AU596800B2 - Improvements in or relating to organic compounds - Google Patents

Description

r COMMONWEALTH OF AUSTRALIA PATENT ACT 1952 Q l COMPLETE SPECIFICATION (Original) FOR OFFICE USE Class Int. Class Application Number: 5 3 /3 Lodged: Complete Specification Lodged: Accepted: Published: Priority: S*-Related Art: 4 S 0 N o a 1 5 t Name of Applicant: Thi document contains the anendments made mudcr Scctin 49 arid is correct fur i Prining.

SANDOZ LTD.

Address of Applicant: Sii CH-4002 Basle,

SWITZERLAND.

o o 0 0 0**6 Actual Inventor(s): Ingolf MACHER Frank Michael UNGER Christian R. H. RAETZ a o Address for Service: "°Address for Service: DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete Specification for the invention entitled: "IMPROVEMENTS IN OR RELATING TO ORGANIC COMPOUNDS" The following statement is a full description of this invention, including the best method of performing it known to us ]A-900-9430 NEW SACCHARIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM The invention relates to saccharides, their preparation, pharmaceutical compositions containing them and their use as pharmaceuticals.

In particular the invention provides new compounds of formula I

HO-.

I I -0 OH w z H0-.0. 1 I (0O .3 4y x I

II

RR2

I

2 00 0 00 *0 S 000 5 0 0 0 0 0 0 0 0 S 09 0 0 0 0 5 00 0 08 8 0 5 0 000 S 5 990 0 8 9 0 5 08 9 00 0 0 0 5 0 wherein X, Y, W and Z are independently 0 or NH and R 1

R

2

R

3 and

R

4 are independently an acyl group of 14 carbon atoms, optionally monosubstituted in the 3 position by an OH or an acyloxy of 14 carbon atoms with the proviso that when X and Z are NH and W and Y are 0 then Ri to R 4 are not all (R)-3-hydroxytetradecanoyl; in free form or in form of an acid addition salt.

A second aspect of the invention provides a method of modulating antimicrobial resistance, of enhancing immune response and unspecific immunity, of preventing endotoxin shock, of treating malignant tumors or of treating inflammation which method comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula I in free form or in pharmaceutically acceptable acid addition salt form.

0 In a further aspect the inven'ion provides a process for oi 0 the preparation of compounds of formula I as defined above which comprises enzymatically condensing a compound of formula II -0o I HO- O H

OH

-O.P

OH

SI o w z R R" 3 4 3 wherein W and Z are as defined above, R3 and R4with the exception of hydrogen have the significance indicated above for R3and R4and U is uridine, with a compound of formula III

HO-.

0 OH H0. P

OH

Rio Rif 1 2

III

wherein X and Y are 0 or NH and R" and R" are independently an acyl of 14 carbon atoms, optionally monosubstituted in the 3 position by OH, or acyloxy of 14 carbon atoms with the proviso that 1) X and Y are not both NH; and e S C It C a S *t a C 5, 5*a S 5S S a S.

S S S S S

S

'Ct

C

t S S C S .5 S..

55 5 2) when X is NH and Y is O, then R" and R' are not both (R)-3-hydroxytetradecanoyl, and if desired, hydrolyzing the resulting product to a corresponding compound of formula I wherein, when X, Y, W and/or Z denote oxygen, then R 1

R

2

R

3 and/or R 4 denote hydrogen or, if desired, submitting a resulting product to an acylamidase reaction to obtain a compound of formula Ia wherein, when X, Y, W and/or Z denote imino, then R 1

R

2

R

3 and/or R 4 denote hydrogen, and recovering the compound of formula I in free form or in the form of an acid addition salt.

The enzymatic condensation may e.g. be effected in a buffered system, e.g. in tris-buffer at pH 7. The reaction may be effected at room or elevated temperature, e.g. at 30 C. An extract obtained from gramnegative bacteria, preferably from E. coli strains (Raetz, J.Biol.Chem. 259 [1984] 4852) may be used as the enzyme preparation. Strains which have been induced by genetic manipulation to overproduce the desired enzyme are preferred.

The optional hydrolysis may be effected in a manner analogous to methods described in the literature.

The acylamidase reaction may be effected in a manner analogous to that described by C.R.Verret et al. in J.Biol.Chem. 257 [1982] 10.222.

The products of the process of the invention may be isolated from the reaction mixture and if desired purified in accordance with known procedures.

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

C C 00 3 f t "C C001 15. ow6 th^6 0 e 1 u Il i i 900-9430 R1, R 2

R

3 and R 4 preferably are identical.

They may e.g. be substituted in the 3 position by a hydroxy, acetoxy or acyloxy group as defined above. The configuration at the carbon atom in the 3 position may be or Thus R 1

R

2

R

3 and R4 may be present in a compound of formula la in achiral, or racemic form. This also applies to compounds of formula II, III and IV. The configuration remains unchanged during the process of the invention, i.e. when a compound in or racemic form is used as starting material a corresponding or respectively, racemic end product is obtained.

Preferred are the compounds of formula 1 wherein R 1

R

2

R

3 and R 4 are an optionally substituted acyl group. Further preferred are the compounds of formula la wherein, when R 1

R

2

R

3 and/or R 4 are hydrogen, then X, Y, W and/or Z are oxygen.

Compounds of formula I preferably are obtained in the form of an acid addition salt. For the increase of the solubility in water preferably hydrophilic basic compounds are used, e.g. tris(hydroxymethyl)aminomethane or L-lysine.

Compounds of formula II may be obtained in known manner by reaction of a compound of formula III with activated a oJ t a I a 4 C 0 0 OS 0 0 0i S 0i 0 0 0 0 000 0 00 0 0 e *3 0 0 *0 0 *i a i «i 6 900-9430 Compounds of formula III may be obtained by a process comprising a) for the production of compounds of formula IlIa -0 Ia

OH

I I 1 2 wherein ROand ROare as defIined above and either X' and Y' are oxygen or Y' is iminv and X1 is oxygen, converting~ in compounds of formula IV

RO-.

I I RIO RIO 1 2 wherein R, 1 X' and Y' are as defined above and R is a protecting group, the unprotected hydroxy group to a protected phosphate group or 7 900-9430 b) for the production of compounds of formula IlIb

HO-.

OH

HO- O.P IIIb

OH

*I

I

0 NH I I R R 2 wherein and R are as defined above, acylating corresponding compounds of formula IIIc

RO-.

RO_

o wherein R" and R are as defined above and 0 0OR o -2 R' is a protecting group, o and thereafter splitting off the protecting groups.

Process variant a) may e.g. be effected by dissolving a compound of formula IV in an inert solvent, e.g. a cyclic ether such as tetrahydrofurane and reacting at lower temperature, e.g. at -700C, with butyllithium in an aliphatic hydrocarbon such as hexane, and thereafter adding dibenzylphosphorochloridate. The product may be recovered from the reaction mixture and if desired purified in known manner. The free hydroxy groups of the

I

i ii 8 900-9430 phosphate residue are protected, e.g. by benzyl. The splitting off of the protecting groups may also be effected in known manner. For example, a protecting group may be cleaved in conventional manner under acidic conditions, e.g. with an aqueous acid (ion-exchanger), or hydrogenolytically.

Process variant b) may e.g. be effected by dissolving a compound of formula IIIc in an inert solvent, e.g. in a chlorinated hydrocarbon such as methylene chloride, together with an acylating agent, appropriately with addition of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and Sletting the reaction take place at lower temperatures, e.g. at about 4 0

C.

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

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

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

HO-.

HO/ -OH 0 oo HO NH 2 H acylation HO-. \.o HO_/ \OH HO NHR2 sp- Mo 900-9430 RO" oH \0 HO NHR" 2 protection (pos. 4 6) protection 44 40 o 4 o 4 444444 4 0 04 00 4 0 4 0040 4 04 4 4 444 4 RO- 1.

\-O

H

TBDMS.O NHR" 2 RO-4_

OR'

RO' O/

R'

TBDMS1c 2qHR

RO-

OR'

HO H1 2li 0 04 4 4044 o oo o o 4 0 44 04 4 04 0 "4 40 0 4 00 0 TBDMS tert.-butyldimethylsilyl The compounds of formula IV may be obtained according to the following reaction schemes, whereby the hydroxy groups not participating in the reaction depicted may appropriately be in protected form. The substituents have the following meanings: R, R' protecting groups R5 both independently have the significance indicated above for RP, R 2

R

3 or R4 Ac acetyl TBDMS tert.-butyldimethiylsilyl 10 0 -900-9430 1. Prepart'aion of compounds of formula IVa: 02 N N 3 43 34 3 43443 4 4 44 44 4 4 4 4 3 34 3 4 44 434 4 4 444 4 II 4 3 1 4 44 4 34 4 4 4 4344 4.4 43 .4 444 44 4 4 44 o 04

HO-

controlle H0- .OH rd u ct ion O 2N NH2 HO. protection (pos. 2)

OH

0 2 N NHR HO0. '-yl reduction HO-! \OH H 2N NHR HO 4\ ac ylation H0-/ \OH

R

5 -HN NHR HO- deprotection (ros.

.0

\OH

HOHN 2 acylation HO-. \oH R 5 HN NHR 4 v protection (pos.

-O OH R 5HN IaNHR Ia (both R 5 different) d AcO \_o HcO! \-OHC HO- Y H- OH O 2N

NH

HO redcytion H0. prtcin(o.46 H0/ \-OH 5 /N NH NH bt 2 4+6) 11 900-9430 2. Preparation of compounds of formula IVb: 44 00 00 0 0 0 4 0~ 04 0 0 0 0 0 0000 0 0 00 0 4 0* V 4 1 o 0* 4 0 4004 '4 *14 0 044 94 4 4 04 0 04

HO-

HO- -OH *N 3% h RO 11 rotection (pos. 4+ RO- N 3 of RO prtetin(ps.1 i-0 N3YO 1.roection 1 2. aclto RO- \.-O.TBDMS

N

3 OH 1.roedction (ps 1 RHN. OR 5 RO..y eooecio (os 6) 12 900-9430 3. Preparation of compounds of formula IVc: AcO.

ACO/ ~OAc ACO OAc AcO VI protection (pos. 1) X-0 Ac 0. \OCH CC1 AcO~ /2 3 AcO CAc HO-. \deorotection (pos. 2, 3, 4, 6) HO-! \O-CH 2CCl HO QH 0 0 0 0 2-00 00 0 0 0 00 0 0 0 5 40 4 0 00 0 4 I ~0t 4 4 40 0 0 00~ 0 40 4 4~ 0 04 00 0 02004 0.0 o 44* 00 4 04 0 44 RO protec RO- 0. CH CCl /2 3 HO OH RO acyla 0. CH CCl 2 3 RO. R 50IR depro RO O R 5

_R

IVc: tion (pos. 4 6) tion tection (pos. 1) 13 900-9430 Insofar as the preparation of any particular starting material is not particularly described this is known or may be effected in conventional manner or in analogous manner to that described in the Examples.

