LU86491A1 - NOVEL DISACCHARIDES, THEIR PREPARATION AND THEIR USE AS MEDICAMENTS - Google Patents

Description

I- - 1 -

The present invention relates to new disaccha-wrinkles, their preparation and their use as medicaments.

The invention relates in particular to the disaccharides of formula I 5 HO—.

\ _0 HO ”/ \ —O — CH- \ / ^ i i / —0 0H i 10 VJ Z HO- / \ -O.P ^

Il \ / ΓθΗ K K i — i 0

TV

"i S

15 in which

R1 ', R2'> R3 'and R4 * represent, independently of each other, hydrogen or an optionally substituted acyl group, and W, X, Y and Z represent, independently of each other, oxygen or an imino group, with the proviso that a) at least one of the symbols Ri ', R2', R3 'and R41 represents an optionally substituted acyl group, and b) when Z and X represent an imino group and W and Y represent sense oxygen, a) Ri 'and R2' do not simultaneously signify an (R) -3-hydroxytetradecanoyl group, provided that R3 'and R4' are identical and represent an (R) -3-hydroxy-tetradecanoyl group or that R41 represents a group (R) -3-30 dodecanoyloxytetradecanoyl and R3 'a group (R) -3-tetra-decanoyloxytetradecanoyl, ß) R2 * is other than hydrogen or that a group (R) -3-hydroxytetradecanoyl , provided that Ri 'and R3' represent hydrogen and R4 'represents a group 35 (R) -3-hydroxytétradécanoyl, and w * - 2 - /) R3' is other than a group - (C0) 2 ~ CH2- (CH0H ) 4-CH20H, provided that the other substituents represent the hydrogen gene, and their salts.

5 The invention also relates to a process for the preparation of the compounds of formula la

HOHO— / \ -— O - CH

10 \ - \ \ __ O OH V? HO_ / \ _0./

^ 3 K \ _ / t0B

I I

15 R1 K2 in which X, Y, W and Z have the meanings given above, and

R1, R2, R3 and R4 represent, independently of each other, hydrogen or an optionally substituted acyl group, with the proviso that at least one of the symbols Ri, R2, R3 and R4 represents an optionally substituted acyl group, and their salts,

process by which a compound of formula II is condensed enzymatically

HO— ^

/ - \ ηΏ '° η OH

HO- /, - ° * PN I τγ

\ _. / δ 0 'ï - D

30] | O

w z

I I

Am t> m R3 4 le - 3 - in which W and Z have the meanings given above, R3 "and R4" have the meanings given above for R3 and R4 except hydrogen, and U represents uridine,

5 with a compound of formula III

HO—

'__O OH

HO- / \ _0.pC. 111

\ __ / è ° H

10 1 1 - T? R "R" 1 2 · in which X and Y have the meanings given above, and Ri "and R2" have the meanings given above for Ri and R2, with the proviso that at least one of the two substituents represents an acyl group optionally substituted and when X or Y represents an imino group, the substituent Ri "or R2" linked to the imino group is other than hydrogen, and, if desired, the resulting compound is hydrolyzed to the corresponding compound of formula la wherein, when X, Y, Z and / or W represent oxygen, Ri, R2, R3 and / or R4 represent hydrogen, or if desired, the resulting compound is subjected to an acylamidase reaction , which gives the compound of formula la in which, when X, Y, Z and / or W represent an imino group, Ri, R2, R3 and / or R4 represent hydrogen, and the compounds of formula la are recovered free form or in the form of salts.

The enzymatic condensation can, for example, be carried out in a buffer system, for example in the tris buffer at pH 7. It can be carried out at temperatures equal to or higher than ambient temperature, for example at 30 ° C. A preparation obtained from a 35 gram-negative bacterium, preferably from E. coli-4 strains, can be used as the enzyme extract.

* - i * <[Raetz, J. Biol. Chem. 2 £ 9 (1984) 4852]. The preferred strains are those produced by genetic manipulation with a view to overproduction of the desired enzyme.

The optional hydrolysis can be carried out according to the 5 methods described in the literature.

The acylamidase reaction can be carried out in a manner analogous to that described by C.R. Verret et al. in J. Biol. Chem. 257 [1982] 10 222.

The compounds of formula I thus obtained can be isolated and, if desired, be purified according to known methods.

The compounds of formula la and III can exist in free form or, optionally, in the form of salts. A free form can be transformed into salt according to the usual methods, and vice versa.

The symbols Rj_, R2, R3 and R4 are preferably identical. They preferably represent hydrogen or an acyl group preferably containing from 4 to 20, more particularly from 12 to 16 carbon atoms, especially 14 carbon atoms. They can, for example, be substituted in position 3 by a hydroxy, acetoxy or acyloxy group as defined above. The configuration of the carbon atom in position 3 can be (R) or (S). The symbols Ri, R2, R3 and R4 can therefore be present in a compound of formula la in achiral form or in (R), (S) or racemic form. This also applies to the compounds of formula II, III or IV. The configuration remains unchanged during the process of the invention, which means that when a starting product is used in (R), (S) or racemic form, a final product (R), (S) or corresponding racemic.

The preferred compounds are the compounds of formula la in which R 1, R 2, R 3 and R 4 represent an optionally substituted acyl group. Other preferred compounds are the compounds of formula la in which, when R 1, R 2, R 3 and / or R 4 represent hydrogen, Y, X, W and / or Z represent oxygen.

* "- 5 -

The compounds of formula la are preferably obtained in the form of salts. To increase the solubility in water, hydrophilic basic compounds are preferably used, for example tris- (hydroxymethyl) aminomethane or L-lysine.

The compounds of formula II can be obtained according to known methods by reacting a compound of formula III with activated uridine-5'-monophosphate.

The compounds of formula III in which a) Y represents oxygen and X represents an NH group with the condition that a) when R] / 1 represents an (R) -3-hydroxytetra-decanoyl group, R2 "is other than '(R) -3-hydroxytetradecanoyl or (R) -3-hexadecanoyloxy-tetradecanoyl group, B) Ri "is other than hydrogen, and Y) Ri" and R2 "do not simultaneously mean an acetyl group or - C0 (CH2) 10CH3, b) Y and X represent oxygen or c) Y represents an NH group and X represents oxygen, with the proviso that at least one of the two substituents R ^ "and R2" represents a group acyl and when X or Y represents an imino group, the substituent R] _ "or R2" linked to the imino group is other than hydrogen, are new and also form part of the present invention.

The compounds of formula III can be obtained according to a process comprising a) for the preparation of the compounds of formula IIla HO_

\ _O OH

30 HO_ / ^ __ O.P ^ IHa: \ _ / i · i ·

I I

Rü R2 t '- 6 - in which

R1 "and R2" have the meanings given above and i either X1 and Y 'represent oxygen, or Y1 represents an imino group and X' represents oxygen, the transformation of the free hydroxy group into a compound of

formula IV

RO—

/ ’° \ IV

RO- / \ - OH

10 \ / • - ·

I. I

Y ’X '

I I

Rï R5 15 in which

R1 ", R2", X 'and Y' have the meanings given above, and R signifies a protective group, in a protected group in the form of phosphoric ester, or else b) for the preparation of the compounds of formula IIIb

H0- \ OH

. · - ° x Illb

\ / 0 0H

25 * ---- I I.

ο 1IH

1 ”R” R1 R2 30 in which Ri "and R2" have the meanings given above, the acylation of the corresponding compounds of formula IIle - 7 - RO— \ 0 i / \ / 0R 'RO— · “—0 · Ρ >. TTTf \ _ / 1110 5 i i

HO NH

D "R2 in which R2" and R have the meanings given above, and R1 represents a protecting group, and the subsequent removal of the protecting groups.

Process a) can for example be carried out by dissolving the compound of formula IV in an inert solvent, for example a cyclic ether such as tetrahydrofuran, and reacting at low temperature, for example at -70 ° C, with butyllithium in an aliphatic hydrocarbon such as hexane, then adding dibenzyl phosphorochloridate. The product obtained can be isolated and optionally purified according to the usual methods. The free hydroxy groups of the phosphate residue are protected, for example by benzyl groups. Removal of protecting groups can also be carried out according to known methods. A protective group can for example be removed according to conventional methods under acidic conditions, for example with an aqueous acid (ion exchanger), or by hydrogenolysis.

Process b) can for example be carried out by dissolving the compound of formula IIl in an inert solvent, for example in a chlorinated hydrocarbon such as methylene chloride, with an acylating agent, advantageously with the addition of dicyclohexyl-carbodiimide and of 4-dimethylaminopyridine, and operating at low temperature, for example at about 4 ° C. The product can be isolated from the reaction mixture and purified, if desired, according to the usual methods. The protective groups present are then removed according to known methods.

.¾.

- 8 - m <

As the protecting group, any group usually used as the protecting group can be used in the chemistry of saccharides. The two substituents R can for example c both represent a benzylidene or isopropylidene group.

As protecting groups for the phosphate residue, known protecting groups such as the benzyl group can also be used.

The compounds of formula IIle are new and can be obtained according to the following reaction scheme: HO—.

\ _O

HO— / \ —OH

\ __ / / \ HO NH2 acylati on HO— \ lo

HO— / \ _OH

\ __ /

Ho "NHR ^ R0_. Protection (pos. 4 + 6)

RO- / '-Λ -OH

/ \ HO NHR "protection y * RO— y \ \ __ 0 \ RO_ / \ _OH \ V_ / \ TBDMS.O NHR" \ RO— · RO— J> _o “· \ _o y RO— / \ _O.P ( _V RO— / '_O.Pn „,

Λ_ / * \ __ / fcN0R

TBDMS.O \ jHR "HO \ lHR" IIIc TBDMS = terts-butyldimethylsilyl - 9 -

The compounds of formula IV can be obtained according to the following reaction schemes, the hydroxy groups which do not take part in the reaction advantageously being in a protected form. aged. The substituents have the following meanings: 5 R, R1 = protecting groups R5 = the two symbols have, independently of one another, the meaning given for R \, R2, R3 or R4 Ac = acetyl TBDMS = tert. butyldimethylsilyl.

ί 1. Preparation of the compounds of formula IVa: - 10 - t /—\_.°_\-o Ao0 - \ ._ 0

^ 'o— / \ -OH - * AcO / \ -0AO

\ _ / oJ V ° 2N '| N3 HO— i HO__ / - ° x

\ reduction HO— / \ - OH

y — o ______— \ ./

HO- / \ -OH ^ rnntrnlÔP 'X

\ / controlled o2n N3 O or \ h reduction 2n wh2 HO—. y

H0— \ I protection (pos. 2) H0 _ / _ OH

\ -0 \ _ / HO— / OH / \ \ / H-N NH- / \ ^ ^, 02N NHR ho — acylatiorr

HO—. y HO _ / _ 0 \ _OH

\ _0 ’N __ / HO- / \ _ OH / \ \ / R.HN NHR.

H, N7 Nnhr ro_. otection (pos. 4 + 6) ΗΠ 2,. \ Jl_0

H0— ψ acylation. R0 __ / \ _0H

'__O \ _ / HO_ / \ _OH / \ \ / RcHN NHRc / r \ 5 5

Rg.HN NHR ^ R identical) Ù of protection. (pos. 2) xva vies z 5 H0 --._ 0

HO- / \ - OH

\ / • - · / 4 \

RgHN NH2 H0 — ’v ^ YQ acylation

HO— '\ -OH

• —— ”· - / \

RcHN NHR ,.

RO—— ^

Yq protection (pos. 4 + 6)

RO— / \ _ OH / "“ N

R.HN ~ NHR-5 D

(the 2% different) -> - 2. Preparation of the compounds of formula IVb - 11 - »*«! HO__

^ __ O

'- HO - / \ -OH

i i n3 oh- protection (pos. 4 + 6) RO - y

X - O

RO / \ —OH

• "MM ·

I I

N3 oh. protection (pos. 1)

RO- · *., · · Y

\ __ o

R0-J \ -0. TBDMS

s __ / i i N, OH r J 1. reduction • · 2. acylation RO --- \ Io

RO_ / \ -0. TBDMS

\ _ / / \ r hn: or_ 5 5 RO __ ^ \ f deprotection (pos. 1)

RO_ / \ -0H

\ / • '· / \ R5HN. ORg IVfa y? 3. Preparation of the compounds of formula IVc - 12 - b "

Co-.

