NL8601551A - NEW SACCHARIDES, THEIR PREPARATIONS AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. - Google Patents

Description

. ^ * r

New saccharides, him 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 a new compound of the formula I, wherein R 1, R 1, R 1, and R 1 independently of each other are hydrogen or an optionally substituted acylic group and ¥, X, Y and Z independently of one another or imino, provided that a) at least one of the groups Rj - is an optionally substituted acylic group and B] when Z and X are imino and ¥ and Y are oxygen, and «). Rj and R ^ are different from Both (R) -hydroxytetrade-canoyl, insofar R ^ and R ^ are identical and (R) -3-hydroxytetrade-canoyl or R | is (R) -3-dodecanoyloxytetradecanoyl and R ^ is 15 (R) -3-tetradecanoyloxytetradecanoyl, g) R ^ is other than hydrogen or (R) -3-hydroxytetradecanoyl, insofar R 'and R ^ are hydrogen and R ^ is (R) -3-hydroxy-tetradecanyl and γ) R ^ is other than a group - (CO ^ -C ^ -CCHOH) ^ - 20 CB ^ OH, insofar as the other substituents are hydrogen.

In a further aspect, the invention provides a process for the preparation of a compound of the formula la, wherein X, Y, ¥ and Z have the meanings given previously and R <1> is independently hydrogen or an optionally substituted acylic group, provided that at least one of the groups R ^ _ ^ is an optionally substituted acyi group, enzymatically a compound of the formula II, wherein ¥ and Z have the previously given meaning, R R and R ^, with the exception of hydrogen, have the 3Q meaning indicated above for R 1 and R 1 and R 1 is uridine, immediately condenses a compound of the formula III, wherein X and Y have the meanings shown and R "j and R ^ have the meanings indicated above. 80'551 - 2 - i $ iÉ> for R 2 and R 2, provided that at least one of the two substituents is an optionally substituted acyl group and when X or Y is imino, the substituent R "or R" bonded to the imino group is other than hydrogen, and optionally hydrolyzes the compound obtained to t is a corresponding compound of the formula la, wherein when X, Y, Z and / or W is oxygen, R 1, R 4, R 1 and / or R is hydrogen, or optionally 1 L 5 4 subjects a resulting compound to an acylamidase reaction to obtain a compound of the formula la, wherein JO is X, Y, Z and / or im imino, then R 1, R 1, R 1, and / or R 1 is hydrogen.

For example, the enzymatic condensation can be accomplished in a buffered system, for example, in a tris buffer, at pH 7. The reaction can be accomplished at room temperature or elevated temperature, for example, at 30 ° C. A preparation obtained from grams of negative bacteria, preferably from D. coli strains (Raetz, J. Biol. Chem. 259 {1984} 4852} can be used as an enzyme extract.

Strains induced by genetic engineering to overproduction of the desired enzyme are preferred.

The optional hydrolysis can be accomplished in a manner analogous to methods described in the literature.

The acylamidase reaction can be accomplished in a manner analogous to that described by C.R, Verret 25 et al. In J. Biol. Chem. 257 {1982} 10 222.

The products of the process of the invention can be isolated from the reaction mixture and purified, if desired, according to known procedures.

The compounds of formulas Ia and III can be hestane in a free form or, if appropriate, in acid addition form. A free form can be converted to a salt form in a conventional manner and vice versa.

Rj_ ^ are preferably identical. They are preferably hydrogen or an acyl group with, for example, 4-20, 35 preferably 12-16, especially 14 carbon atoms.

8601551 d a - 3 -

For example, they can be substituted in the 3-position by a hydroxy, acetoxy or acyloxy group as defined above.

The 3-position carbon atom configuration can be (R) or (S). thus may be present in a compound 5 of the formula la in achiral, (R), (S) or racemic form.

This also applies. for a compound of formula 2, 3 and 4.

The configuration remains unchanged during the process of the invention, ie when a compound in (R), (S) or racemic form is used as starting material, a corresponding (R), (S) or racemic end product is obtained .

Preferred compounds of the formula Ia, wherein R 1, is an optionally substituted acyl group. Also preferred are the compounds of formula Ia wherein, when R 1, R, R 1, and / or R, 12 3 4 is hydrogen, Y, X, W and / or Z is oxygen.

A compound of the formula la is preferably obtained in the form of an acid addition salt. Hydrophilic basic compounds, for example tris (hydroxymethyl) -20 aminomethane or L-lysine, are preferably used to increase the water solubility.

A compound of the formula II can be obtained in a known manner by reacting a compound of the formula III with activated uridine 5'-monophosphate.

A compound of the formula III, wherein 25 Al Y is oxygen and X is NH. provided «} when R” is (R} -3-hydroxytetradecanoyl, R ”is other than (R) -3-hydroxytetradecanoyl or (R] -3-hexadecanoyl-oxytetradecanoyl, g) R" is other than hydrogen and 3Q γΐ Rj and R | J are different from both acetyl or the group -CHOCCH ^ jqCRj, b) Y and X are oxygen or c) Y is NH and X is oxygen, provided that at least one of the two substituents is an acyl group and 6δδ1551? * s - 4 - when X or Y is imino, the substituent R "or R" attached to the imino group is other than hydrogen, it is new and also forms part of the invention.

A compound of the formula III can be obtained according to a process, wherein already for the production of a compound of the formula IIIa in which R 'and R' have the previously given meaning, either X 'and Y' are oxygen, or Y 'is imino and X 'is oxygen, in a compound of the formula IV, wherein IQ R ", R 1, R', X 'and Yr have the meaning given earlier and R is a protecting group, converting the unprotected hydroxy group into a protected phosphate ester group or b) acylates to produce a compound of the formula Illb, wherein R 1 and R 6 have the previously given meaning, acylates a corresponding compound of the formula IIIc, wherein R 1 and R have the previously given meaning and R 'is a protecting group , and then split the protection groups.

Process variant a) can be effected, for example, by dissolving a compound of the formula IV in an inert solvent, for example a cyclic ether such as tetrahydrofuran and reacting it at a lower temperature, for example at -70 ° G, in a butyl lithium in a aliphatic hydrocarbon, such as hexane, and then adding dibenzylphosphoric chloride. The product can be recovered from a reaction mixture and, if desired, purified in known manner.

The free hydroxy groups of the phosphate residue are protected, for example, by benzyl. The cleavage of the protecting groups can also be effected in a known manner.

For example, a protecting group can be cleaved off in a known manner under acidic conditions, for example with aqueous acid (ion exchanger 1 or hydrogenolytic).

Process variant b) can be effected, for example, by dissolving a compound of the formula IIIc in an inert solvent, for example in a hydrocarbon SS ö 15 3 f -5.-

Jt * 5 such as methylene chloride together with an acylating agent suitable with addition of dicyclohexylcarhodiimide and 4-dimethylaminopyridine, the reaction is allowed to proceed at lower temperatures, for example at about 4 ° C. The product can be isolated from the reaction mixture and, if desired, purified in known manner. The protective groups present are then split off in a known manner.

As the protecting group, one can use any group commonly used for protection in the saccharide industry JO. Both R substituents can, for example, together form the benzylidene or isopropylidene group. Known protecting groups, such as benzyl, can also be used as protective groups for the phosphate residue.

The compounds of formula IIIc are new and can be obtained according to the reaction scheme A on the formula sheet.

The compounds of formula XV can be obtained according to the reaction schemes B, C and D on the formula sheet, wherein the hydroxy groups which do not participate in the indicated reaction may suitably be in protected form. The substituents have the following meanings: R, RT = protecting groups R ^ = both independently the meaning indicated above for Rj, R ^, R ^ or R ^ 25 Ac = acetyl TBDMS = tertiary butyldimethylsilyl J. Preparation of the compounds of the formula XVa: see reaction scheme B.

2. Preparation of the compound of formula IVb: see 3Q reaction scheme C.

3. Preparation of the compound of formula IVc; see reaction scheme D.

Insofar as the preparation of any particular starting material has not been specifically described, it is known or can be carried out in a conventional manner or in an analogous manner as described in the examples.

The following examples illustrate the invention. All temperatures are in degrees Celsius. EDTA means ethylenediaminetetraacetic acid, DTA is 1,4-dithioerythrite, 5 UDP stands for uridine phosphate, and tris is tris (hydroxymethyl) aminomethane.

Example 1: 6-Q- {2-deoxy-3-0 - {(B.) - 3-hydroxytetradecanoyl] 2- {(R)} -3-hydroxytetradecanoylamido} —GD-glucopyrano-syl} -2,3- di-0 - {(R)} - 3-hydroxytetradecanoyll · 1-0-jq phosphono qD-glucopyranose

2.03 mg of UDP-2-deoxy-3-0 - {(R) -3-hydroxytetradecanoyl} -2 - {(R) -3-hydroxytetradecanoylamido} -1-O-phosphane-aD-glycopyranose and 1.42 mg 2,3-di-0- {(R) _- 3-hydroxytetradecanoyl} - 1-0-phosphono-aD-glycopyranose both in the form of tris salts

^ in 10 mM tris buffer from PI. 7 with 0.2 mM ETA and 0.2 mM DTE

and incubate with 60 µl of enzyme extract at 30 ° C.

The enzyme extract can be prepared as follows:

E. coli JB 1104 is cultivated in L-broth (culture + 10 µg ampicillin / ml at 3 ° C overnight), main culture at 30 ° C stirred incubator, starting from 0D = 0.1 to 0D, .cn = 1). The cells are 550nm 550nm

collected by centrifugation at 10,800 g (10 min./4 C), the pill suspended in 10 mM tris buffer of pH 7.0 + 0.2 mM EDTA

and 0.2 mM DTE and centrifuged again (6000 g, 10 min / 4 ° C).

25

The pill is then resuspended in the above buffer and ultrasonically fragmented (5 x 20 sec / 30 sec break at 120 watts}. The cell fragments are separated by centrifugation (12000 g, 10 min / 4 ° C) and the solution is centrifuge (150,000 g, 90 min / 7 ° C)

Or} upper 2/3 of the supernatant is recovered. This fraction is subjected to fractional ammonium sulfate precipitation. The range of 10-40% ammonium sulfate is used. The obtained pill is suspended in the above buffer.

860 1551 * * i - 7 -

After the reaction is complete (checked by thin layer chromatography), the solution is acidified to pH 2 with cironic acid and centrifuged. The pill is suspended in chloroform / methano1 / acetic acid / water (65/15/5/2) and chromated. matographed on silica gel using the same eluent. The fractions containing the product are combined and evaporated until no organic solvent remains and the remainder is lyophilized.

The lyophilisate is dissolved in tetrahydrofuran / water 8/2) and converted into the tris salt in that solvent over a tris-form cation exchanger. The tetrahydrofuran is evaporated and the residue lyophilized. All values are determined on silica gel plates. (in chloroform / methanol / acetic acid / water 65/25/5/5) * 0.48.

Example 2: 6-0- £ 2-deoxy3-0 ~ {(R) -3-hydroxytetradecanoyl] 2- ((R) -3-hydroxytetradecanoylamido} -gD-glucopyronosyl} -2-deoxy-3-0 - {(S) -3-hydroxytetradecanoyl} - 2 - {(S) -3-hydroxytetra-decanoylamido} -1Q-phosphane-qD-glucopyranose 20

20 is allowed to fill. parts of a 5 mM solution of 2-deoxy-3-0 - {(S) -3-hydroxytetradecanoyl} -2 - {(S) -3-hydroxytetra-decanoylamino}} - 1-0-f os f ct-D-glycopyranose in 10 mM tris / HCl buffer (pH 7) with Q, 2 mM EDTA and 0.2 mM DTE, 20 parts of a 5 mM solution of UDP-2-deoxy-3-0 - {( R)} - 3-hydroxytatra-decanoyl) -2 - ((R) -3-hydroxytetradecanoylamido} -1-0-phosphono-a-D-glycopyranose in the same buffer and 20 parts of a 100 mM tris / HCl buffer ( pH 7) with 2 mM EDTA and 0.2 mM DTE reacting with 40 parts of an enzyme preparation (prepared as described in Example 30) is 10 mM tris / HCl buffer (pH 7) with 2 mM EDTA, 2 mM DTE and 0.1% Triton X-100 at 30 ° C for 8 hours, setting the final concentration of Triton X 100 to 0.2% The reaction mixture is lyophilized, dissolved in about 1/3 of the initial volume in the eluent for the following

-35%!