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

C 0 0 0 r 111~

MMMM

14- 900-9430

C

66n 66 S 661P 66 6 66 6I Example 1: 6-O-[2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanolamido]-B-D-qlucopyranosyl]-2,3-di-O-[(R)-3-hydroxy tetradecanoyl]-1-O-phosphono-a-D-qlucopyranose 2.03 mg UDP-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamido]-1-O-phosphono-a-D-glucopyranose and 1.42 mg 2,3-di-O-[(R)-3-hydroxytetradecanoyl]-l-O-phosphono-a-D-glucopyranose both in the form of tris-salts are dissolved in 10 mM tris-buffer, pH 7 with 0.2 mM EDTA and 0.2 mM DTE and incubated with 60 lp1 enzyme extract at 300.

The enzyme extract may be obtained as follows: E. coli JB 1104 is cultured in L-broth (proculture 10 ig ampicilline/ml at 300 overnight; main culture at 30° in agitation incubator, starting from OD550nm 0.1 up to OD550nm The cells are collected by centrifugation at 10.800g (10 minutes/40), the pellet suspended in 10 mM tris-buffer, pH 7 0.2 mM EDTA and 0.2 mM DTE and again centrifuged (6000g, minutes/40). The pellet is then resuspended in above buffer and ultrasonically fragmented (5x20 seconds/30 seconds pause with 120 watts).

The cell fragments are separated by centrifugation (12.000g, 10 minutes/40) and the solution ultracentrifuged (150.000g, 90 The top 2/3 of the supernatant is recovered. This fraction is submitted to fractional ammonium sulfate precipitation. The range 10-40% ammonium sulfate is used. The resultant pellet is suspended in the above buffer.

After the reaction has ended (check with thin-layer chromatography) the solution is acidified to pH 2 with citric acid and centrifuged. The pellet is suspended in chloroform/methanol/acetic acid/water (65/15/5/2) and chromatographed over silicagel using the same eluent. The fractions containing the product are pooled and evaporated until no organic solvent is left and the remaining part lyophilized. The lyophilisate is dissolved in tetrahydrofurane/water and converted in that solvent into the tris salt over a cation exchanger in tris-form. The tetrahydrofurane ist evaporated and the residue lyophilized. All Rf values are determined on silicagel plates.

6 6 66 66 6-i, 6d 6 I x 15 900-9430 Rf (in chloroform/methanol/acetic acid/water 65/25/5/5) 0.48.

Example 2: 6-O-12-deoxy-3-0-[(R)-3-hydroxytetradecanoyll-2-(R)-3-hydroxy tetradecanoylamido]-0-D-qlucopyranosyl]-2-deoxy-3-O-[(S)-3-hydroxytetradecanoyl-2-[(S)-3-hydroxy tetradecanoylamido]-1-O-phosphono-a-D-qlucopyranose parts by volume of a 5 mM solution of 2-deoxy-3-O-[(S)-3-hydroxytetradecanoyl]-2-[l(S)-3-hydroxytetradecanoylamido]-1-O-phosphono- a-D-glucopyranose in 10 mM tris/H-Cl-buffer (pH 7) with 0.2 mM EDTA and 0.2 mM DTE, 20 parts of a 5 mM solution of UDP-2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamido]-1-O-phosphono-a-D-glucopyranose in the same buffer and 20 parts of a 100 mM tris/HCI-buffer pH 7) with 2 mM EDTA and 0.2 mM DTE are reacted with 40 parts of an enzyme preparation (prepared as described in Example 1) in 10 mM tris/HCI-buffer (pH 7) with 2 mM EDTA, 2 mM DTE and 0.1 Triton X-100 at 300 for 8 hours, whereby the final concentration of Triton X-100 is adjusted to 0.1 The reaction mixture is lyophilized, dissolved in about 1/3 of the initial volume in the eluent for the subsequent chromatography for separation according to molecular weight (Sephadex LH 20) (eluent: pyridine/acetic acid/water 98/1/1) and filtered. The volume of the column is about 20 times larger than the sample volume. The fractions with the pure product 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 pellet is dissolved into a mixture of chloroform/methanol filtered and the solvent removed under reduced pressure. For the obtention o of the mono-L-lysine salt the calculated stoichiometric amount of L-lysine (free base) is added in the form of an aqueous 100 mM solution to an aqueous suspension of the residue and the mixture again lyophilized.

Rf (chloroform/methanol/acetic acid/water 65/25/5/5) 0.44.

16 900-9430 Example 3: 6-0-[2-deoxy-3-0O4(R)-3-hydroxytetradecanoyll-2-[(R)-3-hydroxy te trade-canoy lam ido]-G-D -q luc opy ranosy -2dpox-3-0P[(R)-3-hydroxytetradecanoy 11-1 -0-phosphono-2-[(R)-3 -te tradpcanoy loxy te tradecanoy lam i do]-ac-D-glucopy ranose 1 I 04 $141 Is 4~ 0 0 I'll I 0 0 II I 01 0 0 I 5 o 00 0000 o 0e 00 0 00 0 000 04 4 o o I parts by volume of a 5 mjM solution of 2-deoxy-3-O-[(R)-3-hydroxy tetradecanoyl]-1 -C-phosphono-2-t(R)-3-te tradecanoy loxy te tradecanoy lam idol- a D-glucopyranose in 10 mM tris/HCI-buffer (pH 7) with 0.2 mM EDTA and 0.2 mM DTE, 20 parts of a 5 mM solution of UDP-2-deoxy-3-O-i(R)-3hydroxy tetradecanoy l]-2-[(R)-3-hydroxytetradecanoylamidol-1-0-phosphono-a-D-glucopyranose in the .1me buffer and 20 parts of a 100 mM tris/HCI-buFfer (pH 7) with 2 mM1 tDTA and 0.2 mM DTE are reacted with 40 parts of enzyme extract (prepared as described in Example 1) in 10 mM tris/HCI-buffer (pH 7) with 2 m-M EDTA, 2 mM DTE and 0.1 Triton X-100 at 300 for 48 hours, whereby the final concentration of Triton X-100 is adjusted to 0.1 The mixture is lyophilized, treated with pyridine and filtered, the filtrate concentrated under reduced pressure and chroMatographed over silicagel using chloroform/methianol/water/2phenethylpicolonium bromide 800/175/22.5/2.5 as an eluent. The fractions with the pure product are collected and the 2-phenethylpicolonium bromide serving as an ion pair forming agent is removed by agitating with 20 mM H 3 PO0 4 in water. The organic phase is concentrated under reduced pressure and for the obtention of the mono-L-lysine salt the calculated stoichiometric amount of L-lysine (free base) is added and the mixture lyophilized.

Rf (chloroform/methanol/acetic- acid/water 65/25/5/5) 0.53.

In a manner analogous to Examples 1 to 3 the following compounds of formula I are obtained whereby, depending on the compounds of formula I and III used, the amounts of enzyme are adjusted so that the reaction proceeds in preferably about 24 to 48 hours.

17- 7 -900-9430

TABLE

R= (R)-3-hydroxytetradecanoyl RI=R2=R Example No W

Z

4 0 NH NH NH 6 0 0 7 0 NH 8 NH NH 9 0 0

NH

NH

0

NH

0 0 X Rf NH 0.45 NH 0.40 NH 0.49 0 0.39 NH 0.41 o 0.48 Example 10: 6-04[2-deoxy-3-0((R)-3-hydroxytetradecanoyl-24( R)-3-hydroxytetradecanoylamidol-0-D-g lucopy ranosy l-2-deoxy-1 -0-phosphono-3-0tetradecanoyl-2-tetradecanoylam-ido- a-D-glucopyranose The title compound is obtained in a manner analogous to Examples 1 to 3.

Rf (chloroform/m-ethaicl/a-zeic acid/ivate. 65/725/5/5) 0.50.

Example 12: 6-04 2-deoxy-24((R)-3-hydroxytetradecanoylamido-0. D-gluco pyranosy 1 -1-0-phosphono-a~-D-g lucopyranose mg of 6-04[2-deoxy-3-O-L(R)-3-hydroxytetradecanoyl-2-(R)-J-hydroxytetradecanoy Iamidol-B-D-glucopyranosy l)-2,3-di-0-[(R)-3-hydroxytetradecaare dissolved In chloroform/methanol reacted with aqueous 25 ammonia (1/3 of the volume) and allowed to stand overnight. After the reaction is complete the mixture is e 9 4 I 'V 4 C CC t 9 4 C C it 4 44 4 1 4 *41 0 444 440 4* 0 tiki Q @0 4 9 4 0

II!

Q 2~

I

18 900-9430 evaporated to dryness, the residue triturated with diethyl ether in order to remove the fatty acids splitted off, the precipitate filtered off and washed with ether. The residue is dissolved in water and the di-tris salt prepared by addition of the calculated amount of tris. The product is lyophilized.

Rf (chloroform/methanol/acetic acid/water 25/15/2/4) 0.45.

Example 13: 2-deoxy-3-O-[(R)-3-hydroxytetradecanoyll-2-I(R)-3-hydroxytetradecanoy lam ido]-1 -0-phosphono- ot-D-q lucopy ranose a) 4,6-O-benzylidene-3-O-[(R)-3-benzyloxytetradecanoyl-2-I(R)-3-benzyloxy te tradecanoy lam ido]-2-deoxy-1-0-d ibenzylphosphono- a-D-q lucopyranose To a solution of 3.9 g 4,6-O-benzylidene-2-li(R)-3-benzyloxytetradecanoylamido]-2-deoxy-1-O--dibenzylphosphono-ct-D-glucopyranose, 2 g (R)-3-benzyloxytetradecanoic acid and 50 mg 4-dimethylaminopyridine in 20 ml of methylene chloride cooled to -101 are added 2 g of dicyclohexylcarbodiimide and the reaction mixture held at 40 overnight. The mixture is then filtered, the filtrate evaporated to dryness, the residue taken up in a small amount of toluene/ethyl acetate and chromatographed with the samne solvent. M.P. 96-981.

