2-[(R)-3-tetradeca noyloxyte tradecanoylamido]- a-D-qlucopyranose To a solution of 950 mg 4,6-O-benzylidene-2-deoxy-2{(R)-3-tetradecanoyl- oxytetradecanoylamido]-D-glucopyranose in 40 ml of dry tetrahydrofurane cooled to -400 is added dropwise 1.25 ml of butyllithium 1.6 M in hexane.

After 5 minutes a solution of dibenzyiphosphorochloridate in benzene is added dropwise and agitation maintained for 5 minutes at this temperature.

The solution is then neutralised with acetic acid, the mixture evaporated to dryness and the residue extracted with methylene chloride/water. The organic phase is dried over sodium sulfate, evaporated to dryness and the residue chromatographed over silicagel (toluene/ethyl acetate 3/1).

20 = +19.30 Cc = 1, chloroform) Lazy = Rf (toluene/ethyl acetate 1/1) = 0.6.

F) 416.O-benzylidene.2-deoxy-1-O-dibenzylphosphono-2-tetradecanoylami- do-a.O-olucopyranose (for Example 5,) a) 2-deoxy-2-tetradecano ylamido-D-qlucose A mixture of 3 g D.glucosamine-hydrochloride, 4.56 g N-tetradecanoyloxysuccinimide and 2.8 ml of diisopropylethylamine in 40 ml of dry dimethylformamide is kept at room temperature for 24 hours. The product is then filtered off, washed with dimethylformamide and water, dried in vacuo and used for the next step without further purification.

b) 4,6 - O-benzy !idene-2-deoxy-2-tetradecanoylam ido-D-qlucose 4 g 2-deoxy-2-tetradecanoylamido-D-glucose are dissolved at 550 in 320 ml of dried dimethylformamide, 2.6 ml dimethoxytoluene and 400 mg p-toluene sulfonic acid-monohydrate are added and the mixture is kept on the rotary evaporator for 4 hours at 55-60 and 30-40 mbar. Then the solvent is distilled off, the residue washed with dilute sodium hydrogen carbonate solution, water and ethanol, dried in vacuo and directly used in the next step.

c) 4,6-O-benzylidene-3-O-(tert.-buty Idimethy lsilyl)-2-deoxY-2-tetradeca- noylamido-D-qlucopyranose 2.2 g 4,6-O.benzylidene-2-deoxy-2-tetradecanoylamido-D-glucose are dissolved at 600 in 250 ml of dry dimethylformamide, 960 mg imidazole and 2.13 g tert.-butyldimethylchlorosilane are added and the reaction kept 24 hours at that temperature. After addition of 480 mg imidazole and 1 g tert.-butyldimethylchlorosilane and 24 hours more at 600 the starting material is no longer present. The solvent is distilled off, the residue extracted with methylene chloride/water, the organic phase washed once with water, dried over sodium sulfate, and distilled off.The residue is chromatographed over silicagel with a gradient of toluene/ethyl acetate (20/1 to 2/1). Two main fractions are obtained: the title compound (650 mg) and the corresponding 1,3-bis-silyl compound (1.82 g). The bis-silyl compound is dissolved in 25 ml of dry tetrahydrofurane, the solution cooled to -400 and 2.5 ml of a 1N solution of tetrabutylammonium fluoride in tetrahydrofurane is added dropwise. After about 1 hour at -400 the cleavage of the anomeric silyl protecting group is complete. 3 ml of methanol are added, the temperature is brought to room temperature and the solvent then removed in vacuo. The residue is extracted with methylene 'chloride/water, the organic phase extracted once with water, then dried and distilled off.A further amount of 1.52 g of title compound is obtained in the form of an anomeric mixture.