\ - O

x AcO - / \ - OAc \ / • MM · / \

AcO OAc

Co-. I protection (p0s. 1) \ —0

AcO / \ —O. CH0CC1 ^ \ __ / 2 3 / \

AcO OAc HO__j deprotection, (pos. 2, 3, 4, 6)

\ —O

HO / \ O. CH0CC1- \ - / 23

I I

HO O H

I protection (pos. 4 + 6)

RO—. X

\ Jo RO— / \ - O.CH-CC1-, \ / 2 3 • '·

I I HO OH

RO v acylation \ ._ 0 RO — J O.CH-CCl-, \ / 2 3 'I · / \

RgO 0R5 RO.— Y deprotection (pos. 1) V — o

RO - / \ - OH

\ / * - · / \ R5 ° or5 ^ IVc * * - 13 -

When the preparation of any starting material is not described in detail, this product is known or can be prepared according to known methods or by proceeding in a manner analogous to that described in the examples.

The following examples illustrate the present invention without in any way limiting its scope. In these examples, the temperatures are indicated in degrees Celsius. EDTA stands for edetic acid (ethylenediaminetetraacetic acid), DTE stands for 1,4-dithioerythritis, UDP stands for uridine phosphate and tris is an abbreviation for tris (hydroxymethyl) aminomethane.

14 "*

Example 1 c 6 ~ Q ~ Î2-deoxy-3-Q - [(R) -3-hydroxytëtradëcanoyl] -2 - [(R) -3-.

hydroxytétradécanoylamidoI-ß-D-glucopyranosyl] -2,3-di-0-_ [(R) - 3-hydroxytétradécanoyl] -l-0-phosphono-a-D-gluco-5 pyranose

Dissolve 2.03 mg of UDP-2-deoxy-3-0 [(R) "3-hydroxytétradécanoyl] -2 - [(R) -3-hydroxy-téttradêcanoyl-amido] -aD-g1ucopyranose and 1.42 mg of 2,3-di-0 - [(R) - 3-hydroxytétradécanoyl] -lO-phosphono-aD-glucopyranose, both in the form of tris salts, in 10 mM tris buffer, pH 7, containing 0, 2 mM EDTA and 0.2 mM DTE and incubated at 30 ° with 60 µl of enzyme extract.

The enzyme extract can be obtained as follows:

E. coli JB 1104 is cultivated in an L culture (preculture + 10 μg of ampicillin / ml at 30 ° overnight; main culture at 30 ° in a stirring fermenter, starting from ODggonm "" 20 0, 1 up to ^ D550nm = 0n cells received by centrifugation at 10,800 g (10 minutes / 4 °), the centrifugation pellet is suspended in 10 mM tris buffer, pH 7.0 + 0 , 2 mM EDTA and 0.2 mM DTE and centrifuged again (6000 g, 10 minutes / 4 °) The centrifugation pellet is then resuspended in the above buffer and fractionated at ultra-high temperatures. sounds (5 x 20 seconds / 30 seconds pause with 120 watts) The cell fragments are separated by centrifugation (12,000 g, 30 10 minutes / 4 °) and the solution is subjected to ultracentrifugation (150,000 g, 90 minutes / 7 °).

The upper 2/3 of the supernatant is recovered. This fraction is subjected to fractional precipitation 15 9 4 with ammonium sulfate. As an enzyme preparation, the 10-40¾ fraction of ammonium sulfate is used.

The resulting centrifugation pellet is suspended in the above buffer.

When the reaction is complete (verification by thin layer chromatography), the solution is acidified to pH 2 with citric acid and centrifuged. The centrifugation pellet is suspended in the chloroform / methanol / acetic acid / water mixture (65/15/5/2) and is chromatographed on silica gel using the same eluent.

The fractions containing the product are combined and evaporated until complete elimination of any organic solvent, and the residue is lyophilized. The lyophilisate is dissolved in a tetrahydrofuran / water mixture (8/2) and, in this solvent, it is converted into tris salt on a cation exchanger in tris form. The tetrahydrofuran is evaporated and the residue is lyophilized. All Rf values are determined on silica gel plates. Rf = 0.48 (chloroform / methanol / acetic acid / water, 65/25/5/5).

Example 2 6-0- [2-dexy-3-0 - [(R) -3-hydroxytëtradécanoyl] -2 - [(R) -3-hydroxytétradécanoylamido] -ß-D-glucopyranosyl] -2-25 deoxy-3 -0 - [(S) -3-hydroxytëtradécanoyl] -2 - [(S) -3-hydroxy tëtradécanoyl ami do] -1 -0-phosphono-qD-glucopyranose 20 parts by volume of a 5 mM solution of 2- dëoxy-3-0 - [(S) -3-hydroxytétradéca-? nuclei] -2 - [(S) -3-hydroxytétradëcanoylamido] -l-0-phosphono-

30 α-D-glucopyranose in 10 mM tris / HCl buffer

(pH 7) containing 0.2 mM EDTA and 0.2 mM DTE, 20 parts of a 5mM solution of UDP-2-deoxy-3-0 - [(R) -3-hydroxytëtradécanoyl] -2- [ (R) -3-hydroxytêtradëcanoyl- 16 amido] -ct-D-glucopyranose in the same buffer and 20 parts of 100 mM tris / HCl buffer (pH7) containing 2 mM EDTA and 0.2 mM DTE are reacted for 8 hours at 30 ° with 40 parts of an enzyme preparation prepared as described in Example 1, in 10 mM tris / HCl buffer (pH 7) containing 2 mM EDTA, 2 mM DTE and 0.1 % of Triton X-100, the final concentration of Triton X-100 being adjusted to 0.1%. The reaction mixture is lyophilized, dissolved in approximately 1/3 of the initial volume of eluent used for subsequent chromatography for separation according to molecular weight (Sephadex LH 20, the solvent: pyridine / acetic acid / water, 98/1 / 1) and filtered.

The volume of the column is about 20 times that of the volume of the sample. The fractions containing the pure product are combined and evaporated under reduced pressure, suspended in water free from pyrogen and lyophilized. After lyophilization, the Triton X-100 is removed by digestion of the product in diethyl ether. After centrifugation, the centrifugation residue is dissolved in a mixture of chloroform and methanol 1/1, filtered and the solvent is removed under reduced pressure.

To an aqueous suspension of the residue, the stoichiometric amount of L-lysine (free base) is added as a 100 mM aqueous solution, to obtain the monosel of L-lysine, and the mixture is lyophilized again. Rf = 0.44 (chloroform / methanol / s · acetic acid / water / 65/25/5/5).

Example 3 6-0- [2-deoxy-3-0 - [(R) -3-hydroxytétradécanoyl] -2 - [(R) -3-hydroxy-tétradécanoylamido] -ß> -D-glucopyranosyl] -2- deoxy-3-0 - [(R) -3-hydroxytëtradëcanoyl] -l-0-phosphono-2 - [(R) -3-tëtradëcanoyloxy-tétradëcanoylami do] -gD-glucopyranose ** 17 H * 20 parts by volume d 'a 5 mM solution of 2-dexy-3-0 - [(R) -3-hydroxy-tetradecanoyl] -l-0-phosphono-2 - [(R) -3-tëtradecanoy1oxytetradecanoyl-friend do] -α-D -glucopyranose in 10 mM 5 tris / HCl buffer (pH 7) containing 0.2 mM EDTA and 0.2 mM DTE, 20 parts of a 5 mM solution of UDP-2-deoxy -3-0- [ (R) -3-hydroxytétradécanoyl] -2 - [(R) -3-hydroxytétradé-canoylamido] -aD-glucopyranose in the same buffer and 20 parts of 100 mM tris / HCl buffer (pH 7) containing 10 2 mM EDTA and 0.2 mM DTE, are reacted for 48 hours at 30 ° with 40 parts of an enzymatic extract (prepared as described in Example 1) in 10 mM tris / HCl buffer (pH 7) containing 2 mM EDTA, 2 mM DTE and 0.1% Triton X-100, the final Trit concentration on X-100 being set to 0.1%. The mixture is lyophilized, treated with pyridine and filtered. The filtrate is concentrated under reduced pressure and chromatographed on silica gel using, as eluent, a mixture of chloroform / methanol / water / 2-phenethylpicolonium bromide 800/175 / 22.5 / 2.5. The fractions containing the pure product are combined and the 2-phenethylpi colonium bromide serving as an ion pair generating agent is eliminated by stirring with 20 mM HgPO 4 in water. The organic phase is concentrated under reduced pressure, the stoichiometric amount of L-lysine base is added in the form of a 100 mM aqueous solution to form, the mono salt of L-lysine, and we? freeze-dry the mixture. Rf = 0.53 (chloroform / methanol / acetic acid / water 65/25/5/5).

By proceeding in a manner analogous to that described in Examples 1 to 3, the following compounds of formula I are obtained, the amounts of enzyme being chosen, according to the compounds of formula II and III

18 used, so that the reaction preferably proceeds for about 24 to 48 hours.

* BOARD

5 R1 = R2 = R3 = R4 = (R) “3“ hydro * ytétradécanoyl

Example ’w__z__Y__X__Rf 4 0 NH NH NH 0.45 10 5 NH NH NH NH 0.40 6 O O O NH 0.49 7 O NH NH O 0.39 8 NH NH O NH 0.41 9 O O O O 0.48 15 c,

Example 10 6-0- [2-deoxy-3-0 - [(R) -3-hydroxytétradëcanoyl] -2 - [(R) -3-hydroxytétradécanovl ami do] -ß-D-g1ucopyranosyl] -2-deox, y- 1-Q-phosphono-3-0-tëtradécanoyl -2-tëtradécanoylamid oaDg 1ucopyranose The title compound is obtained analogously to that described in Examples 1 to 3.

Rf = 0.50 (chloroform / methanol / acetic acid / water 65/25/5/5).

Example 11 25 6-0- [2-dexy-3-0 - [(R) -3-hydroxytëtradécanoyl] -2 - [(R) -3- hydroxytétradécanoylamido] -ß-D-glucopyranosyl] -2-acëtamido-2 -deoxy-3-0 - [(R) -3-hydroxytëtradëcanoyl] -1 -0 -phosphono-aD-glucopyranose

The title compound is obtained analogously to that described in Examples 1 to 3.

Rf = 0.32 (chloroform / methanol / acetic acid / water / 65/25/5/5).

19 *

Example 12 6-Q- [2-deoxy “2 - [(R) -3-hydroxytétradécanoylamido] -ß-D-glucopyranosyl1-1-Q-phosphono-g-D-glucopyranose

Dissolve 5 mg of 6-0 - [(2-deoxy-3-0-5 [(R) -3-hydroxytétradëcanoyl] -2 - [(R) -3-hydroxytétradéca-noylamido] -ß-D-glucopyranosyl] -2,3-di-0 - [(R) -3-hydroxy-tetradecanoyl] -10-phosphono-aD-glucopyranose in an equal mixture of chloroform and methanol, reacted with an aqueous ammonia solution 10 to 25¾ (1/3 of the volume) and left to stand overnight. The reaction complete, the mixture is evaporated to dryness, the residue is triturated in diethyl ether in order to remove the fatty acids formed by cleavage, filtered the precipitate and washed with ether. The residue is dissolved in water, the calculated amount of tris is added to form the tris di-salt, and the product is lyophilized. Rf = 0.45 ( chloroform / methanol / acetic acid / water 25/15/2/4).

Example 13 20 2-Deoxy-3-0 - [(R) -3-hydroxytëtradécanoyl] -2 - [(R) -3- hydroxytëtradécanoylami do 1-1-O-phosphono-aD-glucopyranose a) 4.6-0 ; benzylidene-3-0 - [[R] -3-benzyloxytétradêcanoyl] -_?: E [^): 3: benzyl: Oxytétradécanoylamido] = 2 = deoxy-l-0- ^ 1! ·! 22? Υΐ2!] 22Ε!} 202ΐ9ίΐ9: 2ΐ222βΗ22222 25 A solution cooled to -10 ° and consisting of 3.9 g of 4,6-0-benzylidene-2 - [(R) -3-benzyloxytëtradécanoylamido] -2-dëoxy-l-0 -dibenzyl-phosphono-aD-glucopyranose, 2 g of (R) -3-benzyloxytétradëcanolque acid and 50 mg of 4-dimethyl-30-aminopyridine in 20 ml of methylene chloride, 2 g of dicyclohexylcarbodiimide are added and maintained the reaction mixture at 4 ° overnight. The mixture is then filtered, the filtrate is evaporated to dryness, the residue is taken up in a small amount of a toluene / ethyl acetate v 8/2 mixture and chromatography with the same solvent.