. ______. The volume of the column is approximately 20 times larger 8601551.

- 8 - then let sample volume. The pure product fractions are collected, concentrated under reduced pressure, suspended in pyrogen free water and lyophilized.

After lyophilization, the Triton X 100 is removed by digestion with diethyl ether. After further centrifugation, the pill is dissolved in a mixture of chloroform / methanol (1 / L), filtered and the solvent is removed under reduced pressure. To obtain the mono-L-lysine salt, the calculated stoichiometric amount of L-lysine (free base) 10 is added in the form of an aqueous 100 mM solution to an aqueous suspension of the residue and lyophilizes the mixture again. R ^ (chloroform / methanol / acetic acid / water 65/25/5/5) = 0.44. Example 3: 6-0- {2-deoxy-3-0 - {(R) -3-hydroxytetradecanoyl} -2- {(R) -3-hydroxytetradecanoylamido} -gD-glucopyrano-15 syl} -2-deoxy- 3-Q - {(R) -3-hydroxytetradecanoyl} -1- Q-phosphono-2— [(R) -3-tetradecanoyloxytetradecanoyl-amido} -QD-glucopyranose

20 is allowed to fill. parts of a 5 mM alloy of 2-deoxy-3-Q - {(R) -3-hydroxy-tetradecanoyl} -1-Q-phosphono-2- 20 {(R) -tetradecanoyloxytetradecanoyl-amido} -a-D-glucopyranose

in IQ mM tris / HCl buffer (pH 7) with 0.2 mM EDTA and 0.2 mM

DTE, 20 parts of a 5 mM solution of UDP-2-deoxy-2-0— {(R) -3-hydroxytetradecanoyl} -2 - {(R) -3-hydroxytetradecanoylamido} ~ 1-0-phosphono-ct -D-glucopyranose in the same buffer and ^ 20 parts of a 100 mM tris / HCl buffer (pH 7) with 2 mM EDTA and Q, 2 mM DTE react with 40 parts of enzyme extract (prepared as described in Example 1} in 10 mM tris / HCl buffer (pH 7) with 2 mM EDTA, 2 mM DTE and 0.1% Triton X 1Q0 at 30 ° C for

48 hours, making the final concentration of Triton X 100 3Q

set to 0.1%. The mixture is lyophilized, treated with pyridine and filtered, the filtrate concentrated under reduced pressure and chromatographed on silica gel using chloroform / methanol / water / 2-phenethylpicolonium bromide 800/175 / 22.5 / 2.5 as eluent.

35 8601551 * "§r" - 9 -

The pure product fractions are collected and the 2-phenethylpicolonium bromide, which serves as an ion pair

molding agent is removed by stirring with 20 mM

phosphoric acid in water. The organic phase was concentrated under reduced pressure and to obtain the mono-L-lysine salt, the calculated stoichiometric amount of L-lysine (free base} is added and the mixture lyophilized.

R_ (chloroform / methanol / acetic acid / water 65/25/5/5) = 0.53. r

In a manner analogous to Examples 1-3, the following compounds of the formula I are obtained, wherein, depending on the compounds of the formula II and III used, the amounts of enzyme are adjusted such that the reaction proceeds in preferably 24-48 hour.

TABLE

15 Rj = = (R} ~ 3 ~ hydroxytetradecanoyl yoorheeld No. WZYX Rf 4 0 NH NH NE 0.45 5 NE NH NE NE 0.40 20 6 0 Ö Q NH 0.49 7 Q NH NH 0 0.39 8 NH NE Q NH 0.41 9 0 0 0.0 0.48 25 Example 10: 6-Q- {2-deoxy-3-Q - {(R) -3-hydroxytetradecanoyl} -2- {(R) -3- hydroxytetradecanoylamido} -gD-glycopyrano-sy13 ~ 2-deoxy-1-0-fo s phono-3-0-tetradecanoy1-2-te-tradecanoylamldo-aD-glucopyranose

The title compound is obtained in a manner analogous to Examples 1-3. R ^ (chloroform / methanol / acetic acid / water (65/25 / 5r5) = Q.50.

yoorheeld 11: 6-0- {2-deoxy-3-0 - {(R) -3-hydroxytetradecanoyl} -2- {(R) -3-hydroxytetradecanoylamido} -gD-glucopyrano-sy1} -2-acetamido-2 -deoxy-3-0 - {(R) -3-hydroxytetra- 8601551 ï 4 · ® * · - 10 - decanoyl} -J-0-fo-sphοοη-α-D-glycopyranose The title compound is obtained in a manner analogous to examples 1 - 3. R ^ (chloroform / methanol / acetic acid / water 65/25/5/5) = 0.32 Example 12: 6.0- {2-deoxy-2 - {(R) - 3-hydroxytetradecanoylamido} - gD-glucopyranosyl} -1-0-phosphono-aD-glycopyranose

5 mg of 6-0- {2-deoxy-3,0 - {(R) -3-hydroxytetradecanoyl} -2 - {(R) -3-hydroxytetradecanoylamido} -GD-glycopyranoysl} -2, are dissolved 3-di-Ö - {(R) -3-hydroxytetradecanoyl} -1-0-phosphono-aD- jq glucopyranose in chloroform / methanol (1/1), react with aqueous 25% ammonia (.1 / 3 of the volume) and let alone during the night. After the reaction is complete, the mixture is evaporated / the mixture is dried, the residue is digested with diethyl ether to remove the cleaved fatty acids, the precipitate is filtered off with suction and washed with ether, the residue is dissolved in water and the di-tris salt is prepared. by adding the calculated amount of tris. The product is lyophilized.

R ^ hloroform / metbano1 / acetic acid / water 25 / J5 / 2/4) = 0.45.

Example 13; 2-deoxy-3-0 - {(R) -3-bydroxytatradecanoyl} -2- {(R) -20 benzyl-oxytetradecanoylamido} -2-deoxy-IQ-dihenzylphosphono-qD-glucopyranose a) 4,6- 0-henzyliden-3-0- {(R) -3-benzyloxytetradecanoyl} - 2- {(R) -3-benzyl-oxytetradecanoylamido} -2-deoxy-1,0-dibenzylphosphono-qD-glucopyranose 25 solution of 3.9 g of 4,6-0-benzyliden-2-Γ (R) -benzyloxytetradecanoyl-amido} -2-deoxy-1 -0-dib enzyme 1 phosphono-aD-glycopyranose, 2 g (R) 3 -henzyloxytetradecanoic acid and 50 mg of 4-dimethylaminopyridine in 2 ml of methylene chloride, cooled to -10 ° C, 2 g of dieyclohexylcarbodizmide are added and the reaction mixture is kept at 4 ° C during the night. The mixture is then filtered, the filtrate is evaporated to dryness, the residue is taken up in a small amount of toluene / acetic acid ester (8/2) and chromatographed with the same solvent. Melting point 96-98 ° C.

8601551 - 11 - 20 o {α} ^ = +33.3 (c = 1 chloroform) B. £ (toluene acetic acid 2/1) = 0.5 B) 2-deoxy-3-0- {(R) -3 -hydroxytetradecanoyl} -2- {(R) - 3-hydroxytetradecanoylamido] -1-0-phosphono-α-glycopyranose 4.2 g of 4.6-0-Benzyliden-3-0 - {(R) - 3-

Benzyloxytetradecanoylamido} -2-deoxy-1-Q-diBenzylphosphono-cHD-glucopyranose in 900 ml tetrahydrofuran / water (85/15) and hydrogenate with 1.5 g 10% palladium ropropole for two hours at 10 Bar and 4 ° C. The catalyst is then filtered off, the tetrahydrofuran evaporated and the aqueous suspension lyophilized.

{a} ^ = + 28.5 ° (c = 0.2, chloroform + 1 drop of methanol) R. £ (chloroform / methanol / acetic acid / water 125/75/10/20) = 0.56

Further purification can be accomplished as follows:

10 g of 2-deoxy-3-0 - {(R) -3-hydroxytetrade-canoyl} -2 - {(R) -3-hydroxytetradecanoylamido} -1-phosphono-a-D-glycopyranose and 1.7 g are treated tris in 150 ml of methanol for 10 min. At 45 ° C in an ultrasonic bath. The suspension is centrifuged and the clear supernatant is decanted from the cake. 1.7 g of tris (dissolved in 20 ml of methanol) are added to this solution, the solution is seeded and allowed to crystallize at room temperature. The di-tris salt of the title compound is obtained.

25 The supernatant is evaporated to dryness.

The residue and cake are welded together in a mixture of 300 ml chloroform, 600 ml methanol and 240 ml pyrogen free water. An additional 300 ml of chloroform and 300 ml of 0.1 N hydrochloric acid are added, the mixture is stirred gently and the phases are allowed to separate. The chloroform phase is separated off and evaporated to dryness. The residue

It consists of crude 2-deoxy-3-0 - {(R) -3-hydroxytetradecanoyl} -2- (R) -3-hydroxytetradecanylamido} -1-Ofosfono-α-D-glucopyranose.

The product obtained is ultrasonically treated with tris and methanol as described above and centrifuged.

660 1 5 5 f ► ** »'' 'v - 12 -

The solution is chromatographed on a Sephadex LH 20 column and eluted with methanol. The corresponding fractions are combined and evaporated to dryness. The mono-tris salt of the title compound is obtained.

o 5.1 g of this product are dissolved at 45 ° C in 40 min. methanol in the ultrasonic bath, a solution of 0.74 tris in 10 ml of methanol is added, the solution is seeded and allowed to crystallize first at room temperature and then at 4 ° C. Further crystalline amounts of di-tris salt of the title compound are obtained. The residue of the supernatant and the centrifugation cake can be subjected to a further round of purification.

SM.Pt: 183-185 ° (dec.)

2Q

{«} P - + 15 ° (c = 1, Q in water) 15 Example 14: 3-deoxy-2-Q - {(R) -3-hydroxytetradecanoyl} -3 - {(R) - 3-hydroxy-tetradecanoylamido } -1-0-phosphono-qD-glycopyranose a) 2-0 - {(R) -3-benzloxytetradecanoy} -3 - {(R) -3-benyloxy-20 tetradecanoyl-amido} -3-deoxy-1, 0-dibenzylphosphono-4,6,0-iso-pronylidene-A-glucopyranose

To a solution of 4.85 g of 2—0 - {(R) -henzyloxytetra-decanoyl} -3 - {(R) -3-benzyloxytetradecanylamido} -3-deoxy-4,6-0-isopropylides-D-glycopyranose 40 ml of absolute tetrahydrofuran, cooled to -7 ° C, 8 ml of 1.6 M butyl lithium in hexane are added. After 5 minutes, a solution of 3.8 g of dibutylphosphoric chloridate in 10 ml of benzene is added at the same temperature. Stirring is continued at -70 ° C for 10 min., 0.3 ml of acetic acid is added and the solution is concentrated 30 to 1/4 of the initial volume. The solution is diluted with 200 ml of methylene chloride, extracted with 50 ml of water, 50 ml of yerdunde sodium bicarbonate solution and 50 ml of sodium chloride solution, dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (toluene / acetic acid ester 7/3).