Rf (toluene/ethyl acetate 1/1) = 0.42.

d) 4,6-O-benzylidene-3-O-(tert.-butvldimethvlsilYI)-2-deoxY-l-O-dibenzYI- phosphono-2-tstradecanoy lamido-a-D-olucoyranose To a solution of 2.46 g 4,6-O-benzylidene-3-O-(tert.-butyldimethylsilyl)-2- deoxy-2-tetradecanoylamido-D-glucopyranose in 100 ml of dry tetrahydrofurane cooled to -600 are added dropwise 3.12 ml of 1.6 M butyllithium in hexane. After 5 minutes at this temperaturç a solution of dibenzylphosphorochloridate in benzene is added dropwise. Agitation is maintained further at -600 for 5 minutes, then the solution is neutralised with acetic acid and evaporated to dryness. The residue is taken up in methylene chloride and extracted with water. After working up and chromatography over silicagel using toluene/ethyl acetate (3/1) as an eluent the title compound is obtaind.

Rf (toluene/ethyl acetate 7/4) = 0.75.

e) 4,6-0-benzylidene-2-deoxy-1-0-dibenzylphosphono-2-tetradecanoylami- do-α-D-glucopyranose To a solution of 2.05 g 4,6-O-benzylidene-3-O-(tert.-butyidimethylsilyl)-2- deoxy-1.O-dibenzylphosphono.2-tetradecanoylamido-α-D-glucopyranose in 40 ml of dry tetrahydrofurane at -10 are added dropwise 2.5 ml of a 1N solution of tetrabutylammonium fluoride in tetrahydrofurane and agitation maintained for 2 hours at 00. Then 3 ml of methanol are added, the solution is evaporated to dryness and the residue extracted with methylene chloride/water. After working up as usual and chromatography over silicagel using tcluene/ethyl acetate (3/2) as an eluent the title compound is obtained.

Rf (toluene/ethyl acetate 1/1) = 0.35.

G) 4,6-O-benzylidene-2,3-di-O{(R)-3-tetradecanoyloxytetradecanoyl]-D-- glucopyranose (for Example 8) a) trichloroethyl 4,6-O-benzvlidene-2,3-dí-O{(R)-3-tetradecanoyloxytetra- dcancylj-0-D-slucopyranoside The title compound is obtained analogous to Example Bc) using (R)-3 tetradecanoyloxytetradecanoic acid as acylating agent and toluene/ethyl acetate (98/2) as an eluent.

Rf (toluene/ethyl acetate 9/1) = 0.75.

b) 4,6-O-benzylidene-2,3-di-O4(R)-3-tetradecanoYloxytetradecanoYI]-D- qiucopyranose The title compound is obtained analogous to Example Bd) after chromatographic purification over silicagel using toluene/ethyl acetate (15/1) as an eluent.

Rf (toluene/ethyl acetate 9/1) = 0.25.

I4i)synthesis of uridinephosphate-2,3-diacYl-hexoses 1.4 g uridine-5'-phosphoromorpholidate N,N1-dicyclohexylcarboxam idine salt are dissolved in 25 ml of anhydrous pyridine and dried twice by evaporation and redissolution in 25 ml of dry pyridine. To this solution is added a solution of 1 g of a 2,3-diacyl-hexose-1-phosphate in 25 ml of pyridine which has been pretreated in the same way and the mixture is kept for 24 hours at 370, then cooled with ice. Then 50 ml of chloroform and 12 ml of 90 % formic acid are added. This solution is chromatographed over silicagel and unreacted starting material is eluted with chloroform/pyridine/90 % formic acid (30/30/7).Pyridine is removed from the column with chloroform/methanol (9/1) and then the uridinephosphatederivative is eluted with chloroform/methanol/water (66/33/4). Chloroform and methanol are evaporated from the peak fraction in vacuo and the remaining aqueous suspension filtered. The precipitate is dissolved in 100 ml of tetrahydrofurane/watsr (2/1) and the solution agitated for 2 hours with Dowex AG50WX8 (in tristhydroxymethyl]aminomethane form).

The ion exchanger is filtered off, the bulk of the tetrahydrofurane distilled off and the aqueous solution lyophilised. The lyophilisate is used in the next step without further purification.