20 F = 96-98 °. [a] D = + 33.3 ° (c = 1, chloroform).

"5 Rf = 0.5 (toluene / ethyl acetate 2/1).

b) 2-deoxy-3; 0; [[R] -3-hydroxytétradécanoyl] -2 - [(R} -3-hydroxytétradécanoylamidolzl ^ O-ghosphono-a-D-gluço-pyranose_

4.2 g of 4.6-0-benzylidene-3-0-10 [(R) -3-benzyloxytëtradëcanoyl] -2 - [(R) -3-benzyloxytetra-decanoylamido] -2-deoxy-1 are dissolved. 0-di benzylphosphono-aD-g1ucopyranone in 900 ml of a tetrahydro-furan / water mixture (85/15) and hydrogenated for 2 hours at 40 ° and under 10 bar, in the presence of 1.5 g of palladium carbon at 10%. The catalyst is then removed by filtration, the tetrahydrofuran is evaporated and the aqueous suspension is lyophilized.

20 [a] p = + 28.5 ° (c = 0.2, chloroform + 1 drop of methanol). Rf = 0.56 (chloroform / methanol / 20 acetic acid / water 125/75/10/20).

Subsequent purification can be carried out as follows: 10 g of 2-dexy-3-0 - [(R) -3-hydroxytëtra-dëcanoyl] -2 - [(R) -3-hydroxytétradécanoylamido] -l-0-phos- 25 phono-aD-glucopyranose and 1.7 g of tris in 150 ml of methanol are treated with ultrasound for 10 minutes at 45 ° in an ultrasonic bath. The suspension is centrifuged and the clear supernatant and the pellet are separated by decantation. To this solution is added 1.7 g of tris (dissolved in 20 ml of methanol), the solution is primed and allowed to crystallize at room temperature. The tris di-salt of the title compound is obtained.

• J "21

The mother liquors are evaporated to dryness. We. dissolve the residue and the centrifugation residue together in a mixture of 300 ml of chloroform, 600 ml of methanol and 240 ml of water (free of pyrogen). To this solution, another 300 ml of chloroform and 300 ml of 0.1 N hydrochloric acid are added, the mixture is stirred carefully and the phases are allowed to separate. The chloroform phase is separated and evaporated to dryness. The residue consists of 2-deoxy-10 3-0 - [(R) -3-hydroxytétradécanoyl] -2 - [(R) -3-hydroxytétra-décanoylamido] -l-0-phosphono-a-D-glucopyranose impure.

The resulting product is treated with ultrasound with tris and methanol as described above and is then centrifuged. The solution is then chromatographed on a Sephadex LH 20 column with methanol as the eluent. The fractions containing the product are combined and evaporated to dryness. The tris mono-salt of the title compound is obtained.

5.1 g of this product are dissolved * at 45 ° in 40 ml of methanol in the ultrasonic bath, a solution of 0.74 g of tris in 10 ml of methanol is added, the solution is primed and leaves to crystallize first at room temperature and then at 4 °. This gives additional amounts of crystals of the tris di-salt of the title compound.

The residue from the mother liquors and the centrifugation pellet can be subjected to another purification test.

20 30 F = 183-185 ° (decomposition); [a] D = + 15 ° (c = 1.0 in water).

I * 22

Example 14 3-dëoxy-2-0 - [(R) -3-hydroxytétradécanoyl] -3 - [(R) -3-hydroxyi tëtradëcanoylamido] -! - O-phosphono-aD-glucopyranose a) 2: 0: [{ R): 3: benzYloxytétradécanoyl] -3 - [(R} -3: benzyloxy ^ 5 tétradécan ° ylamido] -3-deoxy ~ l; 0: dibenzyiphosphono; 4,6-0-isogr ° py! Idene-aD = glucopyranose To a solution cooled to -70 ° and consisting of 4.85 g of 2-0 - [(R) -3-benzyloxytétra-decanoyl] -3 - [(R) -3-benzyloxytétradécanoylamido] -3-10 deoxy-4 , 6-0-isopropylidene-D-glucopyranose in 40 ml of anhydrous tetrahydrofuran, 8 ml of 1.6 M butyllithium is added in hexane. After 5 minutes, a solution of 3.8 g is added at the same temperature of dibenzyl phosphorochloridate in 10 ml of benzene, stirring is continued at -70 ° for 10 minutes, 0.3 ml of acetic acid is added and the solution is concentrated to a quarter of the initial volume.

The solution is diluted with 200 ml of methylene chloride, extracted with 50 ml of water, 50 ml of a dilute solution of sodium hydrogencarbonate and 50 ml of a solution of sodium chloride, <bn dried over sodium sulfate, evaporated to dryness and the residue is chromatographed (toluene / ethyl acetate 7/3). Rf = 0.58 (toluene / ethyl acetate 2/1).

b) 3-deoxy-2-0 - [[R) -3-hydrox ^ tetradëcano ^ 1] -3 - [[R); 3; hydroxytétradécanoylamidol-l-O-ghosghono-a-D-çjluco-gyranose_

A solution of 980 mg of 2-0 - [(R) -3-30 benzyloxytétradécanoyl] -3 - [(R) -3-benzyloxytétradê- canoylami do] -3-dëoxy-l-0-di benzylphosphono-4,6 -0-i sopropylidene-aD-gl ucopyranose in 100 ml of tetra-hydrofuran, is hydrogenated for approximately 5 hours il * 23 under normal pressure, in the presence of 300 mg of charcoal pal 1 adie at 10%. The catalyst is then removed by filtration, water (10 ml) and Dowex AG50WX8 H + are added and the mixture is stirred at room temperature until the isopropylidene group is completely split. The ion exchanger is separated by filtration, the solution is neutralized with tris and the tetrahydrofuran and water are removed by evaporation under reduced pressure. The residue is dissolved in methanol and chromatographed on Sephadex LH 20. Rf = 0.65 (chloroform / methanol / acetic acid / water 125/75/10/20).

Example 15 2,3-di-0 - [(R) -3-hydroxytëtradëcanoyl] -l-0-phosphono-a-15 Dg 1ucopyranose a) § »* 2-di- 0; [[R]; 3; benzj (loxytêtra; décanoyl] -l-0-dibenzylphosphono-aD-glucopyranose To a solution of 1 g of 4,6-0-benzy-1i dène-2,3-di-0 - [(R) -3-benzyloxytétradécanoyl] -D-20 glucopyranose in 10 ml of anhydrous tetrahydrofuran cooled to -70 °, 0.9 ml of 1.6 M butyl lithium in hexane is added dropwise. After 5 minutes, dropwise added to same temperature "a solution of 420 mg of dibenzyl phosphorochloridate 25 in 4 ml of benzene. Stirring is continued for 10 minutes at -70 °, the solution is neutralized with acetic acid and evaporated to The residue is chromatographed (silica gel, hexane / toluene / ethyl acetate 4/4/1).

30 Rf = 0.65 (tolene / ethyl acetate 6/1).

b) 2,3-di-0 - [(R) -3-hydroxytétradëcanoylH-T-0-phosphonO2_ 24

230 mg of 4.6-0-benzylidene-2,3-di-0-c [(R) -3-benzyloxytétradécanoyl1-1-0-dibenzylphosphono- α-D-glucopyranose are dissolved in a tetrahydrofuran / water mixture 9 / 1 and hydrogenated for about 5 hours under normal pressure in the presence of 80 mg of 10¾ palladium on carbon. The catalyst is removed by filtration, the solution is neutralized with tris and evaporated to dryness. The residue is chromatographed on Sephadex LH 20 with methanol. Rf = 0.65 10 (chloroform / methanol acetic acid / water 125/75/10/20).

Example 16 2-Deoxy-3-0 - [(S) -3-hydroxytëtradécanoyl] -2 - [(S) -3-hy-droxy-tétradécanoylamido] -l-0-phosphono-aD-glucopy-15 ranose a) 4,6-0-benzylene-3-0-C (S} -3-benzyloxytétradécanoyl1-2- [ÎSl: 3: benzyloxytétradêcanoylamido] -2-dëoxy-l-0- ^ ÎȧD? YISlî2§E! 32D2laz9lS ^ u22EY! TËD2se

The compound is obtained analogously to that described in Example 13a). Rf = 0.44 (toluene / 4/1 ethyl acetate).

b) 2-deoxy; 3-0 - [[S) -3-hydroxytétradécanoyl]; 2; [[S]; 3-! 3y2L2ïyi§ÏLÊ ^ ec2D2Ylam1 ^ 2]: l: 9 “PhosEhono" “: 9: 2luco : p ^ ran2§e 25 The title compound is obtained in a manner analogous to that described in Example 13b).

F = 150-200 ° (with decomposition). Rf = 0.5 (methylene chloride / methanol / ammonia 1/1/1; lower phase).

Example 17 2-Deoxy-3-0 - [(R) -3-hydroxytëtradécanoyl] -l-0-phosphono- 2- [(R) -3-tetradëcanoyloxytëtradëcanoyl amido3-aD-gl ucopyranose 25 a) 4,6- O ^ benzylid | ne23-0 - [[R) -3-benzylox ^ tétradécanoyl] - £ zd§2XYzlz9zi! I!? 2DZYl2! 3222lî2D2zi: zIÎBlz§ZΧÎEadëcanoyl - ~ oxytétradécanoylami do Jz ^ fZ ^ z2 1222 ^

The title compound is obtained analogously to that described in Example 13a). F = 82-95 °.

[a] p ° = + 24.1 ° (c = 1, chloroform). Rf = 0.7 (toluene / ethyl acetate 2/1).

b) 2-deoxy-3: 0Î [R) -3-hydroxytétradécanoyl]: ^ - 0-phosghono: _? _ ”t [Blz? zî§ÎrÊ ^ §2ËD2 ^ l25yî§îrÊ ^ §2ËD2ylÊ! DlÎ2 ^ z2f_0z_ 10 gluç2pyranose

The title compound is obtained in a similar manner to that described in Example 21b).

20 [a] Q = + 14.3 ° (c = 1, tetrahydrofuran / pyridine).

Rf = 0.35 (chloroform / methanol / acetic acid / water 80/25/5/5).

Example 18 2-Deoxy-1Q-phosphono-3-0-tetradëcanoyl-2-tetradecanoyl-amido-gD-glucopyranose a) 1 »§ζ0: ^ §ηζγ1idene-2-deoxY-l-0-dibenzolphosphono-3: 0: 20 tétradécanoyl-2-tétradêcan2ylamidoza jl “Xl_u_c_ojDy_r_a_n_o_s e

The title compound is obtained in a similar manner to that described in Example 13a) using tetradecanolque acid and a toluene / ethyl acetate mixture (4/1) as eluent. F = 123-129 °.

25 Rf = 0.6 (toluene / ethyl acetate 2/1).

b) 2-deoxy-1-0-phosphono-3-0-tëtradécanoyl-2-tétradê-canoylamido-a-D-glucopyranose_

The title compound is obtained in a similar manner to that described in Example 21b). Rf = 0.65 30 (chloroform / methanol / acetic acid / water 125/75/10/20).

J

26

Example 19 2-Deoxy-2 - [(R) -3-hydroxytétradëcanoylamido3-l-0-phospho-no-3-0 - [(R) -3-tétradécanoyl oxytétradécanoyl] -aD-g1uco-pyranose âk 5 a) 4 , 6-0-benzylidene-2 - [[R | -3-benzyloxytëtradécanoylamido] - 2-dëoxY-l-0-dibenzylphosghon ° -3-0 - [[R) -3-tetradecanoYl-oxytetradecanoYlJza ^ D-glucop ^ ranose

The title compound is obtained analogously to that described in Example 13a), using (R) -3-tetradecanoyloxytetradecanoic acid and the toluene / ethyl acetate system (4/1) as 2Ω mixture of solvents. F = 89-90 °. [α] β = + 30.9 ° (c = 1, chloroform / methanol 1/1). Rf = 0.5 (toluene / ethyl acetate 4/1).