35 R ^ (toluene / acetic acid ester 2/1) = 0.58.

8601551 - 13 - b) 3-deoxy-2-0- {(R) -3-hydroxytetradecanoyl 3—3- {(R) - 3-hydroxytetra-decanoylamido} -1 -0-phos phono-a-D-glycopyranose

A solution of 980 mg of 2-0- {(R) -5 3-benzyloxytetradecanoyl} -3- {(R) .- 3-henzyl-oxytetradecanyl-amido} -3-deoxy-1Q-dibenzylphosphono-1 is generated hydro) 6, O-isopropylidene-α-glycopyranose in. 100 ml of tetrahydrofuran for about 1 hour with 30 mg of 10% fs palladium-op-koal under normal pressure. The catalyst is then filtered off, 10 ml of water is added and Dowex AG5GWX8 H + and the mixture is stirred at room temperature until the isopropylidene group has completely separated off. The ion exchanger is filtered off, the solution is neutralized with tris and the tetrahydrofuran and water are evaporated under reduced pressure. The residue is dissolved in methanol and chromatographed on Sephadex LH 20.

R ^ (chloroform / methanol / acetic acid / water 125/75/10/20) = 0.65 Example 15: 2,3-di-0— [(R) -3-hydroxytetradecanoyll — 1-0-phosphono-aD- glucopyranose a) 4,6-O-benzylides-2,3-di-4-f (R) -3-benzyloxytetra-20-decanoyl} -1-0-dibenzylphosphono-a-glueopyranose

To a solution of 1 g of 4,6-0-benzyls-2,3-di-0 - {(R) -3-benzyloxytetradecanoyl} -D-glucopyranose in 10 ml of absolute tetrahydrofuran cooled to -70 ° C is added dropwise 0 .9 ml of 1.6 M butyl lithium in hexane. After 25 minutes at that temperature, a solution of 42 mg of dibenzylphosphorus chloridate in 4 ml of benzene is added dropwise. Stirring is continued for a while. Continue for 10 min at -70 ° C, neutralize the solution with acetic acid and evaporate to dryness. The residue is chromatographed (silica gel, hexane / toluene / acetic ester 4/4/1).

R ^ (toluene acetic acid 6/1) * 0.65 b) 2,3-di-0 - {(R) -3-hydroxytetradecanoyl} -1-Q-phosphono-ct-D-glucopyranose

23Q mg of 4.6-0 benzylides-2,3-di-O- {R) -3-35 8601351 * -14-benzyloxytetradecanoyl} -1-0-1-dibenzylphosphono-α-glycopyranose are dissolved in tetrahydrofuran / water (9/1) and hydrogenates under normal pressure with 80 mg of 10% palladium on carbon for about 5 hours. The catalyst is filtered off, the solution is neutralized with tris and evaporated to dryness. The residue is chromatographed with methanol or Sephadex LH 20 R @ 2 (chloroform / methanol / acetic acid / water 125/75/100/20) = 0.65.

Example 16; 2-deoxy-3,0 - {(S) -3-hydroxytetradecanoyl} -2 - {(S) - 3-hydroxytetradecanoylamido} -1Q-phosphono-aD-JQ glucopyranose a) 4,6-0-benzylides-3 ~ 0 - {(S) -3-benzyloxytetradeca-noyl} -2 - {(S) -3-6enzyl-oxytetradeeanoylamidoi-2-deoxy-1 -Q-dibenzylphosphono-ct-D-glucopyranose

The title compound is obtained in a manner analogous to Example J3a. R toluene acetic acid 4/1) = 0.44 fb) 2-deoxy-3-0 - {(S) -3-hydroxytetradecanoyl} -2 - {(S) - 3-hydroxytetradecanoylamido} -1-0-phosphono-aD- glucopyranose 2Q The title compound is obtained by a procedure analogous to example 13b.

SM.Pt. 150-200 ° C (dec.).

R ^ (methylene chloride / methanol / ammonia 1/1/1; under phase = 0.5 Example 17; 2-deoxy-3-Q - {(R) -3-hydroxytetradecanoyl} -1-0-25 phosphono-2- {(R) -3-tetradecanoyloxytetradecanoyl-amidoj-α-glycopyranose a) 4,6-0-benzylides-3-0 - {(R) -3-benzyloxytetradecanoyl3-2-deoxy-1dd-benzylphosphono-2H [(R) -3-Tetradecanoyloxytetrade-canoylamido} -aD-glycopyranose The title compound is obtained in a manner analogous to Example 13a, SM.Pt. 82-95 ° C,> 2Q / yy +24.1 (c = 1, chloroform). R 2 (tolyenoacetic acid ester 2/1) = 0.7 8601551 r-15-b] 2-deoxy-3-Q- {(R). -3-bydroxytetradecanyl} ~ 1 -0-phosphono-2-ί (Rl-3-tetradecanoylo.xytetradacanoylamido} -g-D-glu-copyranose

The title compound is obtained in a manner analogous to Example 21b.

20 o {a} ^ = +14.3 (c ~ 1, tetrabydrofuran / pyridine) R ^ (chloroform / methanol / acetic acid / water 80/25/5/5/1 = 0.35 Example 18: 2-deoxy ^ 10-phosphono-3-O-tetradecanoyl-2-tetra-decanoyl-amido-qD-glucopyranose 10 al 4,6-Q-benzylides-2-deoxy-1-0-dibenzylphosphono-3-tetradecanoyl-2 -tetradecanoylamido-α-glneopyranose

The title compound is obtained by eluting in an analogous manner to Example 13a using tetradecanoic acid and with toluene / acetic ester 4/1 as the eluent. Melting point 123-129 ° C.

(toluene acetic ester 2/1) = 0.6 b) 2-deoxy-1-O-phospho-ono-3-O-tetradecanoyl-2-tetrade-canoylamido-α-D-glucopyranose

The title compound is obtained in a manner analogous to Example 21b. (chloroform / methanol / acetic acid / water 125/75 ^ 0/201 - 0.65.

Example 19; 2-deoxy-2- {R) -3-bydroxytetradecanoylamido} -1Q- osfono-3-0 - {(R) -3-tetradeeanoylQxytetradecanoyl} - qD-glucopyranose 25 a) 4,6-0-benzylides — 2- {(R) -3-benzyloxytetradecanoyl-am £ do} -2-deoxy-1Q-dibenzylphos £ ono-3-0 ~ {(R) -3-tetradecanoyloxytetradecanoyl} -qD-glucopyranose

The title compound is obtained in an analogous manner to Example 13a using (R) -3-tetra-3Q decanoyloxytetradecanoic acid and with toluene / acetic ester 4/1 as solvent. Melting point 89-90 ° C. {q} ^ = + 30.9 ° (c = 1, chloroform / methanol 1 / l). R ^ (toluene acetic acid ester 4/1) = 0.5.

b) 2-deoxy — 2 - {R} -3-hydroxytetradecanoylamido} -1-Q ~ 860 1 551 • 16 - phosphono-3-0 - {(R) -3-tetradecanoyl} oxytetradecanoyl} -et-D-glucopyranose

The title compound is obtained in a manner analogous to Example 21b. The lyophilisate is dissolved in methanol and chromatographed on Sephadex LH 20 using the same solvent.

R ^ (chloroform / methanol / acetic acid / water 80/25/5/5) = 0.32 Example 20: 2-deoxy-2 - {(R) -3-hydroxytetradecanoylamido} -1-0-phosphono-3-0 -tetradecanoyl-ct-D-glycopyranose ^ a} 4? 6-O-benzyliden-2 - {(R) -3-benzyloxytetradecanoyl-amldo} T-2-deoxy-1-0-dl-benzylphosphono-3-0-tetra- Decanoyl-α-glycopyranose The title compound is obtained analogous to Example 13a, using tetradecanoic acid and 5 toluene / acetic acid ester (3/1) as the solvent mixture. Melting point 125.5-126.5 ° C.

R ^ (toluene acetic ester 3/2) = Q, 6 B) 2-deoxy-2 - (R) -3-hydroxytetradecanoylamido} -1-O-phosphono-3-O-tatradecanoyl-qD-glyeopyranose 20 Dissolve 354 mg 4,6-O-benzyliden-2 - {(R) -3-benzyl-oxytetradecanoylamido] -2-deoxy-1-O-dibenzylphosphonone-3-Q-tetra-decanoyl-α-glycopyranose in 30 ml of tetrahydrofuran / water (9/1) and hydrogenates with 200 mg palladium-on-charcoal under normal pressure for 10 hours. The catalyst is then filtered off and the solution is adjusted to pH 7.5 with tris (hydroxy-methyl) aminomethane. The tetrahydrofuran is evaporated under reduced pressure and the aqueous solution is lyophilized. The lyophilisate is digested twice with ether and dried.

30 R ^ (chloroform / methanol / acetic acid / water 125/75/10/20) = 0.6

Example 21: 2-Acetamido-2-deoxy-3-0 - {(R) -3-hydroxytetradecanoyl} -1 -0 -phosphon-D-glucopyranos et al.) 2-acetamido-4,6, 0-benzylides-3,0- {(R) -3-benzyl-oxytetradecanoyl} -2-deoxy-1 -0-dibenzylphosphonate-35-D-glycopyranose 860 1 55 1 P v - 17 -

A solution of 3.55 g of 2-acetamido-4,6-Q-benzyliden-2-deoxy-JO-dibenzylphosphono-a-D-glucopyranose and 2.1 g of (R) -3-benzyloxytetradecanoic acid in 15 ml of methylene chloride is cooled and react with 1.32 g of dicyclohexylcarbodiimide 5 and p-dime thylaminopyridine. The reaction is stopped after 2 hours at this temperature. The mixture is filtered, evaporated to dryness and chromatographed on silica gel first with methylene chloride / methanol (50/1) and then with toluene / acetic ester (2/1}.

10 R ^ (chloroform / methanol 20/1} = Q, 7 hl 2-acetamido-2-deoxy-3-Q-I (R) -3-hydroxytetra-decanoyl} -1-0-phosphono-α-D-glycopyranose

2.08 g of 2-acetamido-4,6-0-benzylides-3-0- {(R) -3-henzyloxytetradecanoyl} -2-deoxy-1Q-dihenzylphosphono-α-D-glucopyranose are dissolved in tetrahydrofuran / water (9/1) and hydrogenates with 1 g of 10% palladium-on-carbon under normal pressure for 3 hours. The catalyst is filtered off and the filtrate is first evaporated to dryness and then lyophilized.

The lyophilisate is dissolved in methanol, neutralized with 20 tris (hydroxymethyl laminomethane) and chromatographed with methanol on Sephadex LH 20.

{ct} ^ »+ 43.3 ° (c ** l, methanol)

D

R ^ (chloroform / methanol / acetic acid / water 125/75/10/20) = 0.4 Example 22; J-0-phosphono-2,3-di-0 - {(R) -3-tetradecanoyloxy-25 tetradecanoyl} -aD-glucopyranose a) 4,6-Q-henzylides- ~ 1-dibenzylphosphono-2,3 -di-0 - {(R) -3-tetradecanoyloxytetradecanoyl} -ct-D-glucopyranose

The title compound is obtained analogous to Example 15a under chromatographic purification on silica gel 30 with ether / hexane (1/1) as eluent.

R ^ (ether / hexane 1/1} - 0.5 h) 1-Q-phosphono-2,3-di-0 ~ {(R) -3-tetradecanoyloxytetra-decanoyl3-q-D-glucopyranose

470 mg of 4,6,0-benzylidenes-1-O-dibenzylphosphono-8601551-18-2,3-di-O - {(R) -3-tetradecanoyloxytetradecanoyl.} AD-glycopyranose in 47 ml of tetrahydrofuran and hydrogenates with 230 mg palladium-on-carbon under normal pressure for 1 hour. The catalyst is filtered off, the filtrate is evaporated to dryness and the residue is chromatographed on silica gel (chloroform / methanol / water / triethylamine 15/10/2 / 0.2). The relevant fractions are pooled and evaporated to dryness. The title compound is obtained as di-triethylamine salt. R ^ (chloroform / methanol / acetic acid / water 8Q / 25/5/5 /) = 0.5.