The compounds of formula III are useful because they possess pharmacological activity. They exert a benificial influence on unspecific antimicrobial resistance. This activity may e.g. be shown in following test methods: 1. Determination of endotoxic activity in the limulus-amoebocytes lysate test Endotoxin catalyses the activation of a proenzyme in limulus-amoebocytes lysate. The releasing of p-nitroaniline 'rom a colourless substrate is measured. The extent of this releasing is detected photometrically, whereby the correlation between absorption and endotoxin concentration (or, respectively, endotoxin activity for analogs) in the range from 0.01 to 0.1 ng/ml endotoxin is linear (comparison with the absorption values of a standard endotoxin). From each sample (dissolved in pyrogen-free sterile doubly distilled water) a dilution series 1:10 is prepared.In addition, to 100 p1 of test sample, reference standard or blank sample, 100 1 of limulus amoebocytes lysate are added. After 10 minutes of incubation at 370C the 200 p1 of substrate are added to the reaction mixture. The reaction is stopped with 200 p1 50% acetic acid after a further 3 minutes of incuba tion. After agitation of the sample the absorption is measured in a spectrophotometer at 405 nm, substracting the background value. The endotoxin contents (the endotoxin activity) of the sample in endotoxin units (E.U.) is computed by linear regression with the values of the standard endotoxin.

2. Induction of endotoxin shock in the mouse The test comprises the induction of an endotoxin shock or an analogous lethal clinical condition with LPS-analogous substances in galactosamin (GalN)-sensibilisized mice. Male C57 bl.-mice (6 animals per group) are given simultaneously i.p. 8 mg GalN dissolved in 0.5 ml PBS, and 0.1 g LPS from Salmonella abortus equi (Sigma) dissolved in 0.2 ml of physiological saline. This treatment kills all animals within 6-12 hours (C. Galanos et al., Proc.Natl.Acad.Sci., USA 76 (1979) 5939-5943). In place of LPS in the standard treatment, the test substances are administered in various dosages either simultaneously with GalN, or parenterally, or orally, before or after treatment with GalN.The experiments are evaluated by comparison of the smallest dosages which are lethal for all animals in a group, or by computation of an LD50 using the Spearman-Kärber method.

3. Microbial septicemia in the neutropenic mouse This model allows the determination of a substance-related increase of the immun response in microbially infected, nsutropenic mice. For the induction of neutropenia, groups of 20 female B6D2F1-mice are given once 200 mg/kg bodyweight s.c. of cyclophosphamide dissolved in 0.2 ml of distilled water, on day 0. On day 3, the test substance is administered parenterally (primarily i.p.) or perorally, if possible dissolved in physiological sodium chloride solution, or dissolved in some other way (0.3 ml).

Infection is performed on day 4 by i.v. administration of the relevant inoculate in a volume of 0.2 ml (number of germs, e.g. per mouse: Pseudomonas aeruginosa 412: 1x105; E. colic120: 2x106; Staph. aure us A 113: 1x106; Candida albicans 124: 1x104). The test animals are observed up to day 10 after infection and the deaths registered daily. The following parameters are computed by reference to the infection control or standards, respectively: a) average survival time b) survival rate The compounds of formula iii effect marked improvements in time and rate of survival over untreated infection controls in experimental infections with gramnegative agents (e.g. Pseudomonas and E.coli) as well as in infections with grampositive agents (e.g. Staph.aureus) or yeasts (e.g.

Candida albicans), after perenteral administration.

4. Activation of the oxidative metabolism of human blood neutrophils.

Neutrophils (at least 95 ,ó pure) are incubated with the test substance in 3 different concentrations for 1 to 2 hours at 370 C. The release by the cells of superoxide anions as an indication of activation is then measured in the form of superoxide dismutase inhibition of the reduction of cytochrome C.

