CH672789A5 - - Google Patents

Description

The present invention relates to saccharides and pharmaceutical compositions containing them.

The invention relates to new saccharides of the formula

HO

45

50

\ _

HO_ /

\ _ /

\. \

■ 0 - CH.

\

OH

w

1

Z

1

l

R '

i

R <

3rd

4th

HO-. '

■ —O.P.

/ \

\ / pOH

i I O

y x

1 1

* 2 I

wherein R [', R2', R3 'and R4' are identical or different and 55 each represent hydrogen or an optionally substituted acyl group and W, X, Y and Z are identical or different and each represent oxygen or the imino group, with with the proviso that a) at least one of the substituents Ri ', R2', R3 'and R4' 60 denotes an acyl group and,

b) when Z and X are imino, W and Y are oxygen, a) Ri 'and R2' do not simultaneously mean (R) -3-hydroxytetradeaanoyl if R3 'and R4' are identical and for (R) -3- Hydroxyte-tradecanoyl or R4 'represents (R) -3-dodecanoyloxytetradecanoyl and 65 R3' represents tetradecanoyloxytetradecanoyl,

β) R2 'is not hydrogen or (R) -3-hydroxytetradecanoyl if Rj' and R3 'are hydrogen and R4' are (R) -3-hydroxytetradecanoyl, and

3rd

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y) R3 'does not mean the - (CO) 2-CH2- (CHOH) 4-CH2OH group if the other substituents are hydrogen, in free form or in the form of their acid addition salts.

Disclaimer a) excludes compounds that are partially known and are also of no interest; disclaimer b) also excludes compounds that are described in the literature, but are not obtained by enzymatic synthesis but by chemical synthesis.

The compounds of the formula I are obtained in free form or in the form of their acid addition salts by using a compound of the formula

HO—,

HO— '

\ /

\

\ / '

- O.P.

S

0H OH

0 - P - ü

*

O

II

w

R3

wherein R3 ', R4', W and Z have the above meaning, and U represents a uridine residue, with a compound of the formula

HOHO-. '

\ /

\ / \ —- O. p "

OH

III

\_/ in"

I 1

T *

R "1

R »2

are the same, and are hydrogen or an acyl group having, for example, 4 to 20, preferably 12 to 16 and in particular 14 carbon atoms, which can be substituted in the 3-position by OH, acetoxy or an acyloxy group, as defined above, 5, where the C-3 atom is configured R or S. In the compounds of the formula I, therefore, R 1, R 2, R 3 and R 4 can be in achiral form, in R, S or racemic form. This also applies to the compounds of the formulas II, III and IV. In the reactions described here, the configuration remains unchanged during the course of the reaction, ie, starting from R or S or racemic starting materials, the corresponding R or S are obtained - or racemic end compounds.

Preferred compounds of the formula I are those in which R 1, R 2, R 3 and R 4 represent an optionally substituted acyl group. 15 Also preferred are compounds of the formula I in which, when R 1, R 2, R 3 and / or R 4 are hydrogen, Y, X, W and / or Z is oxygen.

The compounds of the formula I are preferably obtained as acid addition salts, hydrophilic basic compounds, for example tris (hydroxymethyl) aminomethane or L-lysine, preferably being used as the counterion to increase the water solubility.

The compounds of the formula II can be prepared from the compounds of the formula III by reaction with activated uridine-25 5'-monophosphate by methods known per se.

The compounds of formula III are new and also form part of the inventions.

Compounds of formula III can be obtained by 30 a) for the preparation of compounds of formula

HO

wherein R ^ 'and R2 ", X and Y have the meaning given in claim 3, condensed enzymatically and, if desired, subjecting the compounds obtained to saponification to give compounds of the formula Ia, in which, if X, Y, Z and / or W is oxygen, Rh is R2, R3 and / or R4 is hydrogen or, if desired, the compounds obtained are subjected to an acylamidase reaction to give compounds of the formula Ia, in which, if X, Y, Z and / or W are the imino group, Ri, R2 , R3 and / or R4 are hydrogen, and the compounds thus obtained in free form or in the form of their acid addition salts.

The disclaimer excludes connections that e.g. are known from WO 85/4881 or as Lipid-X and Lipid-Y.