Example 23: 2-deoxy-3-Q - {(R ^ -3-hydroxytetradecanoyl} -2- {(S) -3-hydroxytetradecanoylamido} -1 -0-phosphono-ct- a) 4,6-0- benzylides-3,0 - {(R) -3-benzyloxytetradecanoyl} - 2- {(S) -3-benzyl-oxytetradecanoylamido} -2-deoxy-1-Q-dihenzylphosphono-a-glucopyranose

The compound is obtained in a manner analogous to Example 13a. R ^ (toluene acetic ester 4/1) = 0.44 b) 2-deoxy-3-Q— {(R).-3-bydroxytetradecanoyl} -2- {(S) -3-h-hydroxytetradecanoylamido} -Q-phosphono -aD- glucopyranose

The compound is obtained in a manner analogous to Example 13b. R ^ (methylene chloride / methanol / ammonia 1/1/1, lower phase). = Q.5.

To further characterize the invention, a fast atomic boron element (FAB) mass spectroscopy (negative mode) is accomplished (Raetz, J. Bio}, Cbem. 259/4852 {1984}).

"Example No. Mass 1 1325 2 1324 30 3 1534 4 1323 5 1322 6 1325 7 1324 8601551 - 19 - continued table:

Example No. Mass 8 1323 9 1326 5 10 1292 11 1140 12 647

Example No: NMR Spectra 10 y 9.82 (d, 9 Hz, 1H); 6.65 (dd, 8.8 h, 3.5 (C_D_N) Hz, 1H); 6.25 (t, 10 Hz, 1H); 5.83 (t, 10 Hz, 1H); 5.63 (d, 8.8 Hz, IH); 5.35 (dd, 10 µl, 2.5 Hz, IH); 4.0 (t, 10Hz, 1H); 3.83 (m 1H).

5.49 (dd, 3.5 h, 7.5 Hz, 1H); 5.24 (dd, (CD30D 9.5 h, 10.5 Hz, IH}; 4.17 (ddd, 2.5, 3.5 h.

10.5 Hz, IH); 4.0 (m. 3K); 3.90 (dd, 2h.

12 Hz, IH); 3.75 (dd, 5.5 h, 12 Hz, IH); 3.71 (s, Tris); 3.63 (t, 10 Hz, IH).

14, 5.77 (dd, 3.5 h, 7.5 Hz, IH); 4.77 (ddd, (CDCl / 2.5, 3.5 h * 10.5 Hz, IH); 4.38 (dd, 9.5 h.

CD 0D) 10.5 Hz); 3.8 bis 4.1 (m. 4H); 3.73 (dd, 3.5 h, 12 Hz, IH; 3.53 (t, 10 Hz, IH).

5.71 (dd, 3.5 h, 7.5 Hz, IH); 5.48 (t, 10 (CD30D) Hz, 1H); 4.77 (ddd, 2.4 µl, 10 Hz, 1H); 3.9 bis 4.05 (m. 3H); 3.86 (dd, 2 h, 12 Hz, IH); 3.7 (dd, 5.5 h, 12 Hz, IH); 3.67 (s, Tris); 3.58 (t, 10 Hz, IH).

16 5.58 (dd, 3h, 7Hz, IH); 5.22 (t, 10 Hz, 3Q (CDC1 / IH; 4.19 (dt, 3 h, 10 Hz, IH); 3.8 bis 4.1 CD30D) (m, 4H); 3.7 (s , Tris tt.dd, IH); 3.46 (£, 10 Hz, IH).

17 5.46 (dd, 3h, 7Hz, IH); 5.22 (t, 10 Hz, CDCl / IH); 5.17 (m. 1H); 4.19 (dt, 3 h, 10 Hz, 35 CD30D (1H)); 3.9 bis 4.1 (m. 3H); 3.7 (s, Tris); 8601551-20 - 3.65 (dd, IE); 3.51 (t, 10 Hz, 1H).

18 / step a) 7.38 (m, 15H); 5.70 (dd, 3.5 u. 6 Hz, 1H); (protected) 5.63 (d, 9.5 Hz, 1H); 5.50 (s. 1H); 5.26 (CDCl 3) (t, 10 Hz, 1H); 5.08 (m, 4H); 4.38 (m, 5 IH); 4.08 (dd, 5 h, 10 Hz, IH); 3.95 (dt 5 h, 10 Hz, 1H); 3.73 (t, 10 Hz, IH; 3.69 (t, 10 Hz, IH); 2.29 (m, 2H); 1.89 (m, 2H). 19 / step a) 7.2 bis 7.45 (m. 20H); 6.34 (d, 9Hz, IH); (protected) 5.77 (dd, 3.5 u. 6 Hz, IH); 5.49 (s, 1H); 10 (CDCl 3) 5.29 (t, 10 Hz, 1H); 5.14 (quintett, 6 Hz, 1 H); 5.0 (m. 4H); 4.51 h. 4.41 (AB, 12 Hz, 2H); 4.41 (m, IH); 4.08 (dd, 5 h, 10 Hz, 1 H); 3.95 (dt, 5 h, 10 Hz, IH); 3.7 (m. 3H); 2.58 h. 2.47 (ABX, 5.5 7.5 vu 15.5 15 Hz, 2H).

2Q / step a) 7.2 bis 7.45 (m, 20H); 6.29 (d, 9Hz, IH); (protected) 5.74 (dd, 3.5 u. 6 Hz, IH); 5.49 (s, 1H); (CDCl3) 5.30 (t, 10 Hz, 1H); 5.0 (m. 4H); 4.52 h.

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

21 / step a) 5.47 (dd, 3.5 µ, 7.5 Hz, IH); 5.23 (dd, 9.5 (protected) u. 10.5 Hz, 1H); 4.18 (ddd, 2.5, 3.5 h.

(CD30D) 10.5 Hz, IH); 3.90 (m, 3H); 3.85 (dd, 2 h.

12 Hz, IH; 3.72 (dd, 5.5 u. 12 Hz, IH); 3.68 (s, Tris); 3.61 (t, 10 Hz, 1H); 1.94 (s, 3H).

22 / step already 7.3 bis 7.45 (m, 15E); 5.92 (dd, 3.5 h.

(protected) 6.7 Hz, IH); 5.60 (t, 9.5 h, 10 Hz, 1H); 30. (CDCl 3) 5.48 (s, 1H); 5.02 to 5.22 (m, 6H); 4.95 (ddd, 2.5, 3.5 h, 9.5 Hz, IH); 4-10 (dd, 4.5 h, 10 Hz, IH); 3.96 (ddd, 5.9 h, 10 Hz, 1H); 3.67 (t, 9.5 Hz, 2H); 2.60 (m, 2H); 2.38 (d, 6.5 Hz, 2H); 2.24 (t, 7.5 Hz, 2H); 8601531-21-2.12 (t, 7.5 Hz, 2H).

23 / step a) 7.2 bis 7.42 (m, 25H); 6.49 (d, 9Hz, (protected) 1H); 5.77 (dd, 3.5 u. 6 Hz, 1H); 5.45 (CDCl3) (s, 1H); 5.35 (t, 10 Hz, 1H; 5.0 (m, 4H); 5 4.54 h. 4.40 (AB, 11 Hz, 2H); 4.47 h. 4.36 (AB, 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 h 15.5 Hz, 1H); 2.34 (dd, 6 h.

15.5 Hz, 1H; 2.2 (m. 2H).

The compounds used as starting materials can be obtained as follows: A) 4,6-O-benzyliden-2 - {(R) -3-benzyloxytetradecanoylamido} -2-deoxy-10-dibenzylphosphono-qD-glucopyranose (for examples 13 , 19 and 20) 15 a) 2 - {(R) -3-benzyloxytetradecanoylamido} -2-deoxy-D-glycopyranose

A mixture of 5.4 g of D-glycosamine hydrochloride, 10.8 g of N - {(R) -3-benzyloxytetradecanoyloxy succinimide and 5 ml of diisopropylethylamine in 25 ml of absolute 2 D-dimethylformamide for 24 hours at room temperature is stirred.

The solvent is mainly distilled off and the residue is dissolved in a mixture of 150 ml of chloroform, 300 ml of methanol and 120 ml of water. After adding a further portion of 15 ml of chloroform and 150 ml of water, the lower phase is separated and washed twice with an upper phase consisting of 100 ml of chloroform, 100 ml of methanol and 90 ml of water. The solution is evaporated to dryness and dried under high vacuum. The product obtained is used in the next step without further purification.

3Q A small part of crude product is purified by chromatography (toluene / ethanol 9/1) for analysis. Melting point iso ^ c.

{alp = +53 (c = 1, dimethylformamide) S. £ (chloroform / methanol 9/1) = 0.3 35 k) 4,6-Benzylides-2 - {(R) -3-benzyloxytetradecanoyl 8601331 * * - 22 - amido} -2-deoxy-D-glucopyranose

A solution is kept 5.83 g of 2 - {(R) -3-benzyloxy-tetradecanoylamido} -2-deoxy-D-glycopyranose, 3 g of benzaldehydedimethylacetal and 500 mg of p-toluenesulfonic acid monohydrate 5 in 200 ml of absolute dimethylformamide for about 3 hours at 55-60 ° G and 30-40 mbar in a Rotavapor. No starting material remains after that. Most of the dimethylformamide is distilled off, 50mL of methylene chloride added and the solution extracted twice with 200 ml of dilute sodium dicarbonate solution and 200 ml of water.

The solution is dried with sodium sulfate, evaporated to dryness and the residue is chromatographed (toluene / acetic ester 6/4).

Sm. Pt 162-J65 ° C

{a} ^ - + 5.5 ° (c ** 1, chloroform) 15 R ^ (toluene / acetic ester 1/1) = 0.2 c) 4,6-0-benzylides-2 - {(R) - 3-henzyloxytetradecanoyl-amido} -2-deoxy-1-0-dibenzylphosphono-a-D-glucopyranose

To a solution of 4.86 g of 4,6-0-benzylides-2 - {(R) -3-benzyloxytetradecanoyl-amido} -2-deoxy-D-glycopyranose 20 in 40 ml tetrahydrofuran cooled to -70 ° C is added dropwise add 8 ml of 1.6 M butyl lithium in hexane. After 5 minutes at that temperature, a solution of 3.8 g of dihenzylphosphorehloridate in 10 ml of benzene is added dropwise.

After further stirring for 10 min at -7 ° C, 0.3 ml of acetic acid are added and the solution is concentrated to 1/4 of the initial volume. The solution is diluted with 200 ml of methylene chloride, extracted with 70 ml of water, 50 ml of dilute sodium hicaronate solution and 50 ml of sodium chloride solution, dried over sodium sulfate and evaporated to dryness. Chromato-3Q graphs the residue (toluene / acetic acid ester 7/3). Melting point 10254Q5 ° C.

= + 31.7 ° (c 1, chloroform) R ^ (toluene acetic ester 1/1) = 0.32 8601551 - 23 - B) 4,6-Q-benzylideii-2,3-di-0- {(R) -3-benzyloxytetra-decanoyl} -D-glucopyranose (for example 15) a) trichlorethyl 2,3,4,6-tetra-O-acetyl-gD-glu-copyranoside 5 To an ice-cooled solution of 2.34 g of penta-O-acetyl D-glycopyranose in 9 ml trichloroethanol, 0.9 ml horium trifluoride etharate is added and the solution is kept at that temperature for 4 hours. The solution is then 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 bicarbonate solution and 50 ml of water, dried over sodium sulfate and evaporated to dryness. After chromatographic purification (silica gel, toluene / acetic acid ester 4/1), the product crystallizes from acetic acid ester / petroleum ether. Melting point 138-140 ° C.