1 x 106 neutrophiles, either pretreated with test substance or not, are added to a solution containing cytochrorne C (80 Pool) and formylmethionylleucylphenylalanine (1û-6M). The controls contain additionally 50 p9 of superoxide dismutase. After 15 minutes of incubation at 370 C the reaction is stopped by cooling the test tubes in an ice bath. The test tubes are then centrifuged at 400 g for 5 minutes to remove the cells. The reduction of cytochrome C, which is proportional to the amount of released superoxide anion, is measured photometrically at 550 nm. The inhibitory effect of the test substance on PMN activation by LPS is measured as described above, whereby after preincubation of the leucocytes the cells are further incubated with a stimulating concentration of LPS.

5. Carbon clearance test This test is based on the principle that particles of a size from 200 to 250 R (e.g. carbon particles) may be eliminated from the organism only by phagocytosis by macrophages. Following intravenous administration, carbon particles are eliminated from the blood circulation by macrophage phagocytosis in the liver (Kupffer stellate cells) and spleen. The determination of RES-activation of a substance is effected by single or repeated administration in aqueous solution or as a suspension. Testsubstances are administered i.p. or s.c. in 4 daily dosages or once 24 hours befor the start of the test. A suspension containing 10 % carbon black is diluted with a 1 % gelatinous sodium chloride solution to a content of 16 mg carbon/ml.Each mouse receives 0.2 ml/20 g body weight i.v. 25 pl blood are collected by puncture of the orbital plexus 3,6,9,12 and 15 minutes after i.v. administration. The blood is hemolysed in 2 ml distilled water. The carbon concentration is determined photometrically. The animals ars then killed and the weights of livers and spleens determined.

6. Herpes infection (mouse) Intracutaneous herpes infection allows for the determination of a substance-mediated increase in the immune response of mice infected with herpes viruses. The cours of the illness is protracted and allows for the observation of the sequential appearance of several parameters. Herpetic lesions appear at the site of infection, and subsequently ulcerate. Eventually, the adjacent leg becomes paralyzed, and paralysis progressively increases until death. Immunocompetent "naked" (hairless) mice are infected intracutaneously on day 0 by intracutaneous administration of the inoculum under investigation in a volume of 0.025 ml (e.g. 1 x 106 plaque-forming units of Hsrpes simplex type 1/mouse). The test substance is administered l.p. in solution, e.g. in physiological saline (0.1 ml).

Systemic herpes infection also allows for the determination of a substancemediated increase in the immune response of mice infected with herpes viruses. Immunocompetent NMRI-mice are infected on day 0 by i.p.

administration of the inoculum under investigation in a volume of 0.1 ml (e.g. 1.3 x 105 plaque-forming units of Herpes simplex type 1/mouse). The test substance is administered s.c. in solution, e.g. in physiological saline.

The test animals are observed up to day 20 after infection and the appearance of lesions, paralysis and death registered. The following parameters are measured and compared with infection controls or a standard: a) Number of mice with lesions (cumulatively) b) Number of mice with paralysis (cumulatively) c) Average survival time d) Survival rate In the experimental HSV-1-infection, the compounds of formula III cause marked improvements as regards the course of illness, survival time and survival rate over untreated controls. These effects are observed after a single i.p. or s.c. administration between days 0 or -1 and +6.

7. 0SF induction (colony stimulatinq factor) The CSFs are mediators produced by the organism following infections or in response to toxins. Their biological effects are complex; they are detected via stimulation of the proliferation of the hemopoietic system, especially of bone marrow cells. 86D2F1 mice are given the test substances once or repeatedly, parenterally or orally, up to a dosage of 50 mg/kg.

Serum is collected from the test animals at various subsequent time intervals. The CSF-activity titers of the sera are measured in a cell culture assay as the rates of proliferation of bone marrow cells from B6D2F1 mice ID. Mstcalf, The Hemopoietic Colony Stimulation Factors, 1984, Elsevier; T. Mosmann, J. Immunol. Methods, 65 (1983) 55-63; L.M. Green et awl., 3, Immunol. Methods, 70 (1984) 257-2681 The compounds of formula III induce CSFs to various extents in mice, whereby time-kinetic differences in CSF-activities are also observed. These could be advantageous in therapeutic use.