The enzymatic condensation is carried out, for example, in a buffered system, e.g. in the Tris buffer at pH 7 and at room temperature or elevated temperature, e.g. at 30 ° C. A preparation obtained from gram-negative bacteria, preferably from E. coli strains (Raetz, J. Biol. Chem. 259/4852 [1984]), can be used as the enzyme extract. Strains which have been stimulated by genetic manipulation to overproduce the desired enzyme are preferred. The subsequent saponification can be carried out using methods known from the literature, the acylamidase reaction analogously to that described by C.R. Verret et. al. in J. Biol. Chem. 257/10222 (1982). The end products can be isolated from the reaction mixture and, if appropriate, purified by methods known per se.

Ri, R2, R3 and R4 are the same or different, preferably

HO

\ _

/

\ _ Y '

.0 OH

\ O.P.

/

1

purple

R! | R £

wherein R "and R2" have the above meaning and either X 'and Y are oxygen or Y are the imino group and X' is oxygen, in compounds of the formula

RO-.

\

• -

-O

RO_ /

\

\

1

/

mrnm •

I.

1

- Y '1

1

XV

1

* î

R2

. - OH

IV

65

wherein r] ", R2", X 'and Y have the above meaning and R represents a protective group which converts the unprotected OH group to a protected phosphate tester or

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b) for the preparation of compounds of the formula

HO -.

\ • '

HO- / \

° H

■ ° / \ _o.PC

è oh

Illb

/

WH

M

where Rj "and R2" have the above meaning, compounds of the formula

RO-.

\ —— O • I

/ \ /O.R

RO_ / \ -O.P

\ /

IN OR'

IIIc

HO

NH "

1

R "

2nd

in which R2 "and R have the above meaning and R 'stands for a protective group, acylates and then cleaves off the existing protective groups.

The process a) can be carried out, for example, by using a compound of the formula IV in one of the

Inert solvent reaction conditions, e.g. in a cyclic ether such as tetrahydrofuran, and at low temperature, e.g. at -70 ° C, with butyllithium in an aliphatic hydrocarbon, e.g. in hexane, and then 5 dibenzyl phosphorochloridate are added. The product can be isolated from the reaction mixture and, if appropriate, purified by methods known per se. The free OH groups of the phosphate residue are protected, e.g. through benzyl. The protecting groups can be split off using known methods. For example, a protective group is split off as usual, e.g. with an aqueous acid (ion exchanger), or hydrogenolytically.

Process b) can be carried out, for example, by reacting a compound of formula IIIc in a solvent which is inert under the 15 reaction conditions, e.g. in a halogenated hydrocarbon, such as methylene chloride, together with the acylating agent, optionally with the addition of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and at low temperature, e.g. at about 4 ° C. The reaction product can be isolated from the reaction mixture and, if necessary, purified by methods known per se. The existing protective groups are then split off in a known manner.

The protective groups which are customary in saccharide chemistry can be used as protective groups. For example, both R together can denote the benzylidene or the isopropylidene group. Known protecting groups can also be used as protecting groups of the phosphate residue, e.g. the benzyl group.

30 The starting compounds of formula IIIc are new and can be obtained according to the following formula:

ho—

Acylate

Protect ro_.

\

ro_ / \ _oh \ /

V

nhr;

tbdms.o ro—. \

Protect (pos. 4 + 6)

or '

ro_ / \ _o.p ^

J ^ \) R '

TBDMS.c / \ HR! J

ro—

\ n 0R '

• —Q /

ro — j vo.p.,.,

\ / £ N0R

/ \ O

ho nhr £

IIIc

5

672

The starting compounds of the formula IV can be obtained according to the following reaction schemes, where OH groups which do not take part in the respective reaction can optionally be protected. In these reaction schemes, the substituents have the following meanings:

R, R 'protecting groups

R5 both R5 are the same or different and have the same meaning as Ri, R2, R3 or R4 Ac acetyl group 5 TBDMS tert. Butyldimethylsilyl

1. Preparation of compounds of formula IVa

/ \

W // •

\

—0

! _ /

\-Oh

\

/

/

\

02n n3

AcO—.

\ O

AcO - / \ -OAc ho

\--O

ho— / \.

\ _ / / \

Oh

Kon t roXlie ^ 5

'Reduction ho.

o2N

\

nh.

- 0

ho— / \ \ / / s nhr

Protect (Fos. 2) - OH

02n ho

h-n ho

\

•

Jlo

) _ /

\

\

/ "

/

\

\

HO_ / \

/

R ^ -HN

V - O

Reduction nhr

Acylate

\ - oh

/

•

N

nhr

De-heating (pos. 2)

\

° 2N'j "

y n-

h0-

\

ho— / \

02n

\ - oh /

\

n-,

Reduction ho y

\ —0

Ho Oh

\ - /

/ \

nh-

h2N

ho -

x4.