20 1 33 3c' (c 1, chloroform) Rf (toluene/ethyl acetate 2/1) boot.b) 2-deoxy-3-O-[(R)-3-hydroxytetradecanoyll-2-[(R)-3-hydroxytetradecanoylamidoi-1-O-phosphono- ct-D-glucopyranose 4.2 g 4,6-O-benzylidene-3-04(R)-3-benzyloxytetradecanoyl-2-I(R)-3-benzyloxytetradecanoylamido-2-deoxy-1--dibenzylphosphono-a-D-glucopyranose are dissolved in 900 ml of tetrahydrofurane/water (85/15) and hydrogenated with 1.5 g 10 palladium on charcoal for 2 hours at 10 bar and 4f00. The catalyst is then filtered off, the tetrahydrofurane evaporated and the aqueous suspension lyophilized.

[]20 =+28.50 (c 0.2, chloroform 1 drop methanol)

[]D

Rf (chloro form/me thanol/ace tic acid/water 125/75/10/20) 0.56.

j 19 900-9430 Further purification may be effected ?s follows: g 2-deoxy-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamido]-l-O-phosphono-a-D-glcopyranose and 1.7 g tris in 150 ml methanol are sonicated for 10 minutes at 450 in a sonicator bath. The suspension is centrifuged and the clear supernatant decanted from the pellet. 1.7 g tris (dissolved in 20 ml of methanol) are added to this solution, the solution seeded and crystallization allowed to occur at room temperature. The di-tris-salt of the title compound is obtained.

The mother liquor is evaporated to dryness. The residue and the pellet are dissolved together in a mixture of 300 ml chloroform, 600 ml methanol and 240 ml water (pyrogen-free). A further 300 ml of chloroform and 300 ml of 0.1 N hydrochloric acid are added, prudent agitation is effected and the Sphases allowed to separate. The chloroform phase is separated and evapo- Srated to dryness. The residue consists of unpure 2-deoxy-3-O-[(R)-3hydroxy tetradecanoyl]-2-[(R)-3-hydroxy tetradecanoylamido]-1-O-phosphonoa-D-glucopyranose.

The resultant product is tr3ated ultrasonically with tris and methanol as described above and centrifuged. The solution is chromatographed over a O 0 Sephadex LH 20 column with methanol as eluent. The fractions containing product are pooled and evaporated to dryness. The mono-tris-salt of the title compound is obtained.

5.1 g of this product are dissolved at 450 in 40 ml of methanol in the sonicator bath, a solution of 0.74 g tris in 10 ml of methanol is added, the 4 4 solution is seeded and crystallization allowed to occur first at room .o otemperature and then at Further amounts of crystalline di-tris-salt of the title compound are obtained. The residue from the mother liquor and the centrifugation pellet may be submitted to a further round of purification.

M.P. 183-185" (dec.) [a0 +150 (c 1.0, water).

-i-r 20 900-9430 0 0 0 4 0 000444 4 4 00 44 o 0 4 0 4 0044 0 4 4 44 4 0 44 4 0 1 004 0 ~'0 0 0 0 00 04 4 4404 04 0 440 4 44 Example 14: 3-deoxy-2-O-[(R)-3-hydroxytetradecanoyll-3-[(R)-3-hydr-oxytetradec anoy lam idol-1 -0-phosphono- ct-D -q lucopy ran ose a) 2-O-[(R)-3-benzyloxytetradecanoyl]-3-L(R)-3-benzyloxytetradecanoylamido]-.3-deoxy-1-O-dibenzylphosphono-4,6-0-isopropylidene-±-D-glucopyranose To a solution of 4.85 g 2-O-[(R)-3-benzyloxytetradecanoylj-34[(R)-3-benzyloxy te tradecanoylam ido]-3-deoxy-4,6-O-isop ropy lidene-D-g lucopy ranose in ml anhydrous tetrahydrofurane cooled to -701 ar, added 8 ml of 1.6 M butyilithium in hexane. After 5 minutes a solution of 3.8 g dibenzy lphosphoroch lor id ate in 10 ml benzene is added at the same temperature. Agitation is pursued at -700 for 10 minutes, 0.3 ml of acetic acid are added and the solution concentrated to 1/4 the initial volume. The solution is diluted with 200 ml of methylene chloride, extracted with 50 ml of water, 50 ml of dilute sodium hydrogen carbonate solution and 50 ml of sodium chloride solution, dried over sodium Sulfate and evaporated to dryness. The residue is chromatographed (toluene/ethyl acetate 7/3).

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

b) 3-deoxy-2-04[(R)-3-hydroxytetradecanoyll-3-1I(R)-3-hydroxytetradecanoylamidol-1-0-phosphono-a -D-glucopyranose A solution of 980 mg 2-O-[(R)-3-benzyloxytetradecanoyl]-34[(R)-3-benzyloxytetradecanoylamido]-3-deoxy-1-O-dibenzylphosphono-4,6-O-isopropylidene-c±-D-glucopyranose in 100 ml tetrahydrofurane is hydrogenated about 1 hour with 300 mg 10 palladium on charcoal under atmospheric pressure. Then the catalyst is filtered off, water (10 ml) and Dowex H are added and the mixture is stirred at room temperature until the isopropylidene group is completely cleaved. The ion exchanger is filteied off, the solution neutralised with tris and tetrahydrofurane and water evaporated under reduced pressure. The residue is dissolved in methanol and chromatographed over Sephadex LH Rf (chloroform/methanol/acetic acid/water 125/75/10/20) 0.65.

21 1 -900-9430 Example 15: 2,3-di-O-[(R)-3-hydroxytetradecanoyll--0-phosphono-ct-Dglucopyranose a) 4,6-O-be-nzylidene-2,3-di-D-[(R)--3-benzyloxytetradecanoyl]-l-O-dibenzylphosphono-ac-D-glucopyranose To a solution of 1 g 4,6-O-benzylidene-2,3-di-O-[(R)---benzyloxytetradecanoyll-D-glucopyranose in 10 ml of dry tetrahydrofurane cooled to -700 are added dropwise 0.9 ml of 1.6 M butyllithium in hexane. After 5 minutes at that temperature a solution of 420 mg dibenzylphosphorochloridate in 4 ml benzene is added dropwise. Stirring is continued for 10 minutes at 70, the solution is neutralised with acetic acid and evaporated to dryness. The residue is chromatographed (silicagel, hexane/toluene/ethyl acetate 4/4/1).

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

b) 2,3-di-O-[(R)-3-hydroxytetrade-canoyl-1-O-phosphono-c±-D-glucopyranose 230 mg 4,6-O-benzy lidene-2,3-.di-O-[(PR)-3-benzy loxytetradecanoyll-1-O-dibenzylphiosphono-a-D-glucopyranose are dissolved in tetrahydrofurane/water and hydrogenated for about 5 hours under atmospheric pressure with 80 mg of 10% palladium over charcoal. The catalyst is filtered off, the solution neutralised with tris and evaporated to dryness. The residue is chromatographed with methanol over Sephadex LH Rf (chlorof orm/me thanol/ace tic acid/water 125/75/10/20) 0.65.

Example 16: 2-deoxy-3-O-I(S)-3-hydroxytetradeCanoyll-2-I(S)-3-hydroxyteo tradecanoy lam a) 4,6-O-benzy Iid ene-3-O-[(S)-3 -b enzy lox y tetrad ecan oy -benzy-I oxytetradecanoylamido]-2-deoxy-1-O-dibenzylphosphono-ct-D-glucopyranose The title compound is obtained in a manner analogous to Example 13a).

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

22 900-9430 b) 2-deoxy-3-04[(S)-3-hydroxy tetradecanoy 1l-2-[(S)-3-hydroxy te tradeca noy lam idol-1-0-phosphono- a-D-q lucopyranose The title compound is obtained in a manner analogous to Example 13b).

M.P. 150-2001 (decomp.) Rf (methylene chloride/methanol/ammonia 1/1/1; lower phase) Example 17: 2-deoxy--3-O-[(R)-3-hydroxytetradeCanoyl-1-O-phosphono-2- [(R)-3-tetradecanoy loxy tetradecanoy 12M idol- ot-D-glucopyranose a) 4,6-0-benzy Iidene--3-0-[(R)-3 -b enz ylox yte tradpc anoy 1l-2-deoxy-1-O-di benzylphosphono-2-[(R)-3-te tradecanoy lox ytetradecanoy lam idol-L-D-glucopyranose The title compound is obtained in a Manner anaiogous to Example 13a).

M.P. 82-950 =+24.10 (c 1, chloroform)

~~D

Rf (toluene/ethyl acetate 2/1) 0.7.

b) 2-deoxy-3-0-[(R)-3-hydroxytetradecanoyll-1-O-phosphono-24[(R)-3-te- Ctradecanoy loxy te tradecanoy lamnidol- a-D-q lucopyranose C The title compound is obtained in a manner analogous to Example 21b).

+14.30 (c 1, tetrahydrofurane/pyridine) Rf (chlorof orm/methanolI/ace tic acid/water 80/25/5/5) 0.35.

Example 18: 2-deoxy-1-O-phosphono-3-O-tetradecanoyl-2-tetradecanoyl- C amido-ct-D-glucopyranose a) 4,6-O-benzylidene-2-deoxy-1-0-dibenzylphosphiono-30etradecanoyl-2 tetradecanoylam ido- ca-D-gluc opyranose The title compound is obtained in a manner analogous to Example 13a) using t~tradecanoic acid and with toluene/ethyl acetate as eluent.

23 900-9430 M.P. 123-1290 Rf (toluene/ethyl acetate 2/1) =0.6.

b) 2-deoxy -1 -0-phosphono -3-0-te tadecanoy 1-2 -te tradec anoylamido- cc-Dglucopyranose The titel compound is obtained in a manner analogous to Example 21b).