15 R. £ (toluene / acetic acid ester 1/1) = 0.67 b) trichlorethyl 4,6-Q-henzylides-g-D-glucopyrano side

7.13 g of trichlorethyl 2,3,4,6-tetra-D-acetyl-gD-glucopyranoside are dissolved in a mixture of methanol 20 and methylene chloride, the solution is cooled with ice and reacted with a solution of sodium methanolate (60 mg sodium in 27 ml of methanol). After 2 hours at 0 ° C, the solution is neutralized with Dowex AG 50 WXS H +, the ion exchanger is filtered off and the solvent is removed under reduced pressure. The residue is dissolved in 60 ml of benzaldehyde, 4.1 g of zinc chloride is added and the mixture is stirred at room temperature for 10 hours. The mixture is then poured onto ice water, extracted with ether, the ether solution is dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (silica gel, toluene / acetic ester 4/1). (chloroform / methanol 9/1) = 0.60 c) trichlorethyl 4,6-0-benzylides-2,3-di-Q - {(R) -3-benzyloxytetradecanoyl} -g-D-glycopyranoside

To a solution of 800 mg trichloroethyl 4,6-Q-8601551 * λ - 24 - benzylidene-G-glucopyranoside, 1.4 g (R) -3-benzyloxytetradecanoic acid and 20 mg dimethylaminopyridine in 10 ml methylene chloride, cooled to 0 ° C, 930 mg of dicyclohexyl carbodiimide is added and the reaction mixture is kept at 4 ° C overnight. The reaction mixture is then filtered off, the filtrate evaporated to dryness, the residue dissolved in hexane / toluene / acetic ester (12/5/1) and chromatographed on the same solvent mixture.

R ^ (hexane / acetic ester 5/1) = Q, 52 10 d) 4,6-O-benzylides-2,3-di-O - {(R) -3-benzyloxytetradeanoyl] -D-glycopyranose

480 mg of trichlorethyl 4,6-0-benzylides-2,3-di-O- {(R) -3-henzyloxytetradecanoyl} -g'-D-glycopyranoside are dissolved in 50 ml of a mixture of dioxane and acetic acid (1 / 1) and react in portions at room temperature with zinc powder until all starting material has been consumed. The reaction mixture is filtered, evaporated to dryness and the residue is chromatographed (silica gel, hexane / toluene / acetic ester 2/5/1).

R ^ (toluene / acetic ester 9/1) = Q, 32 2Q C) 2-0 - {(R) -3-henzyloxytetracecanoyl} -3 - {(R) -3-benzyloxytetradecanoylamido} -3-deoxy-4 6-0-isopropylides-D-glycopyranose (for example 14) a) 3-azido-3-deoxy-4,6-O-isopropylides-D-glycopyranose 25 1.02 g of 3-azido 3- deoxy D-glucopyranose in 10 ml of absolute dimethylformamide, add 940 µl of 2-methoxy-propylene and a catalytic amount of p-toluenesulfonic acid monohydrate and keep the solution at room temperature for about 2 hours. The p-toluene-sulfonic acid is then neutralized with sodium bicarbonate, the solution is evaporated to dryness and the residue is chromatographed (silica gel, chloroform / methanol 9/1).

R ^ (chloroform / methanol 9/1) = 0.64.

860 1 551 - 25 - b) tert-butyldimethylsilyl-3-azdQ-3-deo: gy-4,6-Q-isopropylides-g-D-glycopyranoside

To a solution of 56Q mg of 3-az-do-3-deoxy-4,6-Q-isopropylides-D-glycopyranose and 33Q mg of imidazole in 5 ml of dry methylene chloride, 345 mg of t-butyldimethylsilylclororide are added and stirred the mixture for 2 hours at room temperature. The excess imidazole chloride is filtered off and the filtrate is shaken twice with 100 ml of water, dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (silica gel, toluene / acetic ester 12/1).

R ^ (tpluenoacetic acid ester 1/3) = Q, 6.

c) tert-butyldimethylsilyl-2-0- {(R) -3-beazyloxyte-tradecanoyl} -3- {(R) -3-benzyloxytetradecanoylamido} -3-deoxy-4,6-Q-isopropylidene-G-glucopyranoside 15 A solution of 3.1 g of tert-hutyl-dimethylsily1-3-az ido-3-deoxy-4,6-Q-isopropylides-gD-glycopyranoside in JQO ml of methanol is hydrogenated for 2 hours with 30 mg of 10% palladium on charcoal. The catalyst is filtered off and the filtrate is evaporated to dryness. The residue is dissolved with 5.35 g of (R} -3-20 benzyloxymyristic acid and 10.0 ml of dimethylaminopyridine in 50 ml of dry methylene chloride, the solution is cooled with ice and 4.1 g of dicyclohexylcarhodiimide are added. After about 3 hours at room temperature, filter the solution is removed, the solvent is evaporated and the residue is chromatographed (silica gel, toluene / acetic ester 9/1).

R ^ (toluea & his acid ester 6/1) = Q, 42 d) 2-0 - {(Rl ~ 3-benzyloxytetradecanoyl} -3 - {(R) -3-henzyloxyt etradec anoylamido} -3-deoxy-4,6 ~ Q -isopropylidenes-D-glucopyTanose 30 To a solution of 5 g of tert-butyldimethylsilyl-2-Q— {(R) -3-benzyloxytetradecanoyl} -3— {(R) -3-benzyloxytetradeanoylamido} -3-deoxy-4 .6-O-isopropylidene-G-glucopyranoside in 10 ml of absolute tetrahydrofuran, cooled to -6 ° C, 5.2 ml of a solution of 3501551-26-tetrabutyl ammonium fluoride in tetrahydrofuran are added dropwise and allowed to warm to -40 ° C and hold at this temperature until all starting material has been consumed (about 30 min.) Then 5 ml of methanol are added, the temperature is brought to room temperature and the solvent is evaporated, the residue is extracted with a mixture of methylene chloride and water. , the organic phase dries over sodium sulfate and the solvent evaporates .. After chromatographic purification (silica gel, toluene / acetic ester 3/1), the title compound is obtained. g as a mixture of anomers.

R. £ (toluene / acetic ester 1/1) = 0.48 and 0.52.

D) 4,6-Q-henzyliden-2 - {(S) -3-benzyloxytetradecanoyl3-2-deoxy-i-b-dihenzylphosphono-q-D-glucopyranose (for example 16) D-glucopyranose

The title compound is obtained in a manner analogous to Example Aa). R ^ (chloroform / methanol 7/1) = 0.37.

b) 4,6-0-benzylides-2 - {(S) -3-benzyloxytetradecanoyl-20 amido} -2-deoxy-D-glucopyranose

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

r, 20 o {a> D = -3.5 (c = 1, chloroform) (toluene / acetic acid 1/1) = 0.37.

C) 4,6-0-henzylides-2 - {(S) -3-benzyloxytetradecanoyl-amido} -2-deoxy-1-O-dlhenzylphosphono-cHD-glucopyranose

The title compound is obtained in a manner analogous to Example Ac). oc \ 30 {«} = + 39.2 ° (c = 1, chloroform) R ^ (toluene / acetic ester 1/1) = 0.46 E) 4.6-0-benzylides-2-deoxy-1-0 -dibenzylphosphono-2- i (R) -3-tetradecanoyloxytetradecanoylamido} -ot-D-glucopyranose (for example 17) 8§1551 - 27 - a.) 2-deoxy-2- {(R) -3-tetradecanoyloxytetradecanoyl- amido} -D — glucose

A mixture of 650 mg of D-glycosamine hydrochloride, 1.9 g of N - {(R) -3-tetradecanoyloxytetradecanoyl-5-axy] succinimide and 0.5 ml of diisopropylethylamine in 15 ml of dimethylformamide is stirred at room temperature for 20 hours.

The solvent is then distilled off and the residue is chromatographed on silica gel using acetic acid ester / methanol (8/1) as eluent.

10 {a} ^ = + 25 ° (c = 1, methanol) R ^ (acetic ester methanol 6/1) = 0.5 b) 4,6-0-benzylides-2-deoxy-2 - {(R) -3 -tetradecanoyl-oxytetradecanoylamido} -D-glyeopyranose

A solution of 5.83 g of 2-deoxy-2 - {(R) -3-tetra-15-decanoyloxytetradecanoylamido} -D-glucose, 3 g of benzaldehyde dimethyl acetal and 500 mg of p-toluenesulfonic acid monohydrate in 200 ml of absolute dimethylformamide is used in the Rotavapor held at 55-60 ° C and 30-40 mbar for 3 hours. The starting material is then consumed. Most of the dimethylformamide is then distilled off, 5 ml of methylene chloride are added and the solution is extracted twice with 200 ml of dilute sodium bicarbonate solution and 200 ml of water. After drying over sodium sulfate and evaporating to dryness, the residue is chromatographed (toluene / acetic ester 6/4). Melting point 162-165 ° C.

{A -1.5 ° (c = 1, chloroform / methanol 1/1) R1 (toluene / acetic ester 1/1) = 0.32.

c) 4,6-0-henzylides-21-deoxy-1-0-dibenzylphosphono-2-{(R) -3-tetra-decanoyloxytetradecanoylamido} -a-D-glycopyranose

To a solution of 950 mg of 4,6-O-benzylides-2-30 deoxy-2- {(R) -3-tradecanoyloxytetradecanoylamido} -D-glucopyrasone in 40 ml dry tetrahydrofuran, cooled to -40 ° G, 1.25 ml of butyl lithium 1.6 M in hexane are added dropwise.

After 5, a solution of dihenzylphosphonochloridate in henzene is added dropwise and stirring is continued for 5 min.

35 at this temperature. The solution is then neutralized 860 1 55! - 28 - with acetic acid, the mixture evaporates to dryness and extracts the residue with methylene chloride / water. The organic phase is dried over sodium sulfate, evaporated to dryness and the residue chromatographed on silica gel (toluene / acetic ester 5 3 / ¾ {αλρ = + 19.3 ° (c = 1, chloroform) K. £ (toluene / acetic ester 1 / 1) = 0.6 F) 4,6-Q-enzymes-2-deoxy-1-Q-dibenzylphosphono-2-tetradecanyl-amido-qD-glycopyranose (for * ® example 18) a) 2-deoxy-2- tetradecanoylamido-D-glucose A mixture of 3 g of D-glucosamine hydrochloride, 4.56 g of N-tetradecanoyloxysuccinimide and 2.8 ml of diisopropylethylamine in 40 ml of dry dimethylformamide-15 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) 4,6-0-benzylides-2-deoxy-2-tetradecanylamido-

4 g of 2-deoxy 2-tetradecanoylamido D-glucose are dissolved at 55 ° C in 320 ml of dried dimethylformamide, 2.6 ml of dimethoxytoluene and 40 mg of p-toluene acetic acid monohydrate are added and the mixture is kept in the Rotavapor for 4 hours at 55-6Q ° C and 3Q-40 mbar. The solution is then distilled off, the residue is washed with dilute sodium bicarbonate solution, water and ethanol, dried in vacuo and used directly for the next step.

c) 5,6-Q-henzyliden-3-0 - (tert-hutyldimethylsilyl) - 2-deoxy-2-tetradecanoylamido-D-glycopyranose

2.2 g of 4,6-Q-benzyliden-2-deoxy-2-tetradecanoylamido-D-glucose are dissolved in 250 ml of dry dimethylformamide at 60 ° C, 960 mg of imidazole-2 g, 13 g of t-butyldimethylchlrosilane are added and the reaction is continued. 24 hours at that temperature. After addition 8601551 - 29 - of 480 mg imidazole and ï g t-butyldimethylchlorosilane

O

and the starting material was consumed for an additional 24 hours at 60 ° C.