8. Induction of Interleukin-1 (IL-1) The determination of a substance-mediated stimulation of cells to produce IL-1 is primarily effected in a tissue culture assay. First, both the rssident and thioglycolate-elicited macrophages are recovered in the form of adherent macrophages. These are incubated in RPMI-medium for 48 hours with various concentrations of test substance and the cell supernatants collected. These are tested for IL-1-activity in cultures of thymocytes from C3H/HeJ mice. The thymocytes of those supernatants containing IL-1 produced by macrophages are induced to proliferate during a 72 hours incubation. Proliferation is measured by incorporation of 3H-thymidine, in -a scintillation counter (J. Gery et al., J.Exp.Med. 136 (1972) 128-141; J.

Oppenheim et al., Cellular Immunol. 50 (1980) 71-81].

The compounds of formula III' possess to various extents (in concentrations of 0.1 - 50 llg/ml) the capacity to induce IL-1 production in macrophages.

9. Induction of LPS(endotoxin) tolerance (lethality tolerance) A so-called tolerance may be induced in mice by parenteral administration of LPS once to thrice a day. This tolerance protects the animals against the lethal effects of LPS following administration of galactosamine (cf.

induction of endotoxin shock in mice above).

The compounds of formula III are eliciting this tolerance already after single i.p. administration of 0.25 mg/mouse. Pretreated (tolerant) mice are exposed to a LPS-challenge at a dosage of 0.01 g LPS + 8 mg galactosamine/mouse i.p. at various times (1 day to 3 weeks) following the last treatment. A larger proportion of animals survive this LPS challengs, especially after repeated administration (3 times), as compared with challenge controls notpretreated.

Furthermore, the compounds of formula III possess antiinflammatory activity, especially in nonspecific, in immunologically-induced, and in hypersensitivity-induced inflammation and in allergic reactions. This acti vity may be demonstrated by various test methods, e.g. by investigation of the influence on prostaglandin synthesis in vitro and in vivo. In vitro, the inhibition of LPS- and zymosan-induced PGE2- and PGF1 -releass is investigated. Thioglycolate-stimulated peritoneal leucocytes from NMRImice are incubated for 24 hours with LPS or test substance. After a change of medium and triple wash of the cells they are stimulated for 2 hours with LPS or for 1 hour with zymosan, respectively. In thess supernatants PGE2 and PGF la are determined.An inhibition is found of LPS- or zymosan.

induced PGE2-production.

The inhibition of LPS-induced PGE2-release by mouse peritioneal leucocytes after pretreatment with test substances is measured in vivo. Groups of 3 NMRI-mice are treated i.p. on days 1,2 and 3 with LPS or, respectively, test substance. On day 4 these animals and a control group are given 1.5 ml thioglycolate i. p.; on day 7, peritoneal mouse leucocytes are collected and stimulated with LPS. A marked reduction in PGE2-release is found as compared with controls.

In a further test, the influence on procoagulant activity (PCA) ist measured. After stimulation with LPS, human endothelial cells synthesize PCA, as detected by a shortening in plasma coagulation time. The coagulation time of recalcified plasma is also shortened by mouse peritoneal macrophages obtained after LPS treatment and by rabbit peritoneal leucocytes after release of the generalized Shwartzmanreaction. To investigate the influence on PCA induced by LPS in vitro, peritoneal mouse leucocytes of B6DvF1-mice stimulated by thioglycolate are treated overnight with LPS alone or with LPS and test substance, respectively, in DMEM medium devoid of fetal calf serum (FCS) ((test design a)].In a test design b), mouse peritoneal leucocytes are treated for 24 hours with LPS or test substance, respectively. After a change of medium, cells are stimulated overnight with LPS. The coagulation time of recalcified human control plasma is measured using the Häkchen-method after addition of the mouse peritoneal leucocytes suspension which has been previously repeatedly frozen and treated ultrasonically.