Acylate s 0

Ho Oh

\ /

• *

/. \

r5hn ro.

nhr,

ro

Protect (pos. 4 + 6)

\] Lo

/ \ _ oh v /

r5hn

\

nhr.

IVa (both Rg the same)

HO— ..

• 0

Ho Oh

\ /

/ IN

RgHN NH2

HO— Y acylation —O

ho '\ -0h

• ammiM /, \

rj-hn nhr «.

RO I

Vn shooters (pos. 4 + 6)

ro— / \ _ oh

\ /

/ \

r5hn nhrs

IVa (both Rg not the same)

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2. Preparation of compounds of formula IVb

6

3. Preparation of compounds of formula IVc ho. ho

\

0

/ \ • <

\

I.

"N.

/

»•

I.

Oh"

-oh ro

V Y X - O

/

\

\

/

i

"Ï

N3

OH

Protect (pos. 4 + 6)

—Oh ro-

Protect (item 1)

V - O

RO / \ -O. TBDMS

\ /

i i

N.

ro. ro.

Oh

RgHNi

1. Reduction

2. Acylate

_ / \ -0. TBDMS / \

ro. ro.

or.

\

Deprotect (item 1)

RjHN.

/ \

.-10

\ -0H /

* \

ORc

IVb

AcO

\.

ACO / Y

\ /

"-O

/

AcO

OAC

•

0

/

\ /

/ N

AcO

OAC

20th

25th

HO 1

\ _ /

i i ho o.h.

• 1

N._Io ro - / \ —o.ch.cc1 ,. \ / 2 3

ho ro

■ OAC

Protect (item 1)

d.ch2cci3

Deprotect (items 2, 3,

4, 6)

o.ch2ccl3

Protect (pos. 4 + 6)

l l ho, oh a \ j i ro y

Acylate

35

40

45

R5O I

RO—. Y

\ O

ro / \ -o.ch-cc1,

\ / 2 3

• mmmmm

/. \

or5

Deprotect (item 1)

0

ro - / \ - oh \ /

/ \

rso ors

IVc

The further starting products are known or can be prepared by known methods or analogously to known methods or analogously as described in the examples.

The compounds of the formulas Ia and III show an influence on the non-specific antimicrobial resistance. This effect can be demonstrated, for example, with the following test methods:

1. Determination of the endotoxic activity using the Limulus amebocyte lysate test

Endotoxin catalyzes the activation of a proenzyme in Limulus amoebicyclic lysate. The elimination of p-nitroaniline from a colorless substrate is measured. The extent of this cleavage is recorded photometrically, the correlation between absorption and endotoxin concentration (or activity in analogs) being linear in the range from 0.01-0.1 ng / ml endotoxin (comparison with the absorption values of a standard endotoxin ). A 1:10 dilution series is prepared from each sample (dissolved in pyrogen-free Aqua bidestillata sterilis). An empty sample is also carried with each series of determinations. 100

p.1 Sample or standard or blank are mixed with 100 jxl Limu-5o lus amoebocyte lysate. After 10 minutes of incubation at 37 ° C., the reaction is mixed with 200 μl of substrate. After a further 3 minutes of incubation, the reaction is stopped with 200 ml of 50% acetic acid. After shaking the sample, the absorption against an empty value is measured in a spectrophotometer at 405 nm. The endotoxin content (endotoxin activity) of the sample as endotoxin units (E.U.) is determined by calculating a linear regression using the values of the standard endotoxin.

60 2. Endotoxin shock induction in the mouse

The test records the generation of an endotoxin shock or a clinically similar state with a lethal outcome by LPS-like substances in galactosamine (GalN) -sensitive mice. Male C57 bl. Mice (6 animals per group) received 65 simultaneously i.p. 8 mg GalN, dissolved in 0.5 ml PBS, and 0.1 (ig LPS from Salmonella abortus equi (Sigma), dissolved in 0.2 ml physiological saline. This treatment leads to the death of all animals within 6-12 hours ( C. Galanos, et al., Proc. Nati.