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

Example 19: 2-deoxy-2-[1(R)-3-hydroxytetradecanoylamidol-1-0-phosphono- 3-0-lj(R)-3-tetradecanoy loxy te tradecanoy a -D-glucopy ranose a) 4,6-0-b enzy lidene-24(R)-3 -benzy loxy te tradecano lam ido]-2-deoxy-1-0dibenzylphosphono-3-04-(R)-3-tetradecanoyloxytetradecanoyll-a-D-glucopyranose The title compound is obtained in a manner analogous to Example 13a), O using (R)-3-tetradecanoyloxytetradecanoic acid and with toluene/acetic ester aS solvent mixture.

0 0 M.P. 89-900.

0000 0 a] 0 +30.91 (c 1, chloroform/methanol 1/1).

Rf (toluene/ethyl acetate 4/1) b) 2-deoxy-2-[(R)-3-hydroxy tetradecanoy lam ido]-1-0-phosphono-3-04[(R)- 3-tetradecanoy loxy tetradecanoy a-D-rglucopyranose 0* The title compound is obtained in a manner analogous to Example 21 b).

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

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

Example 20: 2-deoxy-2-[(R)-3-hydrox ytetradecanoy lam ido]l-0-phosphonoc-D-qlucopyranose 24 4 -900-9430 a) 4,6 -0-b enzy Iidene-2-[j(R)-3 -benz ylox yte tradecan oy lam id]-2-deo5< y-1-0 The title compound is obtained analogous to Example 13a), using tetradeca noic acid and toluene/ethyl acetate as solvent mixture.

125.5-126.50.

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

b) 2-deoxy-2-li(R)-3-hy dro xyte tradecanoy lam ido]-1 -0-phosphono-3-O-te tradecanoyl- a-D-cilucopyranose 354 mg 4,6-O-benzy lidene-2 -benzy loxy te tradecanoy lam ido-2-deoxy- 1-O-dibenzylphosphono-3-O-tetradecanoyl-cz-D-glucopyranose are dissolved in 30 ml tetrahydrofurane/water and hydrogenated with 200 mg palladium on charcoal for 10 hours under atmospheric pressure. The catalyst is then filtered Off and the solution adjusted to pH 7.5 with tris(hydroxyme thylI)amninomethane. The tetrahydro furane is evaporated under reduced pressure and the aqueous solution lyophilised. The lyophilisate is triturated twice with ether and dried.

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

Example 21: 2-ace tam ido-2 -deoxy-3-O-[(R)-3-hydroxy te tradecanoy l-1-Ophshno-cz-D-g luco py ranose a) 2-ac e tam ido-4,6-O-benzy li den e-3.-O-[(R)-3-benzy loxy te tradecanoy 11-2deoxy-1-O-dibenzylphosphono-ac-D-glucopyranose A solution of 3.55 g 2-ace tam ido-4,6-O-benzy lidene-2-deoxy-1-O-dibenzylphosphono-ca-D-glucopyranose and 2.1 g (R)-3-benzyloxytetradecanoic acid in 15 ml methylene chloride is cooled to 00 and 1.32 g dicyclohexylcarbodiimide and p-dimethy lam inopyrid ine are added. After 2 hours at this temperature the reaction is complete. The mixture is filtered, evaporated to dryness and chromatographed over qilicaqeI first with methylene chloride/methanol (50/1) and then with toluene/etv I acetate v~' 4" '4 4 44 '4 C.

.2 L '40 25 900-9430 Rf (chloroform/methanol 20/1) =0.7.

b) 2-c tmd ex doyerdc ny-10 pop,,i a-D-g lucopyranose 2.08 g 2-ace tam ido-4,6-0-benzy lidene-3--[(R)-3-benzy lxyte trdecaloylI]- 2-deoxy-1-O-dibenzylphosphono- c-D-glucopyranose are dissolved in tetrahydrofurane/water and hydrogenated for 3 hours with 1 g 10 palladium on charcoal under atmospheric pressure. The catalyst is filtered off and the filtrate first evaporated and then lyophilised. The lyophilisate is dissolved in methanol, neutralised with tris(hydroxymethyl)aminomethane and chromatographed with methanol over Sephadex LH 43.31 (c 1, methanol) Rf (chloroform/methanol/acetic acid/water 125/75/10/20) 0.4.

o o 0 0 -a o PC 0 1 0 0 0 Example 22: 1-O-phosphono-2,3-di-O-[(R)-3-tetradecanoyloxytetradecanoyl]- a-D-glucopy ranose a) -e z ldn -iez lhsp a o23d- )3ttaea noy loxy te tradecanoyl]- a-D-glucopyranose The title compound is obtained analogous to Example 15a), under chromatographic purification over silicagel with ether/hexane as eluent.

Rf (ether/hexane 1/1) b) 1 -0-phosphono -2,3-di -te tr adecanoy lox ytetrad ec anoy I- (I-D-q lucopyranose 470 mg 4,--ezldn---iezlhopoo23d--()3tta decanoyloxytetradecanoyl-c-D-glucopyranose are dissolved in 47 ml tetrahydrofurane and hydrogenated 1 hour with 230 mg palladium on charcoal under atmospheric pressure. The catalyst is filtered off, the filtrate concentrated to dryness and the residue chromatographed over silicagel (chloroform/methanol/water/triethylamine 15/10/2/0.2). Fractions contai- 26 900-9430 ning product are pooled and evaporated to dryness. The title compound is obtained as di-triethylamine salt.

Rf (chlorof orm/rnethari,-l/ace tic acid! water 80/25/5/5) Example 23: 2-ex---()3hdoyeraeaol--()3hdoye tradecanoy lam id o]-1 -0-phosphono- ct -D-q luc op yranose a) 4,6-O-benzy lidene-3-OAl(R)-3-benzy lox yte tradecanoy -benzyloxytetradecanoylamido]-2-deoxy-1-0-dibenzylphosphono- CL-D-glucopyran-ose The compound is obtained in a manner analogous to Example 13a).

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

b) 2-deoxy--3-O-[(R)-3-hydroxytetradecanoyll-2-i(5)-3-hydroxytetradecanoyl- The compound is obtained in a manner analogous to Example 13b).

Rf (methylene chloride/methanol/ammonia I/1/1, lower phase) 6605 For further characterization of the compounds Fast Atom Bombardment O 0 (FAB) mass spectroscopy (negative mode) was performed (Raetz, J. Biol.

0 Chem. 259/4852 [19841).

Example No. Mass 1 1325 002 1324 0 663 1534 0 064 1323 1322 6 1325 7 1324 8 1323 9 1326 1292 11 1140 12 647 27 7 -900-9430 Example No: NMR-Spectra 1 9.82 9 Hz, 1H); 6.65 (dd, 8,8 a. 3,5 Hz, 1H); 6.25 (C 5 DN) Hz, lH); 5.83 10 Hz-, 1H); 5.63 8,8 Hz, 1H); 5.35 (dd, a. 2,5 Hz, 1H); 4.0 10 Hz, 1H); 3.83 (m 1H).

13 5.49 (dd, 3,5 a. 7,5 Hz, 1H); 5.24 (dd, 9,5 a. 10,5 Hz, 1H); (CD 3 OD) 4,17 (ddd, 2,5, 3,5 a. 10,5 Hz, 1H); 4.0 (in, 3H); 3.90 (dd, 2 a.

12 Hz, 1H); 3.75 (dd, 5,5 a. 12 Hz, 1H); 3.71 This); 3,63 (t, Hz, 1H).

14 5.77 (dd, 3,5 a. 7,5 Hz, 1H); 4.77 (ddd, 2,5, 3,5 a. 10,5 Hz, (CD1 4.38 (dd, 9,5 a. 10,5 Hz); 3.8 to 4.1 (mn, 4H); 3.73 (dd, CD..O 3j 5,5 a. 12 Hz, 1H); 3.53 10 Hz, 1H).

5.71 (dd, 3,5 a. 7,5 Hz, 1H); 5.48 10 Hz, 1H); 4.77 (ddd, 2, (CD 3 OD) 4 a. 10 Hz, 1H); 3.9 to 4.05 (mn, 3H); 3.86 (dd, 2 a. 12 Hz, 1H); 3.7 (dd, 5,5 a. 12 Hz, 1H); 3.67 Tris); 3.58 10 Hz, 1H).

o 16 5.58 (dd, .3 a. 7 Hz, IH); 5.22 10 Hz, 1H); 4.19 (dt, 3 a.

(CDCl 10 Hz, 1H); 3.8 to 4.1 (in, 4H); 3.7 This a. dd, 1H); 3.46 (t, CD 3 0 10 Hz, 1H).

17 5.46 (dd, 3 a. 7 Hz, 1H); 5.22 10 Hz, 1H); 5.17 (in, 1H); (CDC1 3 4.19 (dt, 3 a. 10 Hz, 1H); 3.9 to 4.1 (in, 3H), 3.7 Tris); CD 3 0 3.65 (dd, 1H); 3.51 10 Hz, 1H).

18/step a) 7.38 (mn, 15H); 5.70 (dd, 3,5 a. 6 Hz, 1H); 5.63 9,5 Hz, (protected) 1H); 5.50 1H); 5.26 10 Hz, 1H); 5.08 (in, 4H); 4.38 (in, (CIJC1 3 1H); 4.08 (dd, 5 a. 10 Hz, 1H); 3.95 (dt, 5 a. 10 Hz, 1H); 3.73 10 Hz, 1H); 3.69 10 Hz, 1H); 2.29 (mn, 2H); 1.89 (in, 2H).

2B 900-9430 04 00 0 0 0 0 o 0 000000 4 0 00 00 0 4 4 0 0 04t0 0I 4a.

41.

I

0 00 00 0 0000 0 000 00 0 0 00 19/step a) (protected) (CDC1 3 a) (protected) (CD1 3) 21/step a) (protected) (CD 3

OD)

22/step a) (protected) (enDC 3 23/step a) (protected) (CDC1 3 7.2 to 7.45 20H); 6.34 9 Hz, 1H); 5.77 (dd, 3,5 a. 6 Hz, 1H); 5.49 1H); 5.29 10 Hz, 1H); 5.14 (quintet, 6 Hz, 1H); 5.0 (in, 4H); 4.51 a. 4.41 (AB, 12 Hz, 2H); 4.41 (mn, 1H); 4.08 (dd, 5 a. 10 Hz, 1H); 3.95 (dt, 5 a. 10 Hz, 1H); 3.7 (in, 3H); 2.58 a. 2.47 (ABX, 5,5, 7,5 a. 15,5 Hz, 2H).