The additon of test substance reduces the PCA elicited by LPS as compared to the control value as manifested by an increase in coagulation time. Pretreatment with test substance similarly results in reduction of PCA.

In vivo, the influence of LPS-induced PCA by mouse peritoneal leucocytes after prstrsatment with test substance may be shown as follows: B6D2F1-mice are treated i.p. on days 1, 2 and 3 with LPS or test substance, respectively. On day 3, all animals additionally receive 1.5 ml of thioglycolate i.p. On day 6, peritoneal leucocytes are collected, the sample from each animal adjusted to 1 x 106 cells/ml, and stimulated for 24 hours with LPS in DMEM medium devoid of FCS. The PCA of these cells is determined as described above. Pretreatment with LPS or test substance, respectively, induces a markedly reduced PCA. A similar reduction may be observed in rabbits.The PCA of rabbit peritoneal leucocytes may be reduced after induction of the generalized Shwartzman reaction, and by pretreatment with LPS or the substance, respectively.

To study the inhibition of the local Shwartzman-reaction, groups of 3 chincilla rabbits are pretreated i.v. or i.p. with LPS or test substance, respectively, on days 1, 2 and 3. On day 6, LPS is administered (40, 20, 10, 5, 2.5 and 1.25 p9), intradermally. On day 7, the reaction is triggered with 21rg LPS/kg i.v. The experiment is evaluated semi-quantitatively by examination of the skin necroses. An almost complete inhibiton of Shwartzman-reaction ist observed after pretreatment with LPS or test substance, respectively.

Furthermore, the compounds of formula III possess activity against tumors, as demonstrated in the following tests: 1. Induction of tumor necrosis factor (TNF) In order to stimulate mouse bone marrow macrophages, bone marrow cells from BDF1-mice (ca. 10-13 days old, induced with CSF) are diluted to 1 x 106 cells/ml in medium ERPMl 1640 + 1 % Pen/Strep (5000 U/ml) + 1 % glutamine] and incubated in flat microtiter plates with the test substances in solution in the range of 100 g to 0.1 Wg/ml end concentration (dilution steps 1:10) for 4 hours at 370 C/5 % C02. Alfter filtration through 0.45 ,um filters, the supernatants are frozen at -700 C until needed.

L 929 cells are precultured overnight C370 C, 5 % Cho2) in microtiter plates at 3 x 104 cells/well/l00 pI, then 100 SL1 of each sample are added and the culture further diluted in 1:2 steps. 100 pl of actinomycin 0 (8 pg/ml of medium) are added and incubation continued for a further 18 hours at 370 C/5 % Cho2. After removal of the supernatants the remaining cell layer is stained with Giemsa solution and absorption measured at 620 nm with a Titertek Multiscan Autoreader (Flow!. One unit of TNF is defined as the activity which results in 50 % lysis of the target L 929 cells.

2. B16F1 melanoma test B16F1 melanoma cells are grown for 5 days in vitro. The cells are synchronized one day before the test by disrupting the monolayer into separate cells using EDTA-trypsin and returning the whole amount into the same bottls with fresh medium. The cells are counted and made up to 106 cells/ml of medium. 0.1 ml of cell suspension (105 cells) is intravenously injected into the tail vein of B6D2F1-mice. 21 days later the mice are killed and the number of lung tumors counted. The test substance is brought into solution and injected intraperitoneally on days -6, -4 and -1.

It is found that the compounds of formula III possess immunoprophylactic activity, which reflects in a reduction of the number of B16F1 melanoma metastases in the lungs. As a test of therapeutic activity, mice are treated on day 3, 6, 8, 10, 13 and 15 after inoculation of the tumor cells. Here, too, a marked reduction in the number of metastases is observed.