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Acad. Sci., USA, 76 (1979) 5939-5943). Instead of the LPS in the standard procedure, test substances are administered in different doses simultaneously with GalN or once before or after the GalN treatment once parenterally or orally. The result is assessed on the basis of a comparison of the lowest dose that leads to the death of all animals in a group,

or by calculating an LD50 using the Spearman-Kärber method.

3. Microbial septicemia in the neutropenic mouse

This model allows the examination of a substance-related defense increase in microbially infected, neutropenic mice. To produce neutropenia, 20 female B6D2Fr mice each receive 1 x 200 mg / kg body weight of cyclophosphamid in 0.2 ml of distilled water. solved, s.c. administered on day 0. On day 3, if possible, the test substance is administered in physiological NaCl solution, but otherwise dissolved in another way (0.3 ml) parenterally (primarily i.p.) or orally. The infection occurs on day 4 by i.v. Administration of the respective inoculum in a volume of 0.2 ml (bacterial count per mouse e.g. Pseudomonas aeruginosa A12: lx IO5, E. coli À 120: 2 x IO6, Staph. Aureus A 113: 1 x 106, Candida albicans A124: lx 104 ). The experimental animals are observed until the 10th day after infection and the deaths that occur are recorded daily. In comparison to infection control or standards, the following parameters are evaluated:

a) Average survival b) Survival rate

The compounds of the formulas Ia and III showed survival after parenteral administration both in the experimental infections by gram-negative germs (e.g. Pseudomonas and E. coli) and in infections by gram-positive germs (e.g. staph. Aureus) or yeasts (e.g. Candida albicans) and survival rate significant improvements compared to the untreated infection control.

4. Activation of human blood neutrophil oxidation metabolism

Neutrophils (at least 95% purity) are incubated with the test substance in three different concentrations for 1 to 2 hours at 37 ° C. The release of superoxide anions by the cells as an index for the activation is then measured as a superoxide dismutase-inhibitable reduction of cytochrome C. 1 x 106 neutrophils, pretreated with test substance or not, are added to the cytochrome C (80 µmol), which contains formylmethionylleucylphenylalanine (IO-6 M). The controls also contain 50 jig of superoxide dismutase. After 15 minutes of incubation at 37 ° C, the reaction is stopped by placing the test tubes in an ice bath. They are then centrifuged rapidly at 400xg for 5 minutes to separate the cells. The reduction in cytochrome C, which is proportional to the amount of superoxide anion released, is measured photometrically at 550 nm. The inhibitory effect of the test substances during PMN activation by LPS is measured as described with the proviso that after the preincubation of the leukocytes, the cells are still incubated with a stimulating concentration of LPS.

5. Carbon clearance test

The principle of the test is based on the fact that particles in the range of 200-250 À (2.0-2.5 x 10 ~ nm) (e.g. carbon particles) can only be eliminated from the organism by phagocytosis by macrophages. With intravenous administration of carbon particles, these are eliminated from the blood stream by phagocytosis of the macrophages in the liver (copper star cells) and spleen. The RES activation of a substance is checked by single or repeated administration as an aqueous solution or as a suspension. The amount of substance intended for the treatment is either in 4 daily single doses or once 24 hours before the start of the test i.p. or s.c. applied. Ink suspension with a 10% gas black content is diluted with a 1% gelatin-NaCl solution to a content of 16 mg coal / ml. Each mouse receives 0.2 ml / 20 g body weight administered intravenously. 3, 6, 9, 12 and 15 minutes after the intravenous administration, blood is drawn by puncturing the orbital plexus 25 | il. The blood is distilled in 2 ml. Hemolyzed water. The coal concentration is determined photometrically. The here are then killed and the liver and spleen weights are determined.

6. Herpes infection of the mouse

The model of intracutaneous herpes infection allows the examination of a substance-related defense increase in mice infected with herpes viruses. The course of the disease is prolonged and enables the observation of the sequential occurrence of several parameters. Herpetic lesions appear at the site of infection, ulcerate them, the nearest leg is paralyzed and the paralysis continues until death. Immune-competent hairless mice are infected intracutaneously on day 0 by intracutaneous administration of the respective inoculum in a volume of 0.025 ml (e.g. 1 x 106 plaque-forming units herpes simplex type 1 / mouse). The test substance is dissolved in solution, for example in physiological NaCl, (0.1 ml) i.p. administered.