7.2 to 7.45 (in, 20H); 6.29 9 Hz, IH); 5.74 (dd, 3,5 a. 6 Hz, 1H); 5.49 1H); 5.30 10 Hz, 1H); 5.0 (in, 4H); 4.52 a.

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

5.47 (dd, 3,5 a. 7,5 Hz, 1H); 5.23 (dd, 9,5 a. 10,5 Hz, 1H); 4.18 (ddd, 2,5, 3,5 a. 10,5 Hz, 1H); 3.90 (in, 3H); 3.85 (dd, 2 a. 12 Hz, 1H); 3.72 (dd, 5,5 a. 12 Hz, 1H); 3.68 Tris); 3.61 10 Hz, 1H); 1.94 3H).

7.3 to 7.45 (in, 15H); 5.92 (dd, 3,5 a. 6,7 Hz, 1H); 5.60 a. 10 Hz, 1H); 5.48 1H); 5.02 to 5.22 (mn, 6H); 4.95 (ddd, 2,5, 3,5 a. 9,5 Hz, 1H); 4.10 (dd, 4,5 a. 10 Hz, 1H); 3.96 (ddd, 5, 9 a. 10 Hz, 1H); 3.67 9,5 Hz, 2H); 2.60 (in, 2H); 2.38 (d, Hz, 2H); 2.24 7,5 Hz, 2H); 2.12 7,5 Hz, 2H).

7.2 to 7.42 (in, 25H); 6.49 9 Hz, 1H); 5.77 (dd, 3,5 a. 6 Hz, 1H); 5.45 1H); 5.35 10 Hz, 1H); 5.0 (in, 4H); 4.54 a.

4.40 (AB, 11 Hz, 2H); 4.47 a. 4.36 (AB3, 11 Hz, 2H); 4.45 (in, 1H); 3.9 to 4.1 (in, 2H); 3.6 to 3.8 (in, 4H); 2.58 (dd, 6,5 a.

15,5 Hz, IH); 2.34 (dd, 6 a. 15,5 Hz, 1H); 2.2 (in, 2H).

The compounds used as starting materials may be obtained as follows: A) 4;6-0-b enzy li dpne--[4(R)-3 -ben zy loxy te tradec ano ylam ido]-2-deo xy -1-Odibenzylphosphono-ct-D-glucopyranose (for Examples 13, 19 and a) 2-[(R)-3-benzy lox yte tradecanoy lam ido-2-deox y-D-q lucopyranose 29 900-9430 A mixture of 5.4 g D-glucosamine-hydrochloride, 10.8 g N-[(R)-3-benzyloxytetradecanoyloxy]succinimide and 5 ml diisopropylethylamine in 25 ml dry dimethylformamide is stirred 24 hours at room temperature. The bulk of the solvent is distilled off and the residue dissolved in a mixture of 150 ml chloroform, 300 ml methanol and 120 ml water. After addition of a further portion of 150 ml chloroform and 150 ml water the lower phase is separated and washed twice with the upper phase of 100 ml chloroform, 100 ml methanol and 90 ml water. The solution is evaporated to dryness and dried under high vacuum. The resultant product ist used in the next step without further purification.

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

M.P. 150-158°.

S[c] 2 +530 (c 1, dimethylformamide) Rf (chloroform/methanol 9/1) 0.3.

b) 4,6-O-benzylidene-2-[(R)-3-benzyloxytetradecanoylamido]-2-deoxy-Dglucopyranose *0,t A solution of 5.83 g 2-[(R)-3-benzyloxytetradecanoylamido]-2-deoxy-D-glucopyranose, 3 g benzaldehydedimethylacetal and 500 mg p-toluene-sulfonic acid-monohydrate in 200 ml of anhydrous dimethylformamide is kept for about 3 hours at 55-600 and 30-40 mbar on a rotary evaporator. Thereafter no starting material is remaining. The major part of the dimethylformamide is distilled off, 500 ml of methylene chloride are added S°and the solution is extracted twice with 200 ml of diluted sodium hydrogen carbonate solution and 200 ml of water. The solution is dried with sodium sulfate, evaporated to dryness and the residue chromatographed (toluene/ethyl acetate 6/4).

M.P. 162-1650.

2D 5.50 (c 1, chloroform) Rf (toluene/ethyl acetate 1/1) 0.2.

30 900-9430 0* 4* 0 0 0 0*~ o 0* 0 4 *00 t o o~ 4 o 0 o oo 4 ~o 0 0 0 0 0 00 0 *0 c) 4 ,6-0-benzylidene-2-[(R)-3-benzyloxytetradecanaylamidol-2-deoxy-1-Cdibe-nzylphosphono-ct-D-glucopyranose To a solution of 4.86 g 4,6-O-benzylidene-2.-l(R)-3-benzyloxytetradecanoylamidol-2--deoxy-D-glucopyranose in 40 ml Of anhydrous tetrahydrofurane cooled to -7011 are added dropwise 8 ml of 1.6 M butyllithium in hexane.

After 5 minutes at that temperature a solution of 3.3 g dibenzylphosphorochioridate in 10 ml of benzene ist added dropwise. After further stirring for 10 minutes at -70' 0.3 ml of acetic acid are added and the solution concentrated to 1/4 of its initial volume. The solution is diluted with 200 ml of methylene chloride, extracted with 50 ml of water, 50 ml of diluted sodium hydrogen carbonate solution and 50 ml of sodium chloride solution, dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (toluene/ethyl acetate 7/3).

M.P. 102-105".

[a]20D 31.70 (c chloroform) Rf (toluene/ethyl acetate 1/1) 0.32.

B) 4,6-O-benzylidene-2,3-di-O-(R)-3-benzyloxytetrad-canoyl-D-glucopyranose (for Exeample a) trichloroethyl 2,3,4,6-tetra--acetyl--D-glucopyranoside To an ice-cooled solution of 2.34 g pen ta-O--ace tyl-D-g lucopyranose in 9 ml trichloroethanol is added 0.9 ml of borontrifluoride etherate and the solution is maintained 4 hours at that temperature. Then the solution is poured onto 100 ml of ice-water, extracted with 100 ml of methylene chloride, the organic phase is separated, washed with 50 ml of sodium hydrogen carbonate solution and 50 ml of water, dried over sodium sulfate and evaporated to dryness. After chromatographic purification (silicagel, toluene/ethyl acetate 4/1) the product crystallises from ethyl acetate/petroleumn ether M.P. 138-1400.

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

U_

I 31 900-9430 b) trichloroethyl 4,6-O-benzylidene-B-D-qlucopyranoside 7.13 g trichloroethyl 2,3,4,6-tetra-O-acetyl-B-D-glucopyranoside are dissolved in a mixture of methanol and methylene chloride, the solution is cooled with ice and reacted with a solution of sodium methanolate (60 mg sodium in 26 ml of methanol). After 2 hours at 0° the solution is neutralized with Dowex AG 50 WX8 H the ion-exchanger is filtered off, and the solvent removed under reduced pressure. The residue is dissolved in 60 ml of benzaldehyde, 4.1 g zinc chloride are added and the mixture is stirred for 10 hours at room temperature. The mixture is then poured onto icewater, extracted with ether, the ether solution is dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (silicagel, toluene/ethyl acetate 4/1).

Rf (chloroform/methanol 9/1) 0.60.

c) trichloroethyl 4,6-O-benzylidene-2,3,-di-O-[(R)-3-benzyloxytetradecanoyl]-0-D-qlucopyranoside To a solution of 800 mg trichloroethyl 4,6-O-benzylidene-B-D-glucopyranoside, 1.4 g (R)-3-benzyloxytetradecanoic acid and 20 mg dimethylamino- 0 opyridine in 10 ml of methylene chloride cooled to 00 are added 930 mg Sdicyclohexylcarbodiimide and the reaction mixture kept overnight at U The reaction mixture is then filtered, the filtrate evaporated to dryness, the residue dissolved in hexane/toluene/ethyl acetate (12/5/1) and chromatographed with the same solvent mixture.

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

d) 4,6-0-benzylidene-2,3-di-O-[(R)-3-benzyloxytetradecanoyl]-D-qlucopyranose 480 mg trichloroethyl 4,6-O-benzylidene-2,3-di-O-[(R)-3-benzyloxytetradecanoyl]-B-D-glucopyranoside are dissolved in 50 ml of a mixture of dioxane and acetic acid and reacted in portions at room temperature with zinc powder until all the starting material has been used up. The reaction 32 900-9430 mixture is filtered, evaporated to dryness and the residue chromatographed (silicagel, hexane/toluene/ethyl acetate 2/5/1).

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

C) 2-0-[(R)-3-benzyloxytetradecanoyU-3-(R)-3-benzyloxytetradecanO l amido]..3-deoxy-4,6--O-isopropylidene-D-glucopyranose (for Example 14) a) 3-azido-3-deoxy-4,6-0-isop ropy Ii dene-D-g lucopyranose 1.02 g 3-azido-3-deoxy-D-glucopyranose are dissolved in 10 ml of dry dimethylformamide, 940 p.1 of 2-methoxypropene and a catalytic amount of p-toluenesulfonic acid-monohydrate are added and the solution is kept at room temperature for about 2 hours. The p-toluenesulfonic acid is neutralised with sodium hydrogen carbonate, the solution evaporated to dryness and the residue chromatographed (silicagel, chloroform/methanol 9/1).

Rf (chloroform/mE-thanol 9/1) 0.64.

b) tert.-butyldimethylsilyl 3-azido-3-deoxy-4,6-0-isopropylidene-0-D-glucopyranoside 4 9 4To a solution of 560 mg 3-azido-3-deoxy-4,6-0-isopropylidene-D-glucapyranose and 310 mg imnidazole in 25 ml of dry methylene chloride are added 345 mg tert.-butyldimethylsilyl chloride and the mixture is stirred for 2 hours at room temperature. The excess imidazole chloride is filtered off and the filtrate is shaken twice with 10 ml of water, dried over sodium sulfate and evaporated to dryness. The residue is chromatographed (silicagel, toluene/ehtyl acetate 12/1).