The compounds of formula III are therefore useful as modulators of unspecific antimicrobial resistance, in the systemic enhancement of immune response, and in the enhancement of unspecific immunity. The compounds of formula III i are thus useful in the treatment or supportive treatment (i.e. together with other specific or supportive therapeutic forms) of conditions associated with decreased immune response, especially decreased humoral immune response and/or decreased over sensitivity reaction of the delayed type, and in the treatment of conditions in which generally a modulation of immune response is indicated.In particular, the compounds of formula III are useful in the treatment or supportive treatment of pathological conditions based on idiopathic immunological deficiencies or immunological deficiencies of the type encountered in geriatric patients, or in patients with heavy burns or generalized infections. The compounds of formula III . are also useful in the treatment or supportive treatment of viral illnesses (such as disseminated Herpes, progressive smallpox and disseminated varicella diseases), of Morbus Hodgkin and other malignant tumors. Furthermore, the compounds of formula III are useful for the prevention of endotoxin shock, e.g. in accidents, burns, and surgical interventions, and as antiinflammatory agents.

For the above-mentioned uses the dosage will, of course, vary depending on the compound employed, mode of administration and treatment desired. In general satisfactory results are obtained when administered at a daily dosage, or as a unique administration for the achievement of an adjuvant effect, e.g. in supportive treatment, of from about 0.0015 mg/kg to about 1 mg/kg animal body weight. Administration is e.g. parenterally, preferably i.p. For the~larger mammal an indicated total daily dosage is in the range of from about 0,1 mg to about 70 mg, conveniently given, in the case of a daily dosage, in divided doses 2 to 4 times a day in unit dosage form containing from about 0.025 to about 35 mg of the compounds admixed or in sustained release form.An indicated total single adjuvant dosage is in the range of up to 70 mg of the compounds.

In view cf their immunomodulating activity the compounds of formula III are also useful as adjuvants in vaccines. For this use an indicated dosage is from about 0.5 mg to about 100 mg, preferably about 70 mg, administered on the day of vaccination with appropriately a repetition in the same dosage 2 to 4 weeks thereafter.

Pharmaceutical compositions containing a compound of formula III may be prepared in accordance with standard galenical methods, e.g. by mixing with conventional, pharmaceutically acceptable diluents or carriers.

They may be prepared e.g. in the form of injectable solutions. They are also part of the invention.

The invention also provides a compound of formula III for use as a pharmaceutical, especially as an immunomodulator, as an antiviral agent and as an intiinflammatory agent.

Claims

CLAIMS:

1. Compounds of formula III

wherein X and Y independently are oxygen or imino and R1 and R2 independently are acyl of 14 carbon atoms optionally monosubsti tuted by hydroxy or acyloxy of 14 carbon atoms, with the provisos that 1) X and Y are not both imino and 2) when X is imino and Y is oxygen, then R1 and R2 are not simultaneously (R)-3-hydroxytetradecanoyl, in free form or in form of acid addition salts.

2. A pharmaceutical composition comprising a compound of formula III as defined in claim 1 in association with a pharmaceutical carrier or diluent.

3. A process for the production of compounds of formula III as defined in claim 1 comprising a) for the production of compounds of formula IlIa

wherein R1 and R2, are as defined in claim 1 and either X' and Y' are oxygen or Y' is imino and X' is oxygen, converting in compounds of formula IV

wherein R1 and R2 are as defined in claim 1, X' and Y' are as defined in this claim and R is a protecting group, the unprotected hydroxy group to a protected phosphate group or b) for the production of compounds of formula lIIb

wherein R1 and R2 are as defined in claim 1.

acylating corresponding compounds of formula Ilic

wherein R is as defined in this claim, R2 is as defined in claiml and R' is a protecting group, and thereafter splitting off the protecting groups and recovering the resulting product in free form or in form of acid addition salts.

9. A compound of formula III as defined in claim 1, in free form or in form of pharmaceutically acceptable acid addition salts, for use as a pharmaceutical.