B) 2: deoxy-2; [[R): 3-hydroxYtétradécanoylamido] -l-0-phos; 2222Y220222

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

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

Example 20 2 5 2-deoxy-2 - [(R) -3-hydroxytétradëcanoylami do] -l-0-phospho- no-3-0-tëtradëcanoy1-aD-glucopyranose a) 4.6: 0: benzylidene: 2: [[R]: 3: benzyloxytétradécanoyl amido] -2-deoxy-l.-0-di benzyl phosphono ^ S ^ O ^ tëtradé-2222 ¥ ΐ: ®: 9ι9ΐ222ΒΥΠ2Π2§2 30 The title compound is obtained analogously to that described in Example 13a), using tetradecanolic acid and the toluene / ethyl acetate system (3/1) as a mixture of solvents.

? 27 F = 125.5 - 126.5 °. Rf = 0.6 (toluene / ethyl acetate 3/2).

b) 2-deoxy-2 - [(R] -3; hydroxytétradécanoylaniido] -l; 0-ghos; 2hono = 3-0-tétradécanoyl-aD-glucopyranose * 5 Dissolve 354 mg of 4,6-0-benzylidene- 2 - [(R) -3-benzyloxytétradécanoylamido] -2-deoxy-l-0-dibenzylphosphono-3-0-tétradécanoyl-aD-glucopyranose in 30ml of a tetrahydrofuran / water mixture (9/1) and hydrogenated for 10 hours under normal pressure 10 in the presence of 200 mg of palladium on carbon.

The catalyst is filtered off and the solution is adjusted to pH 7.5 with tris (hydroxymethyl) -aminomethane. The tetrahydrofuran is evaporated under reduced pressure and the aqueous solution is lyophilized. "The lyophilisate is triturated twice in ether and dried. Rf = 0.6 (chloroform / methanol / acetic acid / water 125/75 / 10/20).

Example 21 2-acetami-do-2-deoxy-3-0 - [(R) -3-hydroxytétradëcanoyl] -l-20 O-phosphono-gD-glucopyranose a) 2-acetami do-4,6-0-benzylidene- 3-0-C (R] -3-benzyloxy-tetradecanoyl] -2-deoxy; l-0; dibenzylghosghono = aD = g! Ucopyranose

A solution of 3.55 g of 2-acetamido-4,6-0-benzylidene-2-deoxy-1-0-dibenzylphosphono-aD-glucopyranose and 2.1 g of acid is cooled to 0 °. (R) -3-Benzyloxytetradecano * ic in 15 ml of methylene chloride and 1.32 g of dicyclohexylcarbodiimide and p-dimethylamino-pyridine are added. After 2 hours at this temperature, the reaction is complete. The mixture is filtered, evaporated to dryness and the residue is chromatographed on silica gel first with a methylene chloride / methanol / 28 mixture (50/1) and then with a toluene / ethyl acetate mixture ( 2/1).

Rf = 0.7 (chloroform / methanol 20/1).

b) 2: acetamido: 2: deoxy: 3: 0: [[R}: 3: hydroxytëtradêcanoyl]: 5 l-0-ghosphono-a-D-gluco | 3yranose_

2.08 g of 2-acetamido-4,6-0-benzylidene-3-0 - [(R) -3-benzyloxytëtradécanoyl] -2-deoxy-1-0-dibenzylphosphono-aD-glucopyranose are dissolved in a tetrahydrofuran mixture / water (9/1) and hydrogenated for 3 hours under normal pressure in the presence of 1 g of 10% palladium on carbon. The catalyzing catalyst is separated by filtration, the filtrate is evaporated and lyophilized. The lyophilisate is dissolved in methanol, neutralized with tri s (hydroxymethyl) aminomethane and chromatographed on Sephadex 20 LH 20 with methanol. [a] D = + 43.3 ° (c = 1, methanol). Rf = 0.4 (chloroform / methanol / acetic acid / water 125/75/10/20).

Example 22 20-0-phosphono-2,3-di-0-Ç (R) -3-tetradëcanoyloxytëtra-decanoyl3-aD-glucopyranose a) 4,6-0-benzyl idene-l-0-di benzyl ghosphono- 2,3-di ;; ^ [[R ^ -3-tétradëcanoyloxytëtradêcanoyl3-aD-glucogyra-nose 25 The title compound is obtained analogously to that described in Example 15a), the product being purified by chromatography on silica gel with an equal part mixture of ether and hexane as eluent. Rf = 0.5 (1/1 ether / hexane).

B) 1-0; ghosghono-2,3-di-0 - [[R]; 3; tëtradécanoyloxytétra ^ dëcanoyl] -a-D-g1ucopyranose

470 mg of 4.6-0-benzylidene-1-0-di benzylphosphono-2,3-di-0-C (R) -3-tetradecanoyl-oxytetradecanoyl] -aD-glucopyranose are dissolved in 47 ml 29 of tetrahydrofuran and hydrogenated for 1 hour under normal pressure in the presence of 230 mg of palladium on carbon. The catalyst is filtered off, the filtrate is concentrated to dryness and the residue is chromatographed on silica gel (chloroform / methanol / water / triethylamine 15/10/2/2/2). The fractions containing the product are combined and evaporated to dryness. The title compound is obtained in the form of a di-triethylamami-friend salt.

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

Example 23 2-Deoxy-3-0 - [(R) -3-hydroxytëtradécanoyl] -2 - [(S) -3-hydroxytëtradëcanoylamido] -l-0-phosphono-aD-glucopy-15 ranose a) 4,6- 0-benzylidene-3-0 - [[R ^ S-benzyloxytétradécanoyl] - 2- [[S ^ -3-benzyloxytétradëcanoylamido] -2-deoxy-1-0-

The compound is obtained analogously to that described in Example 13a). Rf = 0.44 (toluene / ethyl acetate 4/1).

b) 2-deoxy-3-0 - [[R] -3-hydroxytêtradêcanoy] J-2 - [(S] · - 3-hydroxytétradécanoylamido] -l-0-phosghono-aD-_ gluc2gyran2se 25 The compound is obtained from analogous to that described in Example 13b). Rf = 0.5 (methylene chloride / methanol / ammonia 1/1/1, lower phase).

Other characteristics of the compounds have been obtained by fast atom bombardment mass spectroscopy (FAB) (negative ions) (Raetz, J. Biol. Chem. 259/4852 [1984]).

30

BOARD

Example No. Mass 1 1325 2 1324 3 1534 4 1323 5 1322 6 1325 7 1324 8 1323 9 1326 10 1292 11 1140 12 647 '' 31 "

Frefliple No: NMR spectrum - 1 9.82 (d, 9 Hz, 1 H); 6.65 (dd £, 8 and 3.5 Hz, 1H); 6.25 (t, 10 Hz, 1 H); (C5D5N) 5.83 (t, 10 Hz, 1H); 5.63 (d, 8.8 Hz, 1H); 5.35 (dd, 1Q and 2.5 Hz, 1H); 4.0 (t, 10 Hz, 1H); 3.83 (m 1H).

13 5.49 (dd, 3.5 and 7.5 Hz, 1H); 5.24 (dd, 9.5 and 10.5 Hz, 1H); "X> OD) 4.17 (ddd '2'5'3'5 and 10'5 H2> 1H) î 4.0 (m' 3H) î 3.90 (dd '2 and 3 12 Hz, 1H) ; 3.75 (dd, 5.5 and 12 Hz, 1H); 3.71 (s, Tris); 3.63 (t, 10 Hz, 1H).

14 5.77 (dd, 3.5 and 7.5 Hz, 1H); 4.77 (ddd, 2,5,3,5 and 10,5 Hi, (CDCW ih); 4.38 (dd, 9,5 and 10,5 Hz); 3.8 to 4.1 (m, 4H); 3.73 (dd, ^ 3} 5.5 and 12 Hz, 1H); 3.53 (t, 10 Hz, 1H).

5.71 (dd, 3.5 and 7.5 Hz, 1H); 5.48 (t, 10 Hz, 1H); 4.77 (ddd, 2, (CD3OD) 4 and 10 Hz, 1H); 3.9 to 4.05 (m, 3H); 3.86 (dd, 2 and 12 Hz, 1H); 3.7 (dd, 5.5 and 12 Hz, 1H); 3.67 (s, Tris); 3.58 (t, 10 Hz, 1H).

16 5.58 (dd, 3 and 7 Hz, 1H); 5.22 (t, 10 Hz, 1H); 4.19 (dt, 3 and ^ Hz> ^ 4.1 ^ H); 3.7 (s, Tris and d, d, 1H); 3.46 3 (t, 10 Hz, 1H).

17 5.46 (dd, 3 and 7 Hz, 1H); 5.22 (t, 10 Hz, 1 H); 5.17 (m, 1H); CDcSÎ 4.19 (<jt ’3 Ä 10 Hz» 1H) î 3.9 to 4.1 (m »3H) î 3.7 (s’ Tris) î 3 3.65 (dd, IH); 3.51 (t, 10 Hz, 1 H).

18 / step a) 7.38 (m, 15H); 5.70 (dd, 3.5 and 6 Hz, 1 H); 5.63 (d, 9.5 Hz, (protected) IH); 5.50 (s, 1H); 5.26 (t, 10 Hz, 1 H); 5.08 (m, 4H); 4.38 (m, (CDC13) m). 4.08 (dd, 5 and 10 Hz, 1H); 3.95 (dt, 5 and 10 Hz, 1 H); 3.73 (t, 10 Hz, 1 H); 3.69 (t, 10 Hz, 1 H); 2.29 (m, 2H); 1.89 (m, 2H).

32 19 / step a) 7.2 to 7.45 (m, 20H); 6.34 (d, 9 Hz, 1H); 5.77 (dd, 3.5 and '(protected) 6 Hz, 1H); 5.49 (s, 1H); 5.29 (t, 10 Hz, 1H); 5.14 (quintet, v (CDCI3) 6 Hz, 1H); 5.0 (m, 4H); 4.51 and 4.41 (AB, 12 Hz, 2H); 4.41 (m, 1H); 4.08 (dd, 5 and 10 Hz, 1H); 3.95 (dt, 5 and 10 Hz, 1H); 3.7 (m, 3H); 2.58 and 2.47 (ABX, 5.5,7.5 and 15.5 Hz, 2H).

20 / step a) 7.2 to 7.45 (m, 20H); 6.29 (d, 9 Hz, 1H); 5.74 (dd, 3.5 and (protected) 6 Hz, 1H); 5.49 (s, 1H); 5.30 (t, 10 Hz, 1H); 5.0 (m, 4H); 4.52 (CDC13) and hA2 (AB> n H2j 2H); 4> 44 1Hj, 4> Q3 (dd} s and 1Q Hz, 1H); 3.94 (dt, 5 and 10 Hz, 1H); 3.7 (m, 3H); 2.15 to 2.35 (m, 4H).

21 / step a) 5.47 (dd, 3.5 and 7.5 Hz, 1H); 5.23 (dd, 9.5 and 10.5 Hz, 1H); (protected) 4.I8 (ddd, 2.5, 3.5 and 10.5 Hz, 1H); 3.90 (m; 3H); 3.85 (dd, 2 (CD3OD) and 12 Hz, 1H); 3.72 (dd, 5.5 and 12 Hz, 1H); 3.68 (s, Tris); 3.61 (t, 10 Hz, 1H); 1.94 (s, 3H).

22 / step a) 7.3 to 7145 (m, 15H); 5.92 (dd, 3.5 and 6.7 Hz, 1H); 5.60 (t, -9.5 (protected) and 10 Hz, 1H); 5.48 (s, 1H); 5.02 to 5.22 (m, 6H); 4.95 (ddd, (αχ13} 2.5, 3.5 and 9.5 Hz, 1H); 4.10 (dd, 4.5 and 10 Hz, 1H); 3.96 (ddd, 5.9 and 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); 2.12 (t, 7, 5 Hz, 2H).