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

C) tert.-butyldimethy Isilyl 2-O-L(R)-3-benzy ox ytetra decano y 1-34t(R)-3b enzy lox y te tradecano ylam ido]-3-deoxy-4,6-- isopropy i dene--D -luc opyranoside A solution of 3.1 g tert.-butyldimethylsilyl 3-azido-3-deoxy-4,6-O-isopro- 33 900-9430 pylidene-B-D-glucopyr~noside in 100 m-l of methanol is hydrogenated for 2 hours with 300 mg 10 palladium on charcoal. The catalyst is filtered off and the filtrate evaporated to dryness. The residue is dissolved with 5.35 g (R)-3-benzyloxytetradecanoic acid and 100 ml of dimethylaminopyridine in 50 ml of dry methylene chloride, the solution is cooled with ice and 4.1 g dicyclohexylcarbodiimide are added. After about 3 hours at room temperature the solution is filtered, the solvent evaporated and the residue chromatographed (silicagel, toluene/ethyl acetate 9/1).

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

d) -benzy loxy tetradecanoy 1-3 -ben zy loxy te tradec anoy Iamido]-3-deoxy-4,6-0-isoprqaylidene-D-jlucopyranose To a solution of 5 g tert.-butyldimethylsilyl 2-0 -[(R)-3-benzy loxy te tradecanoyl1j-3 -L(R)-3-benzy loxy tetradecano ylam idoj-3 deo xy-4,6-0-isopropy liclene-1-D-glucopyranoside in 100 ml of anhydrous tetrahydrofurane cooled to -600 are added dropwise 5.2 ml Of a solution of tetrabutylammonium fluoride in tetrahydrofurane. This solution is allowed to come to -401 and kept at this temperature until starting material is no longer present (about minutes). 5 ml of methanol are then added, the temperature is brought to room temperature and the solvent evaporated. The residue is extracted with a mixture of methylene chloride and water, the organic phase dried over sodium sulfate and the solvent evaporated. After chromatographic puriicaion(silicagel, toluene/ethyl acetate 3/1) the title compound is obtained as a mixture of anomeres.

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

D) 4,6-0-benzylidene-2-1(S)-3-berizyloxytetradecanoyli-2-de-oxy-1-O-dibenzylphosphono-ct-D-glucopyranose (for Examole 16) a) 2-[(S)-3-benzyloxy te tradecanoy lam ido]-2-deoxy-D-glucopyranose The title compound is obtained in a manner analogous to Example Aa).

Rf (chroroform/methanol 7/1) 0.37.

34 4 -900-9430 b) 4,6-0-benzylidene-2-[(S)-3-benzy loxy te tradecanoy lam idol-2-deox y-Dglucopy ranose The title compound is obtained in a manner analogous to Example Ab).

20 -3.50 (c 1, chloroform) Rf (toluene/ethyl acetate 1/1) 0.37.

c) 4,6-0-benzylidene-2-4(S)-3-benzyloxytetradecanoylamido]-2-deoxy-1-0dibe-nzylphosphono- a-D-glucopy ranose The title compound is obtained in a manner analogous to Example Ac).

LaD =+39.20 (c chloroform) Rf (toluene/ethyl acetate 1/1) 0.46.

E) 4,6-0-benzy lidene-2-deoxy-1-0-dibenzyIphosphono-2-[(R)-3-tetradecanoyloxyte-tradecanoylamido]-a-D-glucopyranose (for Example 17) a) 2-deoxy-2-[(R)-3-t-tetradecanoyloxytetradecanoyl2Mido]-D-glucose A mixture of 650 mg D-glucosamine-hydrochloride, 1,9 g N-[(R)-3-tetradecanoyloxytetradecanoyloxylsuccinimide and 0.5 ml diisopropylethylamine in ml dim-ethylformamide is agitated for 20 hours at room temperature.

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

+2-50 (c 1,methanol) [aD Rf (ethyl acetate/methanol 6/1) b) 4,6-0-benzyIi dene-2-deo xy-2 -te tradecanoy loxy tet radecanoy Ia rnidoII-D-q lucopy ranose A solution of 5.83 g 2-deo xy-2-L(R)-3-tetradecanoy loxy te tradec anoy lam ido]-D-glucose, 3 g benzaldehydedimethylacetal and 500 mg p-toluenesulfonic acid-monohydrate in 200 ml of dry dime thyIf ormamide is kept on a rotary *evaporator for 3 hours at 55-600 and 30-40 mbar. After this time 900-9430 starting material is no longer present. Most of the dimethylformamide is then distilled off, 500 ml of methylene chloride are added and the solution extracted twice with 200 ml of dilute sodium hydrogen carbonate solution and 200 ml of water. After drying over sodiurn sulfate and evaporation to dryness the residue is chrornatographed (toluene/ethyl acetate 6/4).

M.P. 162-1650.

Lai 0 (c 1, chloroform/methanol 1/1) Rf (toluene/ethyl acetate 1/1) 0.32.

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

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

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

La]D +19.3" (c 1, chloroform) Rf (toluene/ethyl acetate 0.6.

F) 4,6-O-benzylidene-2-deoxy-1-O-dibenzylphosphono-2-tetradecanoylamido-ca-D-qlucopyranose (for Example 18) 0 0 0 00 alp a) 2-deoxy-2-tetradecanoylamido-D-qlucose A mixture of 3 g D-glucosamine-hydrochloride, 4.56 g N-tetradecanoyloxysuccinimide and 2.8 ml of diisopropylethylamrnine in 40 ml of dry dimethylformamide is kept at room temperature for 24 hours. The product is then filtered off, washed with dimethylformamide and water, dried in vacuo and used for the next step without further purification.

36 900-9430 b) 4,6-O-benzylidene-2-deoxy-2-tetradecanoylamido-D-qlucose 4 g 2-deoxy-2-tetradecanoylamido-D-glucose are dissolved at 550 in 320 ml of dried dimethylformamide, 2.6 ml dimethoxytoluene and 400 mg p-toluene sulfonic acid-monohydrate are added and the mixture is kept on the rotary evaporator for 4 hours at 55-60° and 30-40 mbar. Then the solvent is distilled off, the residue washed with dilute sodium hydrogen carbonate solution, water and ethanol, dried in vacuo and directly used in the next step.

c) 4,6-Q-benzylidene-3-0-(tert.-butyldimethylsilyl)-2-deoxy-2-tetradecanoylamido-D-qlucopyranose 6 2.2 g 4,6-O-benzylidene-2-deoxy-2-tetradecanoylamido-D-glucose are dis- Ssolved at 600 in 250 ml of dry dimethylformamide, 960 mg imidazole and 2.13 g tert.-butyldimethylchlorosilane are added and the reaction kept 6, 24 hours at that temperature. After addition of 480 mg imidazole and 1 g 6 tert.-butyldimethylchlorosilane and 24 hours more at 600 the starting material is no longer present. The solvent is distilled off, the residue extracted with methylene chloride/water, the orranic phase washed once with water, dried over sodium sulfate, and distiled off. The residue is oo° chromatographed over silicagel with a gradient of toluene/ethyl acetate (20/1 to Two main fractions are obtained: the title compound (650 mg) and the corresponding 1,3-bis-silyl compound (1.82 The bis-silyl compound is dissolved in 25 ml of dry tetrahydrofurane, the solution cooled to -400 and 2.5 ml of a IN solution of tetrabutylammonium fluoride in 6 6 tetrahydrofurane is added dropwise. After about 1 hour at -400 the 04, 6 o 66 cleavage of the anomeric silyl protecting group is complete. 3 ml of methanol are added, the temperature is brought to room temperature and the solvent then removed in vacuo. The residue is extracted with methy- .lene chltide/water, the organic phase extracted once with water, then dried and distilled off. A further amount of 1.52 g of title compound is obtained in the form of an anomeric mixture.

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

37 900-9430 d) 4,6-0-benzylidene-3-0-(tert.-butyldime-thylsily)-2-deoxy-1-0-dibpnzylphosphono-2-te tradecanoy lam ido- a -D-q lucopy ranose To a solution of 2.46 g 4, 6-0-b enzylIidene--3-O tyId irnpthylIsi lyl)-2deoxy-2-tetradecanoylamido-D-glucopyranose- in 100 ml of dry tetrahydrofurane coolpd to -60' are added dropwise 3.12 ml of 1.6 M butyllithium in hexane. After 5 minutes at this temperature a solution of dibenzylphosphorochioridate in benzene is added dropwise. Agitation is maintained further at -601 for 5 minutes, then the solution is neutralised with acetic acid and evaporated to dr iness. The residue is taken up in methylene chloride and extracted with water. A\fter working up and chromatography over silicagel using toluene/ethyl acetate as an eluent the title compound is o btaind.

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

e) 4,6-0-benzylide-ne-2--deoxy-1-O-dibenzylphosphono-2-tetradecanoylamido- a-D-cilucopyranose To a solution of 2.05 g 4,6-O-benzylidene-3-O-(tert.-butyldimethylsilyl)-2deoxy -1-0-d ibenzy lphosphono-2-te tradecanoy lam ido- a -D-g lucopy ranose in ml of dry tetrahydrofurane at -101 are added dropwise 2.5 ml of a IN solution of tetrabutylammonium fluoride in tetrahydrofurane and agitation maintained for 2 hours at 01. Then 3 ml of methanol are added, the solution is evaporated to dryness and the residue extracted with methylene chloride/water. After working up as usual and chromatography over silicaget using toluene/ethyl acetate as an eluent the title compound is obtained.

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

C) 2-acE-tamido-4,6-O-benzylidene-2-deoxy-1-O-dibenzylphosphono-ct-Dglucopyranose (for Example 21) a) 2-acetamido-4,6-O-benzylidene-3-O-(tert.-butyldimethylsilyl)-2-deoxy- D-glucbpy ranose 38 900-9430 A solution of 5.17 g 2-acetamido-4,6-0-benzylidene-2-deoxy-D-glucopyranose, 3.4 g imidazole and 6.3 g tert.-butyldimethylchlorosilane in 300 ml of dimethylformamide is kept at 60-70°. After 6 hours 500 mg imidazole and 1 g tert.-butyldimethylchlorosilane are added and the mixture is kept at that temperature until the reaction is complete. The solvent is evaporated off, the residue extracted with methylene chloride/water and purified by chromatography with toluene/ethyl acetate For the cleavage of the anomeric tert.-butyldimethylsilyl group the product is dissolved in 600 ml of dry tetrahydrofurane, the solution is cooled to -400 and 14 ml of a 1M tetrabutylammonium fluoride solution added. After 10 minutes the reaction is stopped by addition of 17 ml of methanol. The mixture is evaporated to dryness, the residue taken up in methylene chloride and extracted with water. The organic phase is dried and the solvent distilled off.