The solvent is distilled off, the residue is extracted with methylene chloride / water, the organic phase is washed once with water with 5 times, dried over sodium sulfate and distilled off.

The residue is chromatographed on silica gel with a gradient of toluene / acetic ester from 20/1 to 2/1.

Two main fractions are obtained: the title compound (650 mg) and the corresponding 1,3-his-silyl compound (1.82 g). Hen 10: Dissolves the bis-silyl compound in 25 ml of dry tetrahydrofuran, cools the solution to -40 ° C and adds 3.5 ml of a 1 N solution of tetrabutylammonium fluoride in tetrahydrofuran dropwise. After about 1 hour, the anomeric silyl protecting group is cleaved at -40 ° C, 3 ml of methanol are added and the temperature is brought to room temperature and the solvent is then removed in vacuo. The residue is extracted with methylene chloride / water, the organic phase is extracted once with water, then dried and distilled off. A further 1.52 g of the title compound are obtained in the form of an anomeric mixture * R- (toluene / acetic ester 1/1) = 0.42 rd) 4,6-0-henzylides-3-Q- (tert-5-butyldimethylsilyi] -2-deoxy-1Q-debenzylphosphono-2-tetradecanoyl-25 amido-ct-D-glycopyranose

To a solution of 2.46 g of 4,6-Q-benzyliden-3-Q- (tert-hutyldimethylsilyl) 2-deoxy-2-tetradecanoylamido-D-glyco-pyranose in 1QQ ml of dry tetrahydrofuran, cooled to -60 ° C 3.12 ml of 1.6 H Butyllithium in 3Q hexane are added dropwise. After 5 minutes at this temperature, a solution of diBenzylphosphorus chloridate in henzene is added dropwise. Stirring is continued for 5 minutes at -60 ° C, the solution is then neutralized with acetic acid and evaporated to dryness. The residue is taken up in methylene chloride 35 and extracted with water. After working up and chromatography on 8601551 * + - 30 - silica gel using toluene / acetic acid ester (3/1) as eluent, the title compound is obtained.

B. £ (toluene / acetic acid ester 7/4) = 0.75.

e) 4,6-0-benzylides-2-deoxy-1-0-dibenzylphosphono-2 -5 tetradecanoyl-amido-ct-D-glycopyranose__

To a solution of 2.05 g of 4,6-0-benzyliden-3 "Q- (tert-butyldimethylsilyi) -2-deoxy-1-dibenzylphosphorio-2-tetra-decanoyl amido-a-glycopyranosis in 40 ml dry tetrahydrofuran is added dropwise at -10 ° C 2.5 ml of a 10 IN solution of tetrabutyl ammonium fluoride in tetrahydrofuran and stirring is continued for 2 hours at 0 ° C. Then 3 ml of methanol are added, the solution is evaporated to dryness and the residue is extracted with methylene chloride / water. After working up as usual and chromatography on silica gel using toluene / acetic acid ester (3/2) as eluent, the title compound is obtained.

B. £ (toluene / acetic ester 1/1) = 0.35 G) 2-acetamido-47-0-benzy 1 -iden-2-deoxy-1 -0-dibenzylphosphono-α-glycopyranose (for example 21) 20 a) 2-Acetamido-4,6-0-benzylides-3-0- (tert-butyldimethylsilyl) -2-deoxy-D-glucpyranose A solution of 5.17 g of 2-acetamido-4,6- is kept Qb.enzylides-2-deoxy-D-glucopyranose, 3.4 g of imidazole and 6.3 g of t-butyldimethylchlorosilane in 300 ml of dimethylformamide at 60-700C. After 6 hours, 500 mg of imidazole and 1 g of t-butyl dimethyl chlorosilane are added and the mixture is kept at that temperature until the reaction is complete. The solvent is evaporated, the residue is extracted with methylene chloride / water and purified by chromatography with toluene / acetic ester (5/1).

30. To cleave off the anomeric t-butyldimethylsilyl group, the product is dissolved in 600 ml of dry tetrahydrofuran, the solution is cooled to -40 ° C and 14 ml of a 1 M tetrabutyl ammonium fluoride solution are added. After 10 minutes, the reaction is stopped by adding 17 ml of methanol.

The mixture is evaporated to dryness, the residue is taken up in methylene-8601551-3-chloride and extracted with water. The organic phase is dried and distilled off.

K. (chloroform methanol) 9/1) = 0.5 b) 2-acetamido-4,6-0-benzylidenes-3-0 (tert-butyldi-5 methylsily1) -2-deoxy-1-0-dihenzylphosphono -aD-glycopyranose

The title compound is obtained in an analogous manner to Example Ec) and after chromatography with toluene / acetic ester (1/1) as eluent.

R. £ (toluene / acetic acid ester 2/1) = 0.58 10 c) 2-acetamido-4,6-0-benzyliden-2-deoxy-1-Q-dibenzylphosphono-ct-D-glycopyranose To a solution 4.4 g of 2-acetamido-4,6-0-benzylidenes-3-0- (tert-butyldimethylsilyl) -2-deoxy-1-0-dibenzylphosphono-a-glucopyranose 200 ml dry tetrahydrofuran cooled with ice, 6.5 ml of a 1 N solution of tetrabutyl ammonium fluoride in tetrahydrofuran is added dropwise and the mixture is kept at 0 ° C for 2 hours.

Then 7 ml of methanol are added and the solution is evaporated to dryness. A solution of the residue in methylene chloride 20 is washed with water, dried and evaporated to dryness.

B. (chloroform / methano 9/1) - 0.65 H) 4.6-0-henzyliden-2,3-di-Q - {(R) -3-tetradecanoyl-oxy-tetradecanoyl} -D-glycopyranose (for example 22) 25 a) trichlorethyl-4,6-0-benzylides-2,3-di-0 - {(R) -3-side

The title compound is obtained analogously to Example Be) using (R) -3-tetradeeanoyloxytetra-30-decanoic acid as acylating agent and toluene / acetic ester (98/2) as an eluent.

(toluene / acetic ester 9/1) = 0.75 b) 4,6-Q-henzyliden-2,3-di-Ö- {(E.) -3-tetradecanoyl-oxytetradecanoyl} -D-glucopyranose

O O

8601551 »* - 32 - D-glueopyranose

The title compound is obtained analogously to Example Bd) after chromatographic purification on silica gel using toluene / acetic ester (15/1) as eluent.

5 (toluene / acetic acid 9/1) = 0.25 I) synthesis of UDP-2,3-diacyl hexoses

1.4 g of uridine-5T-phosphororfolidate-N, N'-di-cyclohexylcarboxamidine salt are dissolved in 25 ml of anhydrous pyridine and dried twice by evaporation and redissolving in 25 ml of dry pyridine. To this solution, 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 manner, is added and left to stand at 37 ° C for 24 hours. The mixture is cooled with ice. Then 50 ml of chloroform and 12 ml of 90% Ts formic acid are added. This solution is chromatographed on silica gel and eluted unreacted starting material with chloroform / pyridine / 90% formic acid (30/30/7). The column is liberated from pyridines with chloroform / methanol 9/1 and the UDP derivative is then eluted with cliloroform / methanol / water (66/33/4). 2Q chloroform and methanol of the pickle fraction are evaporated in vacuo and the remaining aqueous suspension is filtered. The precipitate is dissolved in 100 ml of tetrahydrofuran / water (2/1) and the solution is stirred for 2 hours with Dowex AG50WX8 (in tris-hydroxy-methylaminomethane form). The ion exchanger 25 is filtered off, most of the tetrahydrofuran is distilled off and the aqueous solution is lyophilized. The lyophilisate is used in the next step without further purification.

The compound of the formula II can be obtained in a manner analogous to that described under I) above, starting from the corresponding compound of the formula III.

The compounds of formula Ia and III had j. Pharmacological activity. They are designated for use as pharmaceuticals. They have a beneficial effect on non-specific antimicrobial resistance. This activity can be demonstrated, for example, by the following test methods: 8601551 - 33 - I. Determination of the endotoxic activity in the limulus-amocyocytes lysate test

Endotoxin catalyzes the activity of a proenzyme in lumulus amoebocytes lysate. The cleavage of 5 p-nitroaniline from a colorless substrate is measured.

The extent of this cleavage is monitored photometrically, with the correlation between absorption and endotoxin concentration (or endotoxin activity for analogs, respectively) in the range of 0.10 - 0.1 ng / ml endotoxin being linear (in comparison with the absorption values of a standard endotoxin). A dilution series of 1:10 is prepared from each sample (dissolved in pyrogen-free sterile double-bubble distilled water) and then a blank sample, 100 µl test sample or reference standard or blank sample is reacted with 100 µl limulus amoehocytes lysate. After 10 min.

incubation at 37 ° C, the reaction mixture is reacted with 20 µl of substrate. The reaction is stopped with 200 µl of 50% Ts acetic acid after an additional 3 min incubation. After stirring the sample, the absorbance is measured in a spectrophotometer at 405 nm, subtracting the background value. The endotoxin levels (the endotoxin activity) of the sample in endotoxin units (E.D.) are calculated by linear setback with the values of the standard toxin.

2. Induction of ____endotoxin shock in the mouse. The test consists of inducing an endotoxin shock or an analogous clinical condition with lethal result with LFS analogs in galactosamine (GaIN) sensitized mice. Male C57 B1 mice (6 animals per group) are given intraperitoneally simultaneously 8 mg GalN 3Q dissolved in 0.5 ml PBS and 0.1 µg LPS from Salmonella abortion equi (sigma) dissolved in 0.2 ml physiological saline. This treatment kills all animals within 6-12 hours (C. Galanos et al. Froc. Natl. Acad. Sci, USA 76 (1979) 5939-5943. Instead of LPS in the standard treatment, the 8601551 - 34 standard substances are administered once in different dosages either simultaneously with GalN or parenterally or orally before or after Treatment with GalN The results are scored by comparing the smallest 5 doses, which are lethal to all animals in a group or by Calculation of an LD ^^ using the

Spearman-Karher method.

3. HicroBial septicemia in the neutropenic mouse

This model enables the determination of a substance-related increase in the imune response in microbially infected neutropenic mice. For the induction of neutropenic groups of 20 female B, D „F mice

o L J

20Q mg / kg body weight given once to cyclophosphamide dissolved in 0.2 ml of distilled water on day 0. On the third 15 day, the test is administered stiffly parenterally (primary intra ™ perito & eaaL) or orally as far as possible dissolved in physiological saline or dissolved in any other way (0.3 ml) .. Infection occurs on day 4 by intravenous administration of the relevant inoculate in a volume of 0.2 ml 20 (number of germs For example Psuedomonas aeruginosa, per mouse Δ 12: 1 x 10 ^ jE. coli £ 120: 2 x 10 ^; Staph, aureus 113: 1x10 ^. 4

Canida albicans Δ124: 1 χ 10). The test animals are observed up to the tenth day after infection and the death toll is recorded daily. The following parameters are calculated using the infection control standard respectively: a). average survival time b) survival rate

The compounds of formulas Ia and III show remarkable improvements in survival time and number Above untreated infection blanks in experimental infections with Gram negative agents (eg Pseudomonas and E. Coli) as well as in infections with Gram positive agents ^ Staph, aureus) or yeasts (eg Candida albicans) 880 1 551 - 35 - after parenteral administration.

4. Activation of the neutrophil oxidation metabolism of human blood

Neutrophils (at least 95% pure) are incubated with the test substance in three different concentrations for 1 - 2 hours at 37 ° C. The release of superoxide anions by the cells as an indication of activation is then measured in the form of superoxide dismutase initiation of the reduction of cytochrome C. 1x10 neutrophils, either pre-treated with test substance or not, are added to the cytochrome C (80 pmol) containing formyl-methionyl-leucylphenylalanine (10 M). The biancans further contain 50 µg of superoxide dismutase. After 15 min. Incubation at 37 ° C, the reaction is stopped by placing the test tubes in an ice bath. The test tubes are then centrifuged at 400 g for 5 min to separate the cells.