23 / step a) 7.2 to 7.42 (m, 25H); 6.49 (d, 9 Hz, 1H); 5.77 (dd, 3.5 and (protected) 6 Hz, 1H); 5.45 (s, 1H); 5.35 (t, 10 Hz, 1H); 5.0 (m, 4H); 4.54 (CTCI3) and 4> 40 (AB> u ^ 2H). 4} 47 and 4.36 (ab, u Hz, 2H); 4.45 (m, 1H); 3.9 bis 4.1 (m, 2H); 3.6 to 3.8 (m, 4H); 2.58 (dd, 6.5 ^ and 15.5 Hz, 1H); 2.34 (dd, 6 and 15.5 Hz, 1H); 2.2 (m, 2H).

The compounds used as starting materials can be obtained as follows: - 33 - A) 4,6-0-benz, ylidene-2 - [(R) -3-benzy1 oxytétradécanoy1amido] -2-deoxy-10-dibenzylphosphono-aD -glucopyranose (for examples 13, 19 and 20) a) 2 - [(R) -3-benzyloxytétradécanoylamido] -2-deoxy-D-glucopyranose 5 A mixture of 5.4 g is stirred for 24 hours at room temperature D-glucosamine hydrochloride, 10.8 g of N - [(R) -3-benzyloxytétradécanoyloxy] succinimide and 5 ml of diisopropylethylamine in 25 ml of dry dimethylformamide. The solvent is removed by distillation and the residue is dissolved in a mixture of 150 g of chloroform, 300 ml of methanol and 120 ml of water. After adding an additional part consisting of 150 ml of chloroform and 150 ml of water, the lower phase is separated and washed twice with the upper phase consisting of 100 ml of chloroform, 100 ml of methanol and 90 ml of 'water. The solution is evaporated and dried under high vacuum. The product thus obtained is used in the next step without further purification.

For the purposes of analysis, a small part of the product in the raw state is purified by chromatography (toluene / ethanol 9/1), F = 150-158 °.

20 [of | 20 = + 53 ° (c = 1, dimethylformamide)

Rf (chloroform / methanol 9/1) = 0.3.

b) 4,6-0-benzylidene-2 - [(R) -3-benzyloxytétradécanoylami do] -2- ^ 22 ^: 9: 912222 ^ 222222

A solution of 5.83 g of 2 - [(R) -3-benzyloxytétradécanoyl-25 amido] -2-deoxy-D-glycopyranose, 3 g of benzaldehyde dimethylacetal and 500 g of p-toluene sulfonic acid monohydrate in 200 ml of anhydrous dimethylformamide is maintained for approximately 3 hours at 55-60 ° and under 30-40 mbar in a rotary evaporator. Then there is no starting material left. Most of the dimethylformamide is separated by distillation, 500 ml of methylene chloride are added and the solution is extracted twice with 200 ml of a dilute solution of sodium hydrogen carbonate and 200 ml of water. The solution is dried with sodium sulfate, evaporated to dryness and the residue is chromatographed (toluene / ethyl acetate 6/4).

Ir * - 34 - F = 162-165 °.

[α] £ 0 = + 5.5 ° (c = 1, chloroform)

Rf (toluene / ethyl acetate 1/1) = 0.2.

c) 4,6-0-benzy] idene-2 - [(R) -3-benzyloxytétradécanoylamido] -2- To a solution of 4.86 g of 4,6-0-benzylidene-2 - [(R) - 3-benzyloxytétradécanoylamido] -2-deoxy-D-glucopyranose in 40 ml of anhydrous tetrahydrofuran cooled to -70 °, 8 ml of 1.6 M butyllithium in hexane are added dropwise. After 5 minutes 10 at this temperature, a solution of 3.8 g of dibenzyl phosphorochloridate in 10 ml of benzene is added dropwise. After stirring for 10 minutes at -70 °, 0.3 ml of acetic acid is added and the solution is concentrated to 1/4 of its initial volume. The solution is diluted with 200 ml of methylene chloride, it is extracted with 50 ml of water, 50 ml of a dilute solution of sodium hydrogencarbonate and 50 ml of a dilute solution of sodium chloride , dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (toluene / ethyl acetate 7/3).

F = 102-105 °.

20 [a] 20 = + 31.7 ° (c = 1, chloroform)

Rf (toluene / ethyl acetate 1/1) 3 0.32.

B) 4,6-0-benzylidene-2,3-di-0 - [(R) -3-benzylox.ytétradécanoyl] -D-glucop.yranose (for Example 15) 25 A solution cooled with ice and consisting of 2.34 g of penta-O-acetyl-D-glucopyranose in 9 ml of trichloroethanol, 0.9 ml of boron trifluoroetherate is added and the solution is kept at this temperature for 4 hours. The solution is then poured into 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 purification by chromatography (silica gel, toluene / ethyl acetate, 4/1), the product is crystallized from a mixture of ethyl acetate / petroleum ether.

F = 138-140 °.

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

b) trichloroethyl-4,6-0-benzylid | ne-B-D-g] ucopyranoside_

7.13 g of trichloroethyl-2,3,3,6,6-tetra-0-acetyl-3-D-glucopyranoside are dissolved in a mixture of methanol and methylene chloride, the solution is cooled with ice and 10 reacts with a solution of sodium methanolate (60 mg of sodium in 26 ml of methanol). After 2 hours at 0 °, the solution is neutralized with Dowex AG 50 WX8 H +, the ion exchangers are filtered and the solvent is evaporated under reduced pressure. The residue was dissolved in 60 ml of benzaldehyde, 4.1 g of zinc chloride was added and the mixture was stirred for 10 hours at room temperature. The mixture is then poured into ice water, extracted with ether, the ethereal solution is dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (silica gel, toluene / ethyl acetate 4/1).

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

c) Îll2t)] 9r2lÎhy] -4,6-0-benzylidene-2,3-di-0 - [(R) ^ 3-benzyloxy: _ To a solution consisting of 800 mg of trichloroethyl-4,6-0- benzylidene-BD-glucopyranoside, 1.4 g of (R) -3-25 benzyloxytetradecanoic acid and 20 mg of dimethylaminopyridine in 10 ml of methylene chloride cooled to 0 °, 930 mg of dicyclohexylecarbodiimide is added and the reaction mixture is maintained at 4 ° overnight. The reaction mixture is then filtered, the filtrate is evaporated to dryness, the residue is dissolved in a hexane / toluene / ethyl acetate mixture (12/5/1) and chromatographed with the same mixture of solvents.

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

- 36 - d) 4.6-0: benzylidene: 2.3: di-0: [(R) -3 ^ ^ g1ucopyranose

480 mg of 4,6-0-benzylidene-v 2,3-di-0 - [(R) -3-benzyloxy-tetradecanoyl] -BD-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 the starting material is completely consumed. The reaction mixture is filtered, evaporated to dryness and the residue is chromatographed (silica gel, hexane / toluene / ethyl acetate 2/5/1).

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

C) 2-0 - [(R) -3-benzyloxytétradécanoyl3-3 - [(R) -3-benzyloxytétra- décanoylami do] -3-déox.y-4,6-0-i sopropyli dene-D-qlucopyranose ( for example 14) 15 a) azido-32 deoxy-4,6-0-isopropylidene-D-glucopyr ^

1.02 g of 3-azido-deoxy-D-glucopyranose is dissolved in 10 ml of dry dimethylformamide, 940 μΐ of 2-methoxypropene and a catalytic amount of the p-toluene sulfonic acid monohydrate are added and the solution is maintained at room temperature for about 2 hours. The p-toluenesulfonic acid is neutralized with sodium hydrogen carbonate, the solution is evaporated to dryness and the residue is chromatographed (silica gel, chloroform / methanol 9/1).

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

B) t6rt.-butyldimethylsilyl-3-azido-3-deoxy-4,6-0; To a solution consisting of 560 mg of 3-azido-3-deoxy-4,6-0-isopropylene-D-glucopyranose and 310 mg of imidazole in.

25 ml of dry methylene chloride, 345 mg of tert.-butyldimethylsilyl chloride are added and the mixture is stirred for 2 hours at room temperature. The excess imidazole hydrochloride is filtered, the filtrate is stirred twice with 10 ml of water, dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (silica gel, toluene / ethyl acetate 12/1).

Rf (toluene / ethyl acetate 1/1) = 0.6.

c) tert ^ -butyldlméthylsilyl ^ -O-ÇW - ^ - benzyloxytétradécanoyl] - 5 3-L_ (R) -3-benzyloxytêtradécano ^ lamido3-3; deoxy-4,6-0-

A solution of 3.1 g of tert.-butyldimethyl-silyl-3-azido-3-deoxy-4,6-0-isopropylidene-ß-D-g1ucopyranoside is hydrogenated in 100 methanol for 2 hours with 300 mg of palladium a 10¾ out of 10 coal. The catalyst is filtered and the filtrate is evaporated to dryness. The residue is dissolved with 5.35 g of (R) -3-benzyloxytetra-decanoic acid and 100 ml of dimethylaminopyridine in 50 ml of dry methylene chloride. The solution is cooled with ice and 4.1 g of dicyclohexyl-carbodiimide are added. After approximately 3 hours at room temperature, the solution is filtered, the solvent evaporated and the residue chromatography (silica gel, toluene / acetate ethyl 9/1).

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

d) 2-0 - [(R) -3-benzyloxytétradécanoyl] -3-C (R) -3-benzyloxyté ^ To a solution comprising 5 g of tert.-butyldimethyl-silyl-2-0 - [(R) - 3-benzyloxytétradécanoyl] -3 - [(R) -3-benzyloxytétra-décanoylami do] -3-deoxy-4,6-0-i sopropyli dène-BD-g1ucopyranosi de in 100 ml of anhydrous and cooled tetrahydrofuran at -60e, 5.2 ml of a solution of tetra-butylammonium fluoride in tetrahydrofuran are added dropwise. The temperature of this solution is allowed to rise to -40 ° and it is maintained at this temperature until the starting material disappears (about 30 minutes). 5 ml of methanol are then added, the mixture is heated to room temperature and the solvent is evaporated. The residue is extracted with a mixture of methylene chloride and water, the organic phase is dried over sodium sulfate and the solvent is evaporated. After purification by chromatography (silica gel, toluene / ethyl acetate 3/1), the title compound is obtained in the form of a mixture of anomers.

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

5 0) 4,6-0-benz.ylidene-2 - [(S) -3-benz.yloxytétradécanoyl] -2-deox, yl- O-dibenzylphosphono-ot-D-glucopyranose (for Example 16) a ) 2 - [(S) -3-benzy] oxytétradécanoylamido] -2: deoxy = D-glucopyranose

The title compound is obtained by proceeding in a similar manner to that described in Example Aa).

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

b) 4,6-0-benzy] idene-2 - [(S) -3-benzyloxytétradé ^

The title compound is obtained by proceeding analogously to that described in Example Ab).

15 [a] 20 = -3} 5 ° (c = 1, chloroform).

Rf (toluene / ethyl acetate 1/1) = 0.37.

c) 4,6-0-benzylidene-2 - [(S) ^ 3-benzyloxytêtradécanoylamido] -2- deoxy-l-O-di benzylphosphono-a-D-glucopyranose

The title compound is obtained by proceeding in a manner analogous to that described in Example Ac).

[a ^ 20 = + 39.2 ° (c = 1, chloroform).

Rf (toluene / ethyl acetate 1/1) = 0.46.

E) 4,6-0-Benzylidene-2-deoxy-1Q-dibenzylphosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamido] -aD-glucopyranose (for Example 17) a) 2-deoxy; (RM ;; tétradécanoyloxytétradécanoylamifo ^ glucose

A mixture composed of 650 mg of D-glucosamine hydrochloride, 1.9 g of N - [(R) -3-tetradecanoyloxytetra-30 decanoyloxyüsuccinimide and 0.5 ml of diisopropylethylamine in 15 ml of dimethylformamide is stirred for 20 hours at ambient temperature. The solvent is then removed by distillation and the residue is chromatographed on silica gel with an ethyl acetate / methanol mixture (8/1) as eluent.

[a] 20 = + 25 ° (c = 1, methanol).