Rf (chloroform/methanol 9/1) b) 2-acetamido-4,6-O-benzy lidene-3-O-(tert.-buty ldimethy lsilyl)-2-deoxy- 1-O-dibenzylphosphono-a-D-glucopyranose The title compound is obtained in a manner analogous to Example Ec) and after chromatography with toluene/ethyl acetate as an eluent.

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

c) 2-acetamido-4,6-0-benzylidene-2-deoxy-1-0-dibenzylphosphono-a-Dqlucopyranose Io To a solution of 4.4 g 2-acetamido-4,6-0-benzylidene-3-O-(tert.-butyldimethylsilyl)-2-deoxy-l-O-dibenzylphosphono-a-D-glucopyranose in 200 ml S° of dry tetrahydrofurane cooled with ice are added dropwise 6.5 ml of a IN solution of tetrabutylammonium fluoride in tetrahydrofurane and the mixture is kept for 2 hours at Then methanol (7 ml) is added and the solution 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.

39 900-9430 H) 4,6-O-benzylidene-2,3-di-O-G(R)-3-tetradecanoyloxytetradecanoyll-Dglucopyranose (for Example 22) a) trichloroethyl 4,6-O-benzylidene-2,3-di-O-[(R)-3-tetradecanoyloxytetradecanoyl]-0-D-lucopyranoside The title compound is obtained analogous to Example Bc) using tetradecanoyloxytetradecanoic acid as acylating agent and toluene/ethyl acetate (98/2) as an eluent.

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

b) 4,6-O-benzylidene-2,3-di-O-[(R)-3-tetradecanoyloxytetradecanoyl]-D- S" I glucopyranose 0 0i~ The title compound is obtained analogous to Example Bd) after chromatographic purification over silicagel using toluene/ethyl acetate (15/1) as an 0 eluent.

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

I) synthesis of uridinephosphate-2,3-diacyl-hexoses 0 io0 0 00 1.4 g uridine-5'-phosphoromorpholidate N,N'-dicyclohexylcarboxamidine 0, salt are dissolved in 25 ml of anhydrous pyridine and dried twice by evaporation and redissolution in 25 mi of dry pyridine. To this solution is added a solution of 1 g of a 2,3-diacyl I-hexose-1-phosphate in 25 ml of pyridine which has been pretreated in the same way and the mixture is kept for 24 hours at 370, then cooled with ice. Then 50 ml of chloroform and 12 ml of 90 formic acid are added. This solution is chromatographed over silicagel and unreacted starting material is eluted with formic acid (30/30/7). Pyridine is removed from the column with chloroform/methanol and then the uridinephosphatederivative is eluted with chloroform/methanol/water (66/33/4). Chloroform and methanol are evaporated from the peak fraction in vacuo and the remaining aqueous suspension filtered. The precipitate is dissolved in 40 900-9430 100 ml of tetrahydrofurane/water and the solution agitated for 2 hours with Dowex AG50WX8 (in tris[hydroxymethyl]aminomethane form).

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

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

The compounds of formula Ia and III are useful because they possess pharmacological activity. They exert a benificial influence on unspecific antimicrobial resistance. This activity may e.g. be shown in following test methods: 1. Determination of endotoxic activity in the limulus-amoebocytes lysate test Endotoxin catalyses the activation of a proenzyme in limulus-amoebocytes SOO lysate. The releasing of p-nitroaniline from a colourless substrate is a measured. The extent of this releasing is detected photometrically, whereby the correlation between absorption and endotoxin concentration (or, O respectively, endotoxin activity for analogs) in the range from 0.01 to 0.1 ng/ml endotoxin is linear (comparison with the absorption values of a standard endotoxin). From each sample (dissolved in pyrogen-free sterile S* doubly distilled water) a dilution series 1:10 is prepared. In addition, to So 100 i.l of test sample, reference standard or blank sample, 100 pil of limulus ramoebocvtes lysate are added. After 10 minutes of incubation at 37 0 C the 200 jil of substrate are added to the reaction mixture. The reaction is stopped with 200 lil 50% acetic acid after a further 3 minutes of incubation. After agitation of the sample the absorption is measured in a spectrophotmeter at 405 nm, substracting the background value. The endotoxin contents (the endotoxin activity) of the sample in endotoxin units ,a 41 900-9430 is computed by linear regression with the values of the standard endotoxin.

2. Induction of endotoxin shock in the mouse The test comprises the induction of an endotoxin shock or an analogous lethal clinical condition with LPS-analogous substances in galactosamin (GalN)-sensibilisized mice. Male C57 bl.-mice (6 animals per group) are given simultaneously i.p. 8 mg GaIN dissolved in 0.5 ml PBS, and 0.1 [tg LPS from Salmonella abortus equi (Sigma) dissolved in 0.2 ml of physiological saline. This treatment kills all animals within 6-12 hours Galanos et al., Son Proc.Natl.Acad.Sci., USA 76 (1979) 5939-5943). In place of LPS in the o standard treatment, the test substances are administered in various dosai ges either simultaneously with GalN, or parenterally, or orally, before or after treatment with GalN. The experiments are evaluated by comparison lo,, of the smallest dosages which are lethal for all animals in a group, or by computation of an LD 5 0 using the Spearman-Karber method.

3. Microbial septicemia in the neutropenic mouse t This model allows the determination of a substance-related increase of the 4 o immun response in microbially infected, neutropenic mice. For the inducooo tion of neutropenia, groups of 20 female BD2Fl-mice are given once 200 mg/kg bodyweight s.c. of cyclophosphamide dissolved in 0.2 ml of distilled water, on day 0. On day 3, the test substance is administered SOO parenterally (primarily or perorally, if possible dissolved in physiolo- 4o" gical sodium chloride solution, or dissolved in some other way (0.3 ml).

Infection is performed on day 4 by i.v. administration of the relevant inoculate in a volume of 0.2 ml (number of germs, e.g. per mouse: Pseudomonas aeruginosa A 12: lx105; E. coliA120: 2x106; Staph. aureus 113: 1x10 6 Candida albicans&124: 1x10 4 The test animals are observed up to day 10 after infection and the deaths registered daily. The following parameters are computed by reference to the infection control or standards, respectively: 42 900-9430 a) average survival time b) survival rate The compounds of formula la and III effect marked improvements in time and rate of survival over untreated infection controls in experimental infections with gramnegative agents Pseudomonas and E.coli) as well as in infections with grampositive agents Staph.aureus) or yeasts (e.g.

Candida albicans), after perenteral administration.

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

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

1 x 106 neutrophiles, either pretreated with test substance or not, are added to a solution containing cytochrome C (80 iMol) and formylmethionylleucylphenylalanine (10-6M). The controls contain additionally 50 ig of superoxide dismutase. After 15 minutes of incubation at 370 C the S. reaction is stopped by cooling the test tubes in an ice bath. The test tubes are then centrifuged at 400 g for 5 minutes to remove the cells. The reduction of cytochrome C, which is proportional to the amount of released o superoxide anion, is measured photometrically at 550 nm. The inhibitory effect of the test substance on PMN activation by LPS is measured as described above, whereby after preincubation of the leucocytes the cells "o are further incubated with a stimulating concentration of LPS.

p op o 5. Carbon clearance test This test is based on the principle that particles of a size from 200 to 250 R carbon particles) may be eliminated from the organism only by phagocytosis by macrophages. Following intravenous administration, carbon particles are eliminated from the blood circulation by macrophage phagocytosis- in the liver (Kupffer stellate cells) and spleen. The determination of RES-activation of a substance is effected by single or repeated administra- 43 900-9430 tion in aqueous solution or as a suspension. Testsubstances are administered i.p. or s.c. in 4 daily dosages or once 24 hours befor the start, of the test. A suspension containing 10 carbon black is diluted with a 1 gelatinous sodium chloride solution to a content of 16 mg carbon/ml. Each mouse receives 0.2 ml/20 g body weight i.v. 25 pl blood are collected by puncture of the orbital plexus 3,6,9,12 and 15 minutes after i.v. administration. The blood is hemolysed in 2 ml distilled water. The carbon concentration is determined photometrically. The animals are then killed and the weights of livers and spleens determined.

6. Herpes infection (mouse) SIntracutaneous herpes infection allows for the determination of a substance-mediated increase in the immune response of mice infected with herpes viruses. The cours of the illness is protracted and allows for the observation of the sequential appearance of several parameters. Herpetic lesions appear at the site of infection, and subsequently ulcerate. Eventually, the adjacent leg becomes paralyzed, and paralysis progressively increases until death. Immunocompetent "naked" (hairless) mice are infected intracutane- 4 ously on day 0 by intracutaneous administration of the inoculum under investigation in a volume of 0.025 ml 1 x 10 plaque-forming units of Herpes simplex type 1/mouse). The test substance is administered i.p. in solution, e.g. in physiological saline (0.1 ml).

Systemic herpes infection also allows for the determination of a substance- 4o« mediated increase in the immune response of mice infected with herpes viruses. Immunocompetent NMRI-mice are infected on day 0 by i.p.

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

The test animals are observed up to day 20 after infection and the appearance of lesions, paralysis and death registered. The following parameters are measured and compared with infection controls or a standard: -44 900-9430 a) Number of mice with lesions (cumulatively) b) Number of mice with paralysis (cumulatively) c) Average survival time d) Survival rate In the experimental HSV-1-infection, the compounds of formula la and III cause marked improvements as regards the course of illness, survival time and survival rate over untreated controls. These effects are observed after a single i.p. or s.c. administration between days 0 or -1 and +6.

7. CSF induction (colony stimulating factor) The CSFs are mediators produced by the organism following infections or in response to toxins. Their biological effects are complex; they are detected via stimulation of the proliferation of the hemopoietic system, especially of bone marrow cells. B 6

D

2

F

1 mice are given the test substances once or repeatedly, parenterally or orally, up to a dosage of 50 mg/kg.