The reduction of cytochrome C, which is proportional to the amount of superoxide anion released, is measured photometrically at 550 nm. The inhistoric effect of the test substance PMN 20 activation by LPS is measured as described above, so that after preincubation of the leucocytes the cells are further incubated with a stimulating concentration of LPS.

5. Carbon purification test

This test is based on the principle that 25 particles in a size of 200 - 250 lb (e.g. carbon particles) can be eliminated from the organism only by phagocytosis by macrophages. When administered intravenously, carbon particles are eliminated from the bloodstream by macrophage phagocytosis in the liver (Kupffer stellar cells) and spleen. The determination of SES activation of a substance is accomplished by single or repeated administration in aqueous solution or as a suspension. The amount of a substance provided for the treatment is administered intraperitoneally or subcutaneously in 4 daily doses or all at once 24 hours before the start of the experiment. A suspension containing 10% carbon black is diluted with a 1% gelatinous sodium chloride solution to a content of 16 mg carbon / ml. Each 5 mouse receives 0.2 ml / 20 g body weight. intravenously and 25 µl of blood is collected by puncture of the ocular blood vessels 3,6,9,12 and 15 min after intravenous administration. The blood is hemolyzed in 2 ml of distilled water.

The carbon concentration is determined photometrically. The 10 animals are then killed and the weights of liver and spleen measured.

6. Herpes infection (mouse)

Intracutaneous herpes infection enables the determination of the rate of increase in immune response in mice infected with herpes viruses. The course of the disease is prolonged and allows for the observation of the successive appearance of several parameters. Herpetic lesions appear at the site of infection, which form ulcers, the nearest leg 20 is paralyzed, and the paralysis progressively increases until death follows. Immunocompetent "naked" (hairlo) mice are infected intracutaneously on day 0 by intracutaneous administration of the test vaccine to be tested in a volume g of 0.025 ml (eg 1x10 plaque forming units of Herpes simplex type 1 / mouse). The test substance is administered intrapod> tonally in the solution, for example in physiological saline (0.1 mm).

Systemic herpes infection also allows the determination of the rate of increase in immune response 3Q in mice infected with herpes viruses. Immunocompetent NMRI mice are infected on Day 0 by interperitoneal administration of the test vaccine to be tested in a volume of 0.1 mm (e.g., 1.3 x 10 µ slice forming units of Herpes simplex type 1 / mouse). The test substance is administered subcutaneously in solution, for example, in physiological saline solution.

The test animals are observed up to day 20 after infection and the appearance of lesions, paralysis and death are recorded. The following parameters are measured and compared to infection blanks or a standard: a) number of mice with lesions (cumulative) b) number of mice with paralysis (cumulative) c) average survival time d) survival number 10 The compounds of formula Ia and III show remarkable improvements in disease course, survival and survival over untreated blanks, following single intraperitoneal and subcutaneous administration between days 15 or -1 and +6 in the experimental transmission of HSV-1 infection.

7. CSF induction (colony stimulation factor)

The CSFs are mediators produced in the organism after infections or poisoning effects. Their biological effects are complex, they are detected via stimulation of the proliferation of the hemo-poetic system, in particular of bone marrow cells. The test substances are administered parenterally or orally to B-D-F. Mice once or repeatedly up to a dose of 50 mg / kg.

O Z 1

Serum is collected from the test animals at different subsequent time intervals. Serum CSF activity titer is measured in a cell culture assay by the measures of proliferation of murine bone marrow cells {D. Metcalf, The Hemopoietic Colony Stimulating Factors, 1984, Elsevier; T. Mosmann, J. Immunol ·. Methods, 65, 55-63 (1983); 30 L.M. Green et al., J. Immunol. Methods, 70 (1984) 257-268}.

The compounds of formulas 1a and III induce CSFs of various sizes in mice, also observing time-kinetic differences in CSF activities, which could be beneficial in therapeutic use.

1601551 - 38 - 8. Induction of Interleukin-1 (IL-1)

The determination of whether a substance stimulates cells to IL-1 production is primarily performed in a cell culture assay. First, adherent macrophages from mice are recovered, both resident macrophages and macrophages released with thiogly colate. These are incubated in RPMI medium for 48 hours with different concentrations of test substance and the supernatant cell fluids collected. These are assayed for IL-1 activity in thymocyte cultures from 10 CH / H mice. The thymocytes of these supernatants containing IL-1 supplied by macrophages are induced to proliferate over a 72 hour incubation. Proliferation is measured by incorporation of 3 H-thymidine, in a flicker counter (J, Gery et al., J. Exp. Med. 136 (1972) 128-142; 15 Oppenheim et al., Cellular Immunol. 50 (1980) 71-81).

The compounds of formulas Ia and III in various sizes (in concentrations of 0.1 - 50 µg / ml) have the ability to induce IL-1 production in macrophages.

9. Induction of LPS (endotoxin) tolerance (mortality - 2Q tolerance)

A so-called tolerance can be induced in mice by parenteral administration of LPS one to three times a day. This tolerance protects the animals from the lite effects of the LPS * after administration of galactosamine (compare induction of endotoxin shock jin mice above).

The compounds of formula Ia and III already release this tolerance after a single intraperitoneal administration of 0.25 mg per mouse. Pretreated (tolerant) mice) are exposed to an LPS challenge at a dose of 0.01 µg LPS + 8 mg galactosamine / mouse intraferitoneally at different times (J day to 3 weeks) following the last treatment. A large number of animals survive this LPS challenge, especially when the administration is repeated three times, compared to non-pretreated challenged blanks.

«601551 .k,« - 39 -

Furthermore, the compounds of formulas Ia and III also have anti-inflammatory activity, especially in nonspecific, immunologically induced and hypersensitive induced inflammation and allergic reactions.

This activity can be demonstrated in various test methods, for example by investigating the influence on prostaglandin synthesis in vitro and in vivo. The inhibition of LPS and Zymosan-induced PGE1 and PGF release is investigated in vitro. Thioglycolate-stimulated lot 10 perlecal leueocytes from NMRI mice are incubated for 24 hours with LPS or test substance. After a change of medium and washing the cells three times, they are stimulated for 2 hours with LPS or 1 hour with Zymosan, respectively. In these supernatants 15 PGE 1 and PGF 2 are determined. An inhibition of PGE 4 production by the cells, stimulated by LPS as well as Zymosan, was found. The inhibition of LPS-induced PGE1 release by mouse peritoneal leueocytes was measured in vivo after pretreatment with test substances.

Groups of 3 NMRI mice are treated infcrasperitoneally on days J, 2 and 3 with LPS or test / erb challenge, respectively.

On day 4 these animals and a blank group are given 1.5 ml of thioglycolate intr-aperitoneally, on day 7 peritoneal mouse leucocytes are collected and stimulated with LPS.

25 There appears to be a marked decrease in PGE2 ~ release compared to the blanks.

In a further test, the influence on procoagulent activity (PCA) is measured. After stimulation with LPS, human endothelial cells synthesize PCA, as detected by a shortening of plasma coagulation time. Mouse peritoneal leueocytes after LPS treatment and peritoneal leueocytes (obtained from rabbits) after release of the generalized Shwartzman reaction also shorten the coagulation time of recalculated plasma. To investigate the influence on PCA induced by LPS in vitro, asc 7551 t <- 40 - mouse peritoneal leucocytes stimulated by thioglycolate overnight with LPS alone or with LPS and test substance devoid of fetal, respectively, were treated. calf serum (FCS) (experimental design a). In a pilot design b], mouse peritoneal leucocytes are treated with LPS or test compound for 24 hours and stimulated with LPS overnight after medium change. The coagulation time of recalcified human blank plasma is measured by the Hackchen method after administration of the mouse peritoneal leucocyte suspension, which has been repeatedly frozen and ultrasonically treated.

The addition of test compound reduces the PCA released by LPS compared to the blank value (increase in eoagulation time). Pretreatment with test compound likewise brings a reduction of PCA.

In vivo, the influence of LPS-induced PCA by mouse peritoneal leucocytes after pretreatment with test substance is demonstrated as follows: B, D, F mice are treated intraperitoneally on days 1, 2 and 0 20 3 with LPS or test compound, respectively. All animals further receive 1.5 ml thioglyeolate intraperitoneally on day 3.

On day 6, peritoneal white blood cells are collected, the sample 6 from each animal adjusted to 1x10 cells / ml and stimulated for 24 hours with LPS in DMEM medium weaned from FCS.

The PCA of these cells is determined as described above.

Pretreatment with. LPS or substance, respectively, induces a remarkably reduced PCA. Something similar is found in rabbits. The PCA of rabbit peritoneal leucocytes can be reduced after induction of the generalized Shwartzmann reaction by pretreatment with LPS or with test compound, respectively.

In inhibition of the local Shwartzmann reaction, groups of 3 Chinchilla rabbits are pretreated intravenously or intraperitoneally with 35 LPS and test compound on days 1, 2 and 3, respectively.

8 S C15 51 - 41 -

On day 6, 40, 20, 10, 5, 2.5 and 1.25 µg of LPS are administered intraderm. On day 7, the reaction is triggered with 2 µg LPS / kg intravenously. The valuation is effected semi-quantitatively by examination of the skin necroses.

Almost complete inhibition of the Shwartzmann reaction is observed after pretreatment with IPS and the test compound, respectively.

Furthermore, the compounds of formulas Ia and III also have anti-tumor activity, as shown in the following tests: 1. Induction of tumor necrosis factor (TNF)

To stimulate mouse bone marrow macrophages, mouse bone marrow cells from BDF1 (approx. 10-13 days old, induced with CSF) are diluted to 1x10 cells / ml in medium 15? BPMI 1640 + 1% Pen / Strep (50QQ U / ml) + 1 % glutamine} and incubated in flat microtiter plates with the test compounds in solution in the range of 100 µg to 0.1 µg / ml final concentration (yun dilution steps 1:10) for 4 hours at 37 ° C / 5% CO2. After filtration through 0.45 µm filters, the 2Q supernatants are frozen at -70 ° C until needed for the TNF test.

L 929 cells are pre-cultured overnight (37 ° C, 5% C021 in antiterl plates at 3x10 cells / bead /

100 µl, then 100 µl of each sample is added and the culture is further diluted in 1: 2 steps. 100 µl actinomycin D

(8 µg / ml medium) are added and incubation restarted for an additional 18 hours at 37 ° C / 5% CO. After removal of the supernatants, the remaining cell surface is stained with Giemsa solution and the absorbance 3Q. measured at 620 nm with a Titertek Multiscan Autoreader (flow! One unit? of TNF is defined as the activity leading to a 50% lysis of the target L929 cells.

2. BIGFI melanoma test i § C 1 5 31 - 42 -

BIGFI melanoma cells are grown in vitro for 5 days. The cells are synchronized one day before the run by tearing the monolayer into individual cells using EDTA trypsin and returning the total amount to the same flask with 6 fresh medium. The cells are counted and provided 10 cells / ml medium. 0.1 ml of cell suspension (10J cells) is injected intravenously into the tail vein. 21 days later, mice are killed and lung tumors counted.

BJhF mice are used. The test substance is dissolved and injected intraperitoneally on days -6, -4 and -1.

It has been found that the compounds of formulas Ia and III have iqjinunoprophylactic activity, the number of BIGFI melanoma metastases in the lungs decreases. As a further test of therapeutic activity, the mice are treated on days 3, 6, 8, 10, 13 and 15 after inoculation of the tumor cells. Here, too, a notable decrease in metastasis formation is observed.