Rf (ethyl acetate / methanol 6/1) = 0.5.

b) 4.6-0: benzylidene-2: deoxy-2: Ç (R): 3-tetradecanoy ^ 5 decanoylamido] -D-glucopyranose

A solution composed of 5.83 g of 2-deoxy-2 - [(R) -3-tetra-decanoyloxytétradécanoylamido] -D-glucose, 3 g of benzaldehyde-dimethylacetal and 500 mg of p-toluene acid monohydrate- sulfonic acid in 200 ml of dry dimethylformamide is kept for 3 hours at 55-60 ° and under 30-40 mbar in a rotary evaporator. The starting material then disappeared. The majority of dimethylformamide is then distilled, 500 ml of methylene chloride are added and the solution is extracted twice with 200 ml of a dilute solution of sodium hydrogen carbonate and 200 ml of water.

After drying over sodium sulfate and evaporation, the residue is chromatographed (toluene / ethyl acetate 6/4). F = 162-165 °.

[a] 20 = -1.5 ° (c = 1, chloroform / methanol 1/1).

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

c) 456-0-benzylidene-2-deoxy-l-0-dibenzylphosphono-2 - [(R) -3- 20 2x-yïêtr ad ec ano ^ l ami d °] -ot- Dg 1 uçopy r ano se A a solution consisting of 950 mg of 4,6-0-benzylidene-2-deoxy-C (R) -3-tetradecanoyloxytetradecanoylamido] -D-glucopyranose in 40 ml of dry tetrahydrofuran and cooled to -40 °, added dropwise 1.25 ml of 1.6 M butyllithium in hexane.

After 5 minutes, a solution of di-benzyl pbosphorochloridate in benzene is added and stirring is continued for 5 minutes at the same temperature. The solution is then neutralized with acetic acid, the mixture is evaporated to dryness and the residue is extracted with methylene chloride and water. The organic phase is dried over sodium sulfate, evaporated to dryness and the residue is chromatographed on silica gel (toluene / ethyl acetate 3/1).

[a] 20 = + 19.3 ° (c = 1, chloroform).

Rf ftoluene / ethyl acetate 1/1) = 0.6.

- 40 - F) 4,6-0-benz.ylidene-2-deoxy-1-0-dibenz.ylphosphono-2-tetra-decanoylamido-ot-D-glucopyranose (for Example 18) a) 2-deoxy -2-tet adêcanoylami do-D-glucose

A mixture composed of 3 g of D-gluco-5 samine hydrochloride, 4.56 g of N-tetradecanoyloxysuccinimide and 2.8 ml of άτι sopropylethyl amine in 40 ml of dry dimethylformamide is kept at room temperature for 24 hours. The product is then filtered, washed with dimethylformamide and water, dried under vacuum and used for the next step without further purification. 10 b) 426-03benzylid | ne-2 = deoxy-2-tetradecanoyl ^ ido-D-g] uco

4 g of 2-deoxy-2-tetradecanoylamido-D-glucose are dissolved at 55 ° in 320 ml of dry dimethylformamide, 2.6 ml of dimethoxytoluene and 400 mg of p-toluene sulfonic acid monohydrate are added and maintains the mixture in a rotary evaporator 15 for 4 hours at 55-60 ° and under 30-40 mbar. The solvent is then distilled, the residue is washed with a dilute solution of sodium hydrogencarbonate, water and ethanol, dried under vacuum and used directly for the next step, c) 4a6-0-benzy1idène- 3-0- (tert ^ -buty1dimethy1si2y22 ~ 2-deoxy-2- 20 tetradecanoylamido-D-glucopyranose

2.2 g of 4.6-0-benzylidene-2-deoxy-2-tetradecanoylamido-D-glucose are dissolved at 60 ° in 250 ml of dry dimethylformamide, 960 mg of imidazole and 2.13 g of tert are added .-butyldimethyl-chlorosilane and the reaction mixture is kept at the same temperature for 24 hours. After the addition of 480 mg of imidazole and 1 g of tert.-butyldimethylchlorosilane and an additional 24 hours at 60 °, the starting material has completely disappeared. The solvent is distilled off, the residue is extracted with methylene chloride and water, the organic phase is washed once with water, dried over sodium sulfate and distilled. The residue is chromatographed on silica gel with a toluene / ethyl acetate gradient (20/1 to 2/1). Two main fractions are obtained: the title compound (650 mg) and the corresponding 1,3-bis- - 41 - silyl compound (1.82 g). The 1,3-bis-silylated compound is dissolved in 25 ml of dry tetrahydrofuran, the mixture is cooled.

V

solution at -40 ° and 2.5 ml of an IN solution of tetrabutylammonium fluoride in tetrahydrofuran are added dropwise.

5 After approximately 1 hour at -40 °, the protective group of the anomeric silyl group is removed. 3 ml of methanol are added, the mixture is heated to ambient temperature and the solvent is removed in vacuo. The residue is extracted with methylene chloride and water, the organic phase is extracted once with water, then it is dried and distilled. An additional 1.52 g of the title compound is obtained in the form of an anomeric mixture.

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

d) 4A6-0-benzy] id§ne-3-0- (terti = but ^ ldiméthy2§ilyl) z? z ^ i25 ^ zîz9z ^] ^ êD? ylB! 39§Pb2D9: 2: yîL ^ | çanoylamido- aD-glucopyranose 15 To a solution composed of 2.46 g of 4,6-0-benzylidene-3- 0- (tert.-butyld i methylsi 1yl) -2-deoxy-2-tetradecanoylami do-D-gluco-pyranose in 100 ml of dry tetrahydrofuran and cooled to -60 °, 3.12 ml of 1.6 M butyllithium in Vhexane is added dropwise. After 5 minutes at this temperature, a solution of dibenzyl phosphorochloridate in benzene is added dropwise. Stirring is continued at -60 ° for a further 5 minutes, then the solution is neutralized with acetic acid and evaporated to dryness. The residue is taken up in methylene chloride and extracted with water. After treatment and chromatography on silica gel with a mixture of tuene / ethyl acetate (3/1) as eluent, the title compound is obtained.

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

e) 4.6-02benzylidene-2-deoxy-120-dibenzylphosphono-2-tetra-decanoylamido-aD-glucopyranose 30 A solution cooled to -10 ° and consisting of 2.05 g of 4.6-0-benzylidene- 3-0- (tert.-butyldimethylsilyl) -2-deoxy-1-0-dibenzylphosphono-2-tetradecanoylamido-aD-glucopyranose in 40 ml of anhydrous tetrahydrofuran, 2.5 ml of a IN solution of tetrabutylammonium fluoride in tetrahydrofuran and stirred for 2 hours at 0 °. 3 ml of methanol are then added, the solution is evaporated to dryness and the residue is extracted with methylene chloride and water.

After treatment according to the usual methods and chromatography on silica gel with a toluene / ethyl acetate mixture (3/2) as eluent, the title compound is obtained.

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

G) 2-acetamido-4,6-0-benzylidene-2-deoxy-l-0-dibenzylphosphono-10 oi-D-glucopyranose (for example 21)

a) 2-acetamido: 4,6-0-benzylidene-3-M

üC0P ^ raD9fe

A solution consisting of 5.17 g of 2-acetamido-4,6-0-benzylidene-2-deoxy-D-glucopyranose, 3.4 g of imidazole and 6.3 g of tert.-butyldimethylchlorosilane in 300 ml of dimethylformamide is maintained at 60-70 °. After 6 hours, 500 mg of imidazole and 1 g of tert.-butyldimethylchlorosilane are added and the mixture is kept at the same temperature until the reaction is complete. The solvent is evaporated, the residue is extracted with methylene chloride and water and purified by chromatography with a toluene / ethyl acetate mixture (5/1). For the elimination of the anomeric tert.-butyldimethylsilyl group, the product is dissolved in 600 ml of dry tetrahydrofuran, the solution is cooled to -40 ° and 14 ml of an IN solution of tetrabutylammonium fluoride 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 chloride and extracted with water. The organic phase is dried and the solvent is distilled.

Rf (chloroform / methanol 9/1) = 0.5.

- 43 - b) 2-acetamido-4,6-0 ^ benz ^] idene-3-0- (tert.-butyldimethylsilyl) - "2:! L22XY: l: 9: d! K§D? YlP!] 22P!] 929: 9 :: 0: 9ly22Py22222e

The title compound is obtained by proceeding in a manner analogous to that described in Example Ec) and after chromatography 5 with a toluene / ethyl acetate mixture (1/1) as eluent.

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

c) 2-acetamido-4,6-0-benzylidene-2 - ^^ aD-glucopyranose In an ice-cold solution composed of 10 4.4 g of 2-acetamido-4,6-0-benzylidene- 3-0- (tert.-butyldimethylsilyl) -2-deoxy-1-0-dibenzylphosphono-aD-glucopyranose in 200 ml of dry tetrahydrofuran, 6.5 ml of an IN solution of fluoride is added dropwise tetrabutylammonium in tetrahydro-furan and the mixture is maintained for 2 hours at 0 °. Then 7 ml of methanol are added and the solution is evaporated to dryness. A solution of the residue in methylene chloride is washed with water, dried and evaporated to dryness.

Rf (chloroform / methanol 9/1) = 0.65.

H) 4,6-0-benzylidene-2,3-di-0 - [(R) -3-tetradecano.ylox.ytétra-20 décanoyl] -D-glucopyranose (for example 22) a) trichloroethyl- ^ eO-benzylidene ^^ S-di-O-Ç ^ Rl-S ^ têtra- ^ 22222 ^ ΐ2:! ^: ί: 2Ϊ22θ22222 ^ 13: §: 9: 9ΐ222Ρ ^ Γ222§1ί! 2

The title compound is obtained by proceeding analogously to that described in Example Bc) using (R) -3-tetradecanoyloxytetradecanoic acid as acylating agent and a toluene / ethyl acetate mixture as eluent.

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

b) 4,6-0-benzy] idene-2,3-dÎ-0 - [(R) -3-tét ^ · 022229 ^ 13: 0: 912222 ^ 222222 30 The title compound is obtained by proceeding in a manner analogous to that described in Example Bd) after purification by chromatography on silica gel using a toluene / ethyl acetate mixture (15/1) as eluent.

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

► / * - 44 - I) Synthesis of UDP-2,3-diacy1-hexoses

1.4 g of uridine-5'-phosphoro- salt are dissolved

Ufi morpholidate-N, N'-dicyclohexylcarboxamidine in 25 ml of anhydrous pyridine and dried twice by evaporation and by redissolution in 25 ml of anhydrous pyridine. To this solution is added a solution composed of 1 g of 2,3-diacyl-hexose-1-phosphate in 25 ml of pyridine and which has been previously treated in the same way, the mixture is kept at 37 ° for 24 hours , then it is cooled with ice. Then 50 ml of chloroform and 12 ml of 90% formic acid are added. This solution is chromatographed on silica gel and the starting product which has not reacted is eluted with a chloroform / pyridine / 90% formic acid mixture (30/30/7). The column is washed with a chloroform / methanol mixture (9/1) to remove the pyridine and the UDP derivative is eluted with a chloroform / methanol / water mixture (66/33/4).

The chloroform and methanol from the peak fraction are evaporated in vacuo and the remaining aqueous suspension is filtered. The precipitate is dissolved in 100 ml of a tetrahydrofuran / water mixture (2/1) and the solution is stirred for 2 hours with Dowex AG50WX8 [in tris- (hydroxymethyl) -aminomethane form]. The ion exchanger is filtered, the tetrahydrofuran is removed by distillation and the aqueous solution is lyophilized. The lyophylisate is used in the next step without further purification.

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

The compounds of formulas la and III are distinguished by interesting pharmacological properties and can therefore be used therapeutically as medicaments. They exert a favorable influence on non-specific microbial resistance. This activity can be demonstrated, for example, in the following tests.

s - 45 - υ 1. Determination of endotoxic activity using the litnulus LAL test (lysate of limulus amibocytes) The endotoxin catalyzes the activation of a proenzyme in the lysate of limule amibocytes. The cleavage of p-nitro-5 aniline is measured from a colorless substance. The elimination rate is detected photometrically, the correlation between the absorption and the concentration of endotoxin (or, respectively, the endotoxic activity for the analogs) being linear in the range of 0.01 to 0.1 ng / ml of endotoxin (compare with ab-10 sorption values of a standard endotoxin). From each sample (dissolved in sterile bidistilized water and free of pyrogen), a 1:10 serial dilution is prepared. To 100 µl of a test sample or a standard reference sample or a blank sample, 100 µl of horseshoe crab lysate is added. After 10 minutes of incubation at 37 ° C., 200 μl of substrate are added to the reaction mixture. The reaction is stopped with 200 μl of 50% acetic acid after another 3 minutes of incubation. After stirring the sample, the absorption is measured at 405 nm with a spectrophotometer by subtracting the basic value. The endotoxin contents (endotoxic activity) of the sample are calculated in endotoxin units (EU) by linear regression with the values of the standard endotoxin.