Serum is collected from the test animals at various subsequent time intervals. The CSF-activity titers of the sera are measured in a cell culture assay as the rates of proliferation of bone marrow cells from B D2F mice Metcalf, The Hemopoietic Colony Stimulation Factors, 1984, Elsevier; T. Mosmann, J. 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 CSFs to various extents in mice, whereby time-kinetic differences in CSF-activities are also observed. These could be advantageous in therapeutic use.

8. Induction of Interleukin-1 (IL-1) The determination of a substance-mediated stimulation of cells to produce IL-1 is primarily effected in a tissue culture assay. First, both the resident and thioglycolate-elicited macrophages are recovered in the form of adhereit macrophages. These are incubated in RPMI-medium for 48 hours 900-9430 with various concentrations of test substance and the cell supernatants collected. These are tested for IL-1-activity in cultures of thymocytes from C 3 H/HeJ mice. The thymocytes of those supernatants containing IL-1 produced by macrophages are induced to proliferate during a 72 hours incubation. Proliferation is measured by incorporation of 3H-thymidine, in a scintillation counter Gery et al., J.Exp.Med. 136 (1972) 128-141; J.

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

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

9 0 oo Induction of LPS(endotoxin) tolerance (lethality tolerance) a9 00 A so-called tolerance may be induced in mice by parenteral administration 0 9 of LPS once to thrice a day. This tolerance protects the animals against 0 0 a" the lethal-effects of LPS following administration of galactosamine (cf.

induction of endotoxin shock in mice above).

0 0 oo The compounds of formula Ia und III are eliciting this tolerance already after single i.p. administration of 0.25 mg/mouse. Pretreated (tolerant) o°o mice are exposed to a LPS-challenge at a dosage of 0.01 pg LPS 8 mg galactosamine/mouse i.p. at various times (1 day to 3 weeks) following the last treatment. A larger proportion of animals survive this LPS challenge, s aespecially after repeated administration (3 times), as compared with challenge controls notpretreated.

Furthermore, the compounds of formula Ia and III possess antiinflammatory activity, especially in nonspecific, in immunologically-induced, and in hypersensitivity-induced inflammation and in allergic reactions. This activity may be demonstrated by various test methods, e.g. by investigation of the influence on prostaglandin synthesis in vitro and in vivo. In vitro, the inhibition of LPS- and zymosan-induced PGE 2 and PGFIa-release is investigated. Thioglycolate-stimulated peritoneal leucocytes from NMRImice are incubated for 24 hours with LPS or test substance. After a change 46 900-9430 of medium and triple wash of the cells they are stimulated for 2 hours with LPS or for 1 hour with zymosan, respectively. In these supernatants PGE 2 and PGFl 1 are determined. An inhibition is found of LPS- or zymosaninduced PGE 2 -production.

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

In a further test, the influence on procoagulant activity (PCA) ist 0 measured. After stimulation with LPS, human endothelial cells synthesize PCA, as detected by a shortening in plasma coagulation Lime. The coagulation time of recalcified plasma is also shortened by mouse peritoneal macrophages obtained after LPS treatment and by rabbit oo peritoneal leucocytes after release of the generalized Shwartzmanon reaction. To investigate the influence on PCA induced by LPS in vitro, peritoneal mouse leucocytes of B 6

D

2

F

1 -mice stimulated by thioglycolate are treated overnight with LPS alone or with LPS and test substance, respectively, in DMEM medium devoid of fetal calf serum (FCS) [(test design In a test design mouse peritoneal leucocytes are treated for 24 hours with LPS or test substance, respectively. After a change of o0o medium, cells are stimulated overnight with LPS. The coagulation time of recalcified human control plasma is measured using the Hkchen-method after addition of the mouse peritoneal leucocytes suspension which has been previously repeatedly frozen and treated ultrasonically.

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

PCA.

-I _I~ 47 900-9430 In vivo, the influence of LPS-induced PCA by mouse peritoneal leucocytes after pretreatment with test substance may be shown as follows:

B

6

D

2

F

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

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

Furthermore, the compounds of formula la and III possess activity against tumors, as demonstrated in the following tests: 1. Induction of tumor necrosis factor (TNF) In order to stimulate mouse bone marrow macrophages, bone marrow cells from BDFl-mice (ca. 10-13 days old, induced with CSF) are diluted to 1 106 cells/ml in medium [RPMI 1640 1 Pen/Strep (5000 U/ml) 1 glutamine] and incubated in flat microtiter plates with the test substances in solution in the range of 100 Vg to 0.1 .g/ml end concentration (dilution steps 1:10) for 4 hours at 370 C/5 CO 2 Alfter filtration through 0.45 Vm filters, the supernatants are frozen at -700 C until needed.

i 48 900-9430 00 00 O S a a 0 00 0 0* 0 a o 00 i a *00 o.4 ai 00 a L 929 cells are precultured overnight (370 C, 5 CO 2 in microtiter plates at 3 x 10 4 cells/well/100 l, then 100 ll of each sample are added and the culture further diluted in 1:2 steps. 100 p1 of actinomycin D (8 lig/ml of medium) are added and incubation continued for a further 18 hours at 370 C/5 CO 2 After removal of the supernatants the remaining cell layer is stained with Giemsa solution and absorption measured at 620 nm with a Titertek Multiscan Autoreader (Flow). One unit of TNF is defined as the activity which results in 50 lysis of the target L 929 cells.

2. B16F1 melanoma test B16FI melanoma cells are grown for 5 days in vitro. The cells are synchronized one day before the test by disrupting the monolayer into separate cells using EDTA-trypsin and returning the whole amount into the same bottle with fresh medium. The cells are counted and made up to 106 cells/ml of medium. 0.1 ml of cell suspension (105 cells) is intravenously injected into the tail vein of B 6

D

2

F

1 -mice. 21 days later the mice are killed and the number of lung tumors counted. The test substance is brought into solution and injected intraperitoneally on days -4 and -1.

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

The compounds of formula la und III are therefore useful as modulators of unspecific antimicrobial resistance, in the systemic enhancement of immune response, and in the enhancement of unspecific immunity. The compounds of formula Ia and III are thus useful in the treatment or supportive treatment together with other specific or supportive therapeutic forms) of conditions associated with decreased immune response, especially decreased humoral immune response and/or decreased over- I_ _Lr -49- 900-9430 00(0 4 O 00 o 0 o 00 sensitivity reaction of the delayed type, and in the treatment of conditions in which generally a modulation of immune response is indicated. In particular, the compounds of formula la and [II are useful in the treatment or supportive treatment of pathological conditions based on idiopathic immunological deficiencies or immunological deficiencies of the type encountered in geriatric patients, or in patients with heavy burns or generalized infections. The compounds of formula la and III are also useful in the treatment or supportive treatment of viral illnesses (such as disseminated Herpes, progressive smallpox and disseminated varicella diseases), of Morbus Hodgkin and other malignant tumors. Furthermore, the compounds of formula la and III are useful for the prevention of endotoxin shock, e.g. in accidents, burns, and surgical interventions, and as antiinflammatory agents.

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

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

Pharmaceutical compositions containing a compound of formula Ia or III CEl~iSI;~1 50 900-9430 may be prepared in accordance with standard galenical methods, e.g. by mixing with conventional, pharmaceutically acceptable diluents or carriers.

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

The invention also provides a method for combatting illnesses and infections as described above by administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula la or III or of where appropriate a pharmaceutically acceptable salt thereof.

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

.s e oz 6 os e 0 a as o B, r) O (a v

Claims (4)

1. A compound of the formula HO-. -CH 2 OH W Z HO I I OH 00 S° R R I I o 3 4 x 0 0 wherein X, Y, W and Z are independently 0 or NH and R 1 R 2 R3 and R 4 are independently an acyl group of 14 carbon atoms, optionally monosubstituted in the 3 position by an OH or an acyloxy of 14 carbon atoms 9 with the proviso that when X and Z are NH and W and Y are O then R, to R 4 are not all (R)-3-hydroxytetra- o 4 decanoyl, in free form or in form of an acid addition salt. 0 00

2. A pharmaceutical composition comprising a compound of formula I as defined in claim 1, in free form or as an o" acid addition salt in association with a pharmaceutical carrier of diluent.

3. A method of modulating antimicrobial resistance, of enhancing immune response and unspecific immunity, of preventing endotoxin shock, of treating malignant tumors or of treating inflammation which method comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound of claim 1 in free form or in pharmaceutically acceptable acid addition salt form.

4. A process for the preparation of a compound of formula I as defined in claim 1 comprising enzymatically condensing a compound of formula Il HO-. 0 OH OH HO-/ -O I ii 0 W Z R j h wherein W and Z are as defined in claim 1, R"and R"with the exception of hydrogen have the significance indicated in claim 1 for R3and R 4 and U is uridine, with a compound' or forrnu!a III HO-. C) OH ~OH I 0 III 6 900115,52 3 O~1 4 0 0 4 1 4 0 06 4 0 1 4 4 0i.~4 4 @4.4 0 4 0..b 40 0 p so 4 0* 44 e 6 4~ 4 4 44 p i wherein X and Y are 0 or NH and R" and R are independently an acyl of 14 carbon atoms, optionally monosubstituted in the 3 position by OH, or acyloxy of 14 carbon atoms with the proviso that 1) X and Y are not both NH; and 2) when X is NH and Y is O, then R' and R" are not both (R)-3-hydroxytetradecanoyl, and if desired, hydrolyzing the resulting product to a corresponding compound of formula I wherein, when X, Y, W and/or Z denote oxygen, then R 1 R 2 R 3 and/or R 4 denote hydrogen or, if des red, submitting a resulting product to an acylamidase reaction to obtain a compound of formula Ia wherein, when X, Y, W and/or Z denote imino, then R 1 R 2 R 3 and/or R 4 denote hydrogen, and recovering the compound of formula I in free form or in the form of an acid addition salt. A compound as claimed in claim 1, a pharmaceutical composition thereof or a method of treatment involving a said compound substantially as hereinbefore described with reference to the Examples. S DATED this 15th day of January 1990. SANDOZ LTD. By Its PaRent Attorneys CQ1SIS ESON '4 4 00 l-<B N* 4 G t