The compounds of formulas Ia and III are therefore indicated for use as modulators of unspecified antimicrobial resistance, for the systemic enhancement of immune response and for the promotion of nonspecific immunity. Thus, the compounds of formulas Ia and III are indicated for use in the treatment or supportive treatment (ie, together with other specific or supportive therapeutic forms) of conditions associated with decreased immune response, particularly decreased humoral immune response and / or decreased hypersensitivity reaction of the delayed type, 30. and for the treatment of conditions generally indicating modulation of immune response,

In particular, the compounds of Formula Ia and III are indicated for use in the treatment or supportive treatment of pathologic conditions based on idiopathic immunologic or immunologic deficiencies 8501551-43 - of the type encountered in geriatric patients, or in patients with severe burns or generalized infections. The compounds of formulas Ia and XIX are also indicated for use in the treatment or supportive treatment of viral diseases (such as metastatic herpes, progressive smallpox and metastatic varicella diseases) or Hodgkin's disease and further malignant tumors. Furthermore, the compounds of formulas Ia and III are indicated for use in the prevention of endotoxin shock, IQ for example in accidents, burns and surgical procedures, and as anti-inflammatory agents.

As indicated, a daily dose or a single administration to obtain a contributing effect, for example in supportive treatment, is from about 0.1 mg to about 70 mg, suitably administered, for example, parenterally, preferably intraperitoneally, in the case of a daily dose, in divided doses 2 to 4 times daily in unit dosage form containing about 0.025 - 35 mg of the compound mixed or 20 in slowly released form. An indicated total single adjuvant dose is in the range of up to 70 mg of the compounds.

In view of an immuno-modulating activity, the compounds of formula Ia and III are also indicated for use as additives in vaccines. For this use, an indicated dose of about 0.2 mg to about 100 mg, preferably about 70 mg, is administered on the day of vaccination with an appropriate repeat at the same dose 2 to 4 weeks later.

Pharmaceutical compositions containing a compound of formula Ia or III can be prepared by standard galenic methods, for example, by mixing with conventional pharmaceutically acceptable diluents or carriers. They can be prepared, for example, in the form of injectable solutions. They are also part of $ 60 1 55 1 ψ * - 44 - of the invention.

The invention also provides a method of combating diseases and infections as described above by administering to a patient in need of such treatment 5 a therapeutically effective amount of a formula Ia or III or, where appropriate, a physiologically acceptable salt thereof. The invention also provides the compound of formula Ia or III for use as a pharmaceutical, in particular as an immunomodulator, IQ as an antiviral agent and as an anti-inflammatory agent.

§ & 01h% 1

Claims (9)

A pharmaceutical composition containing a compound of the formula Ia, wherein R independently of 1-4 each other is hydrogen or an optionally substituted acyl group and W, X, Y and Z are independently oxygen or iminc 5 with the proviso that at least one of the groups R 1, is an optionally substituted acyl group, in free form or where appropriate in pharmaceutically acceptable acid addition salt form, together with a pharmaceutical carrier or diluent. 2. A method of modulating antimicrobial resistance, enhancing the immune response and nonspecific immunity, preventing endotoxin shock or treating malignant tumors and inflammation, characterized in that an animal receiving such treatment requires administering a therapeutically effective amount of a compound of claim 1 in free form or where appropriate in pharmaceutically acceptable acid addition salt form. 3. A compound of the formula I, wherein 2Q independently of one another represent hydrogen or an optionally substituted acyl group and W, X, Y and Z have the meaning given in claim J, with the proviso that a) at least one of the groups is an optionally 25 substituted acyl group and L) when Z and X are imino and W and Y are oxygen, a) R 'and R' are different from both i (e) (Rj-3-hydrorytetradecanoyl, provided R ^ and R ^ are identical and (R) -3-hydroxytetradecanoyl or R ^ is (R) -3-dodecanoyloxy-tetradecanoyl and R ^ is (R) -3-tetradecanoyloxytetradecanoyl, g) is other than hydrogen or (R) -3-hydroxytetradecanoyl, to the extent when Rj and are hydrogen and R ^ is (Rj-3-hydroxytetradecanoyl and 8301551 9 - 46 - γ) ΙΙ ^ is other than a group - (COJ ^ -CH ^ -CCHOHJ ^ -CH ^ OH as far as the other substituents hydrogen, in free form or where appropriate in acid addition salt form. Process for preparing a compound of the formula Ia as defined in claim 1, characterized in that a compound of the formula II in which W and Z have the meaning given in claim J, and R 1 with the exception of hydrogen, meaning indicated in claim 1 for R 1 and R 1 and U is uridine enzymatically condenses with a compound of formula III, wherein X and Y have the meaning given in claim 1 and R 1 and R 1 the meaning given in claim 1 for R 2 and R 1 with the proviso that at least of the two substituents is an optionally substituted acyl group and, when X or Y is imino, the substituent R 'or R' 'bonded to the imino group is other than hydrogen, and optionally a obtained compound hydrolyses to a corresponding compound of the formula Ia wherein, when X, Y, Z and / or W is oxygen, R 1, R 1, R 3 and / or R 2 is hydrogen or optionally subject a obtained compound to a acylamidase reaction to to obtain a compound of the formula la in which, when X, Y, Z and / or W is imino, R 1, R 1, R 2 and / or R 2 25 is hydrogen, and the resulting compound is recovered in free form or where appropriate in acid addition salt form. Pharmaceutical composition, characterized in that it contains a compound of the formula III in which X and Y have the meanings given in claim 1, and 30. Rj and R 'have the meanings indicated for Rj and Ri with provided that at least one of the two substituents is an optionally substituted acyl group and, when X or Y is imino, the substituent R 1 or R 2 bonded to the imino group is other than hydrogen, in free form or where appropriate in pharmaceutically acceptable 8601551 Acid addition salt form together with a pharmaceutical carrier or diluent. 6. Methods of modulating antimicrobial resistance, enhancing immune response and nonspecific immunity, endotoxin shock or treating malignant tumors and inflammation, characterized in that an animal in need of such treatment is administering a therapeutically effective amount of a compound of claim 5 in free form or where appropriate IQ in pharmaceutically acceptable acid addition salt form. 7. A compound of the formula III, wherein R 1 and R ”independently of one another are hydrogen or an optionally substituted acyl group and a) Y is oxygen and X is NE with the proviso that when R" is (R) -3- hydroxytetradecanoyl, R ^ is other than (R) -3-hydroxytetradecanoyl or (R) -3-hexadecynoyloxytetradecanoyl, g) Rj is other than hydrogen and 2Q γ} R "and R ^ are different from either acetyl or the group - C0 (CH2) jqCH ^ h) Y and X are oxygen or c} Y is M and X is oxygen, with the proviso that at least one of the two substituents R "and R" is an acyl group and when X and Y is imino the substituent R 'j' or R 'bonded to the imino group is other than hydrogen, in free form or where appropriate in acid addition salt form. Process for the production of a compound 3Q of the formula III as defined in claim 5, characterized in that a) is used to prepare a compound of the formula IIIa in which R 1 and R 2 have the meaning given in claim 5 and both X1 and Y 'are oxygen or YT is imino and X' is oxygen, in a compound of formula IV, wherein 5601551 i * - 48 - R "and R '^ have the meanings given in claim 5, X" and Y 'have the meaning given in this claim and Y is a protecting group, converting the unprotected hydroxy group into a protected phosphate ester group or h) to prepare a compound of the formula IIIh wherein R 1 and R 1 have the meaning given in claim 5 have a corresponding compound of formula IIc, wherein R has the meaning given in this claim, R 'has the meaning given in claim 5 and R' is a protecting group, acylates and then splits off the protecting groups and the resulting compound in free v orm wins or where appropriate in acid addition salt form. A compound of the formula Ia as defined in claim 1 or a compound of the formula III as defined in claim 5, in free form or where appropriate in pharmaceutically acceptable acid addition salt form, for use as a pharmaceutical. G gt Λ Λ 5 * - o öy1 3 5; S 'S Η0 "> ^ Η0 ^ OH HO— \ V — 0 — CHa y — O / nu VY • ^ -o / oh ho-tVo-p;? H wz HO_y \ —ο-p. W vo — p —U R r! Π r0H wzo T R3 ^ yxo '| 0 1, 1, r RR * R, R, Γ I HO— \ Η0Λ ^ "°' \ π Η0Λ-0 -OH 1. ho ^ 3-op (H H0_V “° 'P} \) h r2 rji rf r oh yxo γ x 0 ij Ia R, Re Rl R * IE HO — i HO — v. "° ^ HC H0 ^ / _ o'pr ™ Y 'x' OO NH 0 l 1 II p'1 Li ri Rz 3Ta 1 Ra je b Y ~ f INdr 'ho - \> - oh HO NH 0 HU IO NH I II Rt Wc R (“R» Ί5Γ SANDOZ AG, BASEL, Switzerland 8601551 —Από — λ Vo π ο - \) - οη ΗΟ ΝΗ ^ Υ acy lering ΗΟ-λ / —Ο ΗΟ— ^ V— ΟΗ η / Λ-hr; 'r protection (ü + 6 place) R0> * ho / ^ nhr * protect inq / / \ R0- \ \ \ O \ RO — OH \. / \ \ TBDMS-0, nhr "\ '\ "°> V“ Λ-Ο, or' R0-yr ° -.% * - r ° - & - ° -? <Or, TBDMS-0 NHR "° H0 NHR * O BE c TBDMS - tert-bufytdimethylsilyl SAHDQZ AG, BASEL, Switzerland _ 8601551 - B— / ^ Λ P AcO— \ \\ /) - (V-0 / —O '-' O— (\ —OH - & - AcO— (\ —O Ac Oal / ^ N3 OiN N3 H0A n HO - \ (_ controlled HO- {V-OH -— - HO— (V- OH \ / reduction \ / Ο., Ν '^ Η * o2n' ^^ n3 u protection ( 2 place) .r reduction H0 _ \ _ O ΗζμΛ_η H0 ~ \ _} - 0H HO-Y ^ V-OH ° * N NHR HtN NH, reduction, acylation HO — HO — y HO * ~ y ^ H2N NHR RfHN NHRg- y acylation protection HQ RQ t C ^ 6 place) HO— / ~ VoH RO — P OH RrHN NHR Rj-H N NHRj- ir deprotection / 2 place) ~ Π7 ~ 3 (both Rg-identical) RsHNNHa 0Cy, erm9 RcHN ^ NHRf it6 Place RjHN NHRg-8 6 0 1 55 1 ^ ^ '^ different) GAHDOZ AG, BAGEL, Switzerland * ____ -, ir - C- HO ~ Vo HO — O-0H n3 oh protection ( 4 + 6 place) Ί RO-v RO - \ - OH N3 OH protection (1 place) RO .......... v Vo RO- (\ —O-TBDMS Nj OH 1. reduction RO-, I 2 'acylation RO- / ~ Λ — O-TBDMS R5H ti ^ 0Rs Rq_ f deprotection (1 place) R0 ~ O ~ 0H RrHN ORs- 1Tb 8601551 QAATTW7 fin ώαοπτ r * · - * - D-AcO—. AcO - / ^ ~ \ _OAc AcO O Ac protection (1 place) AcO — v Vo Ac0 — V) -O -CHjC Ci3 AcO o Ac H0 f deprotection (2,3,4,6 place) H ° —0-CHi CCU HO OH ,, protection (4 + 6 place) RO-v 'R0- (3 ~ Ο-0Ηε0013 HO OH RO_ T acylation Vo R0 - () - o-CHjCa, R <rO ORf ,, deprotection (1 place) RO -v RO - / \ —OH R50 OR5 TVc 8 6 0 1 5 5 1 SAEDOZ AG, BASEL, Switzerland