2. Induction of Endotoxin Shock in Mice The test involves inducing endotoxin shock or a similar lethal clinical condition using LPS-like substances in mice sensitized to galactosamine (GalN). C57 bl male mice are administered intraperitoneally. (6 animals per group) simultaneously 8 mg of GalN dissolved in 0.5 ml of PBS and 0.1 μg of LPS of Salmonella abortus equi 30 (Sigma) dissolved in 0.2 ml of physiological saline solution. This treatment kills all animals within 6 to 12 hours [see C. Galanos et al., Proc.Natl.Acad.Sei., USA 76 (1979) 5939-5943]. In place of LPS in standard treatment, the test substances are administered in varying doses simultaneously with GalN, or parenterally or orally before or after treatment with GalN. The results are evaluated by comparing the lowest doses which are lethal for all animals in a group, or by calculating the LD50 according to the Spearman-Kärber method.

5 3. Microbial septicemia in neutropenic mice

This model makes it possible to determine an increase in the immune response provoked by the substance in a neutropenic mouse, infected with microbes. To induce neutropenia, 200 mg / kg of cyclo-10 phosphamide dissolved in 0.2 ml of distilled water, on day 0, are administered at once subcutaneously to groups of 20 female BgD2Fi mice. On day 3, the test substance is administered parenterally (mainly intraperitoneally) or orally, if possible dissolved in a physiological solution of sodium chloride, or dissolved in another way (0.3 ml ). The infection is caused on day 4 by injecting the appropriate inoculum intravenously in a volume of 0.2 ml (number of germs per mouse: for example Pseudomonas aeruginosa 12: 1 x 10 ^; E. coli Δΐ20: 2 x 106;

Staph. aureus Δ. 113: 1 x 10 ^; Candida albicansΔ 124: 1 x 104. The 20 animals are observed until day 10 from the infection and the number of dead animals is recorded every day. The following parameters are calculated with respect to the infected controls or with respect to the standards: a) average survival time 25 b) survival rate.

After parenteral administration, the compounds of formulas la and III markedly improve the duration and survival rate compared to untreated infected controls, both in the case of experimental infections caused by gram negative agents (e.g. Pseudonomas and E. coli strains), only in the case of infections caused by gram-positive agents (for example the Staph. aureus strains) or yeasts (for example the Candida albicans strain).

- 47 - "4. Activation of the oxidative metabolism of human blood neutrophils

Neutrophils (at least 95¾ pure), s are incubated at 37 ° C with the test substance at 3 different concentrations for 1 to 2 hours. The release of superoxide anions by the cells indicates their activation and is measured by the inhibition of superoxide dismutase which catalyzes the reduction of cytochrome C. 1 × 10 6 neutrophils previously treated or not treated with the test substance are added to a solution containing cytochrome C (80 10 dMM), as well as formylmethionylleucylphenylalanine (10 “6m). The controls also contain 50 µg of superoxide dismutase. After 15 minutes of incubation at 37 ° C, the reaction is stopped by cooling the test tubes in an ice bath. These tubes are then centrifuged at 400 xg for 5 minutes to remove the cells. The reduction in cytochrome C, which is proportional to the quantity of superoxide anion released, is measured by photometry at 550 nm. The inhibitory effect of the test substance on the activation of PMN by the LPS is measured as described above, except that after the preincubation of the leukocytes, the cells are continued to incubate with a stimulating concentration of LPS.

5. Carbon clearance test

This test is based on the principle that particles with a size between 200 and 250 A, for example carbon particles, can only be eliminated from the body by phagocytosis by macrophages. After intravenous administration of carbon particles, they are eliminated from the bloodstream by phagocytosis of macrophages in the liver (Kupffer star cells) and in the spleen. RES activation of a substance is determined by administering it once or repeatedly in aqueous solution or as a suspension. The substances to be tested are administered intraperitoneally or subcutaneously at the rate of 4 doses daily or in one portion 24 hours before the start of the test. A suspension containing 10% carbon black is diluted with a gelatinous solution of 1% sodium chloride to a carbon content of 16 mg / ml. Each mouse is injected intravenously 0.2 ml per 20 g of body weight. 25 µl of blood are collected per. puncture into the orbital plexus 3, 6, 9, 12 and 15 minutes after intravenous administration. The blood is hemolysed in 2 ml of distilled water. The carbon concentration is determined by photometry. The animals are then sacrificed and the weights of the liver and spleen are calculated.

6. Herpetic Infection (Mouse) Intra-cutaneous herpetic infection makes it possible to determine the increase in the immune response provoked by a substance in mice contaminated with herpetic viruses. The course of the disease is prolonged and makes it possible to observe the successive appearance of several parameters. Herpetic lesions appear at the site of infection and then begin to ulcerate. Possibly, there is a paralysis of the paw adjacent to the infection, this paralysis progressing progressively until death. Immunocompetent nude mice are infected intracutaneously on day 0 by intracutaneous administration of T inoculate in a volume of 0.025 ml (for example 20 1 × 10 6 units of Herpes simplex type 1 plaque / per mouse). The test substance is administered intraperitoneally in a solution, for example in physiological saline solution (0.1 ml).

Systemic herpes infection also allows the determination of an increased immune response caused by a substance in mice infected with a herpes virus. Immunocompetent NMRI mice are infected on day 0 by intraperitoneal administration of the inoculate in a volume of 0.1 ml (e.g. 1.3 x 10 ^ Herpes simplex type 1 plaque forming units / per mouse). The test substance is administered subcutaneously in a solution, for example a physiological solution of sodium chloride.

• 3 - 49 -

The test animals are observed until day 20 after infection and the appearance of lesions, paralysis and the dead animals are recorded. The following parameters are measured and compared with the infected controls or a standard: ~ 5a) 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 form-10 mule la and III produce a marked improvement in terms of the course of the disease, the duration of survival and the survival rate compared to the untreated controls. These effects are observed after a single intraperitoneal or subcutaneous administration between days 0 or -1 and +6.

15 7. Induction of CSF (colony stimulating factor)

CSFs are mediators produced by the body as a result of infections or in response to toxins. Their biological effects are complex; they are detected by means of stimulation of the proliferation of the hemopoietic system, in particular of the cells of the bone marrow. BgD2Fi mice are administered the test substance one or more times parenterally or orally, at doses up to 50 mg / kg. Animal serum is collected at different intervals. Titers of CSF activity in serum are determined in a cell culture assay as the rate of proliferation of bone marrow cultures from B6D2F1 mice Cv ° ir 0- Metcalf, The Hemopoietic Colony Stimulating Factors, (1984), Elsevier; T. Mosmann, O. Immunol.Methods, 65 (1983) 55-63; L.M. Green et al!., J. Immunol.Methods, 70 (1984) 257-268].

The compounds of formula la and III induce the formation of CSF in mice in different degrees, kinetic differences in CSF activities being also observed. These differences can have advantages in therapeutic use.

- 50 - 8. Induction of Interleukin-1 (IL-1) The ability of a substance to stimulate cells to produce IL-1 is determined in tissue culture tests. Resident macrophages as well as macrophages released by thioglycolate are first recovered in the form of adherent macrophages. These are incubated in RPMI medium for 48 hours with various concentrations of the test substance and the supernatant cells are collected. The IL-1 activity of these cells is demonstrated in cultures of pro-10 thymocytes from C3H / HeJK mice. The supernatant cells containing IL-1 produced by the macrophages are induced to proliferate the thymocytes during an incubation period of 72 hours. The proliferation is measured by incorporation of 3H-thymidine using a scintillation counter [see J. Gery et al., J.Exp.Med. 136 15 (1972) 128-142; J. Oppenheim et al., Cellular Immunol. 50 (1980) 71-81].

The compounds of formulas la and III have to varying degrees (concentrations of the order of 0.1 to 50 μg / ml) the capacity to induce the production of IL-1 in macrophages.

20 9. Induction of tolerance (tolerance of lethal effect) to LPS

(endotoxin)

Tolerance of this type can be induced in mice by parenteral administration of LPS, one to three times a day. This tolerance protects the animal against the lethal effect of LPS after administration of galactosamine (see above under "Induction of shock with endotoxin in mice").

The compounds of formula la and III already ensure this tolerance after administration by the intraperitoneal route at a single dose of 0.25 mg / mouse. The previously treated mice (toier-30 rantes) are subjected to a challenge with LPS at a dose of 0.01 μg of LPS + 8 mg of galactosamine / mouse, administered intraperitoneally at various periods (from 1 day to 3 weeks ) after the last treatment. More animals survive this LPS challenge, particularly after repeated administration (3 times), compared to control animals not previously treated and challenged.

- 51 -

The compounds of formula la and III also have anti-inflammatory activity, in particular in non-specific inflammations, in immunologically induced inflammations, in inflammations induced by hypersensitivity as well as in allergic reactions. This activity can be demonstrated by various test methods, for example by studying the influence of these compounds on the synthesis of prostaglandins in vivo and in vitro. The inhibition exerted in vitro on the release of PGE2 and PGFi induced by LPS and Zymosan was examined. Thioglycolate-stimulated peritoneal leukocytes from NMRI mice are incubated for 24 hours with LPS or the test substance. After changing the medium and triple washing of the cells, the latter are stimulated for 2 hours with LPS or for 1 hour with Zymosan. PGE2 and PGFi are determined in the supernatants. There is an inhibition of the production of PGE2 stimulated by LPS or Zymosan.

The inhibition exerted by the compounds on LPS-induced PGE2 release in mouse peritoneal leukocytes was measured in vivo. Groups of 3 NMRI mice are treated intraperitoneally on days 1, 2 and 3 with LPS or with the test substance. On day 4, 1.5 ml of thioglycolate are administered intraperitoneally to the test animals as well as to the control animals; on day 7, peritoneal leukocytes are collected and stimulated with LPS. There is a clear decrease in the release of PGE2 compared to the control animals.

In another test, the influence of the compounds on the procoagulant activity (PCA) was measured. After stimulation with LPS, ** 30 ’human endothelial cells synthesize PCA, which * can be demonstrated by a reduction in plasma coagulation time. The peritoneal macrophages of mice obtained after treatment with LPS as well as the peritoneal leukocytes (taken from rabbits) after release of the generalized Schwartzman reaction, also reduce the coagulation time of the "recalcified" plasma. In order to determine the influence of PCA induced by LPS in vitro, peri-tonal leukocytes from B5D2F1 mice stimulated with thioglycolate are treated overnight, either only with LPS or with LPS and the substance. to be tested in a DMEM medium devoid of fetal calf serum (FCS) (test a)). In test b), mouse peritoneal leukocytes are treated for 24 hours with LPS or with the substance to be tested. After changing the medium, the cells are stimulated overnight with LPS. The coagulation time of the control recalcified human plasma is determined using the Häkchen method after adding a suspension of mouse peritoneal leukocytes which has previously been frozen several times and treated with ultrasound.

Addition of the test substance reduced the PCA provoked by LPS compared to the control values, as a result of the increased coagulation time. Likewise, pretreatment with the test substance results in a reduction in PCA.

In vivo, the influence of LPS-induced PCA in mouse peritoneal leukocytes after prior treatment with the test substance can be demonstrated as follows: b6d2f1 mice are treated intraperitoneally on days 1, 2 and 3 with LPS or with the substance to be tested. On day 3, all animals additionally received 1.5 ml of thioglycolate intraperitoneally. On day 6, the peritoneal leukocytes are collected, the sample from each animal is adjusted to 1 × 10 6 cells / ml, and they are stimulated for 24 hours with LPS in a DMEM medium free of FCS. The PCA of these cells is determined according to the method described above. Treatment prior to 30 LPS or with the test substance causes a marked reduction in PCA. A similar reduction is observed in the rabbit. The PCA of rabbit peritoneal leukocytes can be reduced after a generalized Schwartzman reaction, and by prior treatment with LPS or the test substance.

T

- 53 - c

To study the inhibition of the local Schwartzman reaction, groups of 3 chinchilla rabbits are treated beforehand by intravenous or intraperitoneal route with LPS or with * the test substance, on days 1, 2 and 3. On the 6th day, we administer 5 intradermally 40, 20, 10, 5, 2.5 and 1.25 µg LPS.

On the 7th day, the reaction is started with 2 μg of LPS / kg intravenously. The test is evaluated semi-quantitatively by an examination of necrosis of the skin. There is an almost total inhibition of the Schwartzman reaction after pre-treatment treated with LPS or with the substance to be tested.

The compounds of formulas la and III also have activity against tumors, as has been demonstrated in the following tests.

1. Induction of tumor necrosis factor (TNF) 15 In order to stimulate macrophages of the bone marrow alone in mice, bone marrow cells are diluted from BDFi mice (aged approximately 10 to 13 days, induced with CSF) so as to obtain 1 × 10 6 cells / ml in a medium [RPMI 1540 + 1% Pen / Strep (5000 U / ml) + 1% glutamine] and incubated for 4 hours at 37 ° C./ 5% CO2 in flat microplates with the substance to be tested in a solution whose final concentration (1:10 dilution steps) is of the order of 100 pg to 0.1 pg / ml. After filtration through 0.45 µm filters, the supernatants are frozen at -70 ° C before use.

L 929 cells are precultured overnight (37 ° C, 5% CO2) in microplates 3 x 10 ^ cells / well / 100 µl, then 100 µl of each sample is added and the culture is further diluted in stages 1: 2. 100 μl of actinomycin D (8 μg / ml of medium) are added and the incubation is continued for a further 18 hours at 37 ° C./5% CO 2. After removing the supernatant cells, the layer of remaining cells is stained with Giemsa solution and the absorption is measured at 620 nm using a Titertek Multiscan Autoreader (Flow) device. One unit of TNF is defined as the activity which produces a lysis of 50¾ of the 35 L 929 target cells.

- 54 - <c 2. B16F1 melanoma test

B16F1 melanoma cells are grown in vitro for 5 days. These cells are synchronized one day prior to performing the assay by separating the monolayer into individual cells using trypsin-EDTA and returning the total amount to the same bottle in fresh medium. The cells are counted and a preparation is made containing 10 ^ cells / ml of medium. 0.1 ml of the cell suspension (105 cells) is injected intravenously into the tail vein of BgD2Fi mice. 21 days later, the animals are sacrificed and the number of lung tumors is counted. The substance to be tested is dissolved and injected intraperitoneally on days -6, -4 and -1.

The compounds of formulas Ia and III have been found to have immuno-prophylactic activity which results in a reduction in the number of metastases of melanoma B16F1 in the lungs. As a test of the therapeutic activity of these compounds, mice are treated on days 3, 5, 8, 10, 13 and 15 after having inoculated the tumor cells. In this case too, there was a marked decrease in the number of metastases.

The compounds of formula la and III can therefore be used in therapy as modulators of non-specific antimicrobial resistance, in systemic strengthening of the immune response and in strengthening of non-specific immunity. The compounds of formula la and III can therefore be used for the treatment or adjunctive treatment (for example with other specific or adjuvant therapeutic forms) of conditions associated with a decrease in the immune response, in particular in particular a decrease in the humoral immune response and / or a decrease in the prolonged hypersensitivity reaction, and for the treatment of conditions in which in general a modulation of the immune response is indicated. In particular, the compounds of formula la and III are useful in the treatment or adjunctive treatment of pathological conditions due to idiopathic immunodeficiencies or immunodeficiencies of the rencon-35 type in elderly patients, or in subjects with severe burns or generalized infections. The compounds of formulas la and III can also be used in the treatment or adjunct treatment of viral diseases (such as disseminated herpes, progressive smallpox and forms of disseminated chickenpox), Hodgkin's disease and other malignant tumors. The compounds of formulas la and III can also be used for the prevention of endotoxin shock, for example in the event of accidents, burns and surgical procedures, and as anti-inflammatory agents.

For the indications mentioned above, the dose to be administered will obviously vary depending on the compound used, the mode of administration and the treatment desired. In general, satisfactory results are obtained by administering these compounds in a daily dose, or alternatively in a single dose as an adjunct effect, for example in the context of adjunct therapy, about 0.0015 mg / kg to about 1 mg / kg of active substance.

Administration can be done, for example, parenterally, preferably intraperitoneally. In human therapy, the appropriate daily dose will be in the range of about 0.1 mg to about 70 mg, preferably administered in divided doses 2 to 4 times a day in unit doses containing from about 0.025 to about 35 mg of active substance, or in a sustained-release form. A single booster dose can be up to 70 mg of active substance.

Thanks to their immunomodulating activity, the compounds of formula la and III can also be used as adjuvants in vaccines. For this use, the appropriate dose will be * between approximately 0.5 and approximately 100 mg, preferably approximately ** 30 70 mg, administered on the day of vaccination, advantageously followed by a booster at the same dose 2 to 4 weeks after.

The invention therefore relates to the compounds of formula la and III in free form or in the form of salts, for the use as medicaments, in particular as immunomodulators, as ** anti-viral agents and as anti-inflammatory agents.

The invention also relates to a pharmaceutical composition comprising a compound of formula la or III, in free form or in salt form, in admixture with pharmaceutically acceptable diluents or carriers. Such compositions can be prepared according to known galenical methods and can be presented, for example, in the form of injectable solutions.

%

Claims (15)

57 * 1. A pharmaceutical composition comprising ^ a compound of formula la tc HO— \ / - ° HO_ / \ —O - CH, \ _ / ^ II> - ° \ (I ·) WZ HO- / \ -OP ^ 10. l X __ / 1 08 3 R4 I I O I ï “i B2 in which R-j9 Rgj R3 and R ^ independently mean hydrogen or an optionally substituted acyl group, and X, Y, W and Z independently mean oxygen or an imino group, provided that at least one of the symbols R ^, R £, R3 and R ^ 20 means an optionally substituted acyl group, in free form or in the form of a pharmaceutically acceptable salt, in association with a pharmaceutically acceptable vehicle or diluent. 2. The compounds of formula I 25 HO- V-O HO __ / \ - O-CH2 \ - / \ __ O ‘OH (I)! i / \ y W f HO- / / - ° * lv t 'R' \ / n 0H 30 R3 R4. - / 0 y x l · R 'R1 2 58 a a. in which Ri '»R21» R3 ‘and R4' represent, independently of each other, hydrogen or an optionally substituted acyl group, and 5 W, X, Y and Z represent, independently of each other, oxygen or an imino group, with the proviso that a) at least one of the symbols Ri ', R2 * "R31 and R4' represents an acyl group optionally above, and 10 b) when Z and X represent an imino group and W and Y represent oxygen, a) Ri 'and R2' do not simultaneously signify an (R) -3-hydroxytetradecanoyl group, provided that R3 'and R4 'are identical and represent a group (R) -3-hydroxy-15 tetradecanoyl or that R4' represents a group (R) -3- dodecanoyloxytetradecanoyl and R3 'a group (R) -3-tetra-decanoyloxytetradecanoyl, ß) R2 * is other than hydrogen or a group (R) -3-hydroxytétradécanoyl, provided that Rj 'and R3' represent hydrogen and R41 represents a group (R) -3-hydroxytétradécanoyl, and R3 'is other than a group - (C0) 2-CH2- (CHOH) 4-CH20H, provided that the other substituents represent hydrogen 7 and their salts. 3. A process for the preparation of the compounds of formula la specified in claim 1, characterized in that a compound of formula II is condensed enzymatically 30 - (formula II see next page) 59 A HO— / '° \ X ° H 1, HO- / \ _o.p '° H "5 Xj — i7 dH w ZII R5 r4 10 in which W and Z are as defined in claim 1, RJ and RJ have the meanings given in resale! Ction 1 for R3 and R ^ except 11 hydrogen and U represents uridine, 15 with a compound of formula III HO— \ - O 'OH (III) / Y / HO- / \ - O.Pv \ / £ OH? o --- 'o άΌ! IYYR ”R" 1 2 · 25 in which X and Y have the meanings given in claim 1, and ^ Ri "and R211 have the meanings given in claim 1 for Rj and R ^, with the condition that at least one of the two substituents represents an optionally substituted acyl group and that when X or 3q Y represents an imino group, the substituent Ri "or R2" linked to the imino group is other than hydrogen, and , if desired, the resulting compound is hydrolyzed to the corresponding compound of formula la in which, when X, Y, Z and / or W 60 represent oxygen, Ri, R2, R3 and / or R4 represent hydrogen, or “if desired, o n subjects the resulting compound to an acylamidase reaction *, which gives the compound of formula la in which, - 5 when X, Y, Z and / or W represent an imino group, Ri, R2, R3 and / or R4 represent hydrogen, and the compounds of formula la are recovered in free form or in the form of salts. 4. A pharmaceutical composition, characterized in that it comprises a compound of formula III HO— \ -O OH H0 \ / 0mh (ΙΠ) ις \ _ / t 0H 15. IT ï R »R« · 1 2 in which X and Y are as defined in claim 1 and 20 R! J and R £ have the meanings given in claim 1 for Rj and R2 with the condition that at at least one of the two substituents represents an optionally substituted acyl group and that when X or Y represents an imino group, the substituent R! or R £ linked to the imino group is other than hydrogen, in free form or in the form of a pharmaceutically acceptable salt, in association with a pharmaceutically acceptable carrier or diluent. 5. The compounds of formula III • · 61 / "HO-. / - /> "(III) t HO- / \ —O.P, \ _ / à oh t? _ R "R" 5 1 2 in which Rlj and R £ independently denote hydrogen or an optionally substituted acyl group and 10 a) Y represents oxygen and X represents an NH group with the condition that a) when Ri "represents a group (R) -3-hydroxytétraécanoyl, R2 "is other than a group (R) -3-hydroxytétradécanoyl ou (R) -3-hexadécanoyloxy-15 tétradécanoyl, ß) Ri" is other than hydrogen, and Ri "and R2" do not simultaneously mean an acetyl group or -CO (CHg) 10CH3, b) Y and X represent oxygen or 2q c) Y represents an NH group and X represents oxygen, with the condition that at least one of the two substituents Ri "and R2" represents an acyl group and that when X or Y represents an imino group, the substituent Ri "or R2" linked to the imino group is other than hydrogen, and their salts. 6. A process for the preparation of the compounds of formula III as specified in claim 4, characterized in that it comprises a) for the preparation of the compounds of formula IIla; * »62 HO— \ _0 OH ^ H ° - {/ - ^ OH 'IIIa); i- 1 · '* 5 IIR "R“ in which R1 "and R2" have the meanings given in claim 4 and either X' and Y 'represent oxygen, 10 or Y' represents an imino group and X 'represents l oxygen, the transformation of the free hydroxy group into a compound of formula IV RQ_ ^ __ O RO- / \ - OH 15 \ __ / i. ï (iv) Y 'X' II Rï R2 20 in which R1 "" R2 "> X1 and Y 'have the meanings given in claim 4, and R signifies a protective group, in a protected group in the form of phosphoric ester, or b) for the preparation of the compounds of formula IIIb HO - \ _0 0H I HO - / * \ _O.PX (111 b) \ __ / oS ° H 30. i O WH! 1 Rï Rj - ^ - 1 - Ί ι.ι ι 11 «- ~ '......' * · I X * 63 in which Ri "and R2" have the meanings given in claim 4,:! $ J acylation of the corresponding compounds of formula IIle; · t. * Ï :: Î "" 5 K0- \ (II le) K RO_ / \ -OP ^ \ / ^ XOR 'ii 10 HO 1JF R2! in which j 15 R2 "and R have the meanings given above, and / '' j R1 represents a protecting group, ~~~ ^ l and the subsequent elimination of the protecting groups, and the resulting compound is recovered in free form or in -i * salt form 20, 7. A compound of formula la as defined in claim 1 or a compound of formula III as defined in claim 4, in free form or in the form of a pharmaceutically acceptable salt, for use as a medicine;; t. 'j 8.- Products and processes in substance as described above with reference to the examples cited. j L j: Ί;' IK r I 1 '