The model of systemic herpes infection allows the examination of a substance-related defense increase in mice infected with herpes viruses. Immune-competent NMRI mice are challenged on day 0 by i.p. Administration of the respective inoculum in a volume of 0.1 ml (e.g. 1.3 x 105 plaque-forming units herpes simplex type 1 / mouse) infected. The test substance is administered subcutaneously in solution, for example in physiological NaCl.

The test animals are observed up to the 20th day after the infection and the lesions, paralyses and deaths that occur are recorded daily. In comparison to infection control or the standard, the following parameters are evaluated:

a) Number of mice with lesions (cumulative)

b) Number of mice with paralysis (cumulative)

c) Average survival time d) Survival rate.

When administered once between day 0 and - 1 and + 6, the compounds of the formulas Ia and III in the experimental HSV-1 infection after i.p. or subcutaneous administration in terms of disease course, survival and survival rate, significant improvements compared to the untreated infection control.

7. Induction of CSF (colony stimulating factor)

CSFs are mediators that are produced in the organism as a result of infections or toxins. Their biological functions are versatile, the evidence is provided by stimulating the proliferation of the hemopoietic system, especially the bone marrow cells. B6D2Fr mice are administered the test substances one or more times up to a dose of 50 mg / kg parenterally or orally. Serum is obtained from the test animals at variable intervals thereafter. The serum content of CSF activity is measured in a cell culture assay using the proliferation rates of bone marrow cells from B6D2F! Mice [D. Metealf, The hemopoietic colony stimulating factors, 1984, Elsevier; T. Mosmann, J. Immunol. Methods 65, 55-63 (1983); L.M. Green et al., J. Immunol. Methods 70: 257-268 (1984)]. Compounds of the formulas Ia and III induce CSFs in mice to varying degrees, with time-kinetic differences in the CSF activities also being observed, which can be advantageous when used therapeutically.

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

The proof of whether substances can stimulate cells for IL-1 production is primarily carried out in cell culture assays. Adherent macrophages are first obtained from mice, both resident and with thioglycolate «elicited macrophages». These are incubated in RPMI medium at various substance concentrations to be tested for 48 hours and the cell supernatants obtained. These supernatants are tested for IL-1 activity in cytocyte cultures of C3H / HeJ mice. If the supernatants of macrophages contain produced IL-1, the thymocytes are stimulated to proliferate during an incubation of 72 hours, which is measured by incorporating 3H-thymidine in the scintilla counter [J. Gery et. al., J. Exp. Med. 136, 128-142 (1972); J. Oppenheim et. al., Cel-lular Immunol. 50: 71-81 (1980)]. Substances of the formulas Ia and III have the ability to induce IL-1 production in macrophages to varying degrees (concentration-related 0.1-50 (ig / ml).

9. Induction of LPS (endotoxin) tolerance (lethality tolerance)

Through one to three times daily parenteral administration of LPS to mice, a so-called tolerance can be generated, which protects the animals against the lethal effects of LPS after administration of galactosamine (see also endotoxin shock induction in the mouse).

With the compounds of the formulas Ia and III, it was possible to use the i.p. Administration of 0.25 mg / mouse triggered such a tolerance. Pretreated (tolerant) mice were challenged at various times after the last treatment (1 day to 3 weeks) with an LPS challenge in a dose of 0.01 µg LPS + 8 mg galactosamine / mouse. subject. A higher percentage of animals survived this LPS exposure compared to the untreated challenge control, especially after three doses.

The compounds of the formulas Ia and III also have an anti-inflammatory effect, in particular in the case of non-specific inflammations, in the case of immunologically induced inflammations, in the case of hypersensitively induced inflammations and in the case of allergic reactions. This effect could be demonstrated by various test methods, for example by examining the influence of prostaglandin synthesis in vitro and in vivo. The inhibition of LPS and zymosan-induced PGE2 and PGFia release was investigated in vitro. Thioglycolate-stimulated:? Eritoneal-M0 from the NMRI mouse was incubated for 24 hours with LPS or test substance. After changing the medium and washing the cells three times, they were stimulated for 2 hours with LPs or 1 hour with Zymosan. PGE2 and PGFia were detected in these supernatants. An inhibition of both the LPS and the zymosan-stimulated PGE2 production of the cells was shown. The inhibition of LPS-induced PGE2 release of mouse Mo after pretreatment with test substances was tested. 3 NMRI mice were given i.p. on day 1, 2 and 3 with LPS or test substance. treated. On day 4 these animals and a control group received 1.5 ml of thioglycolate i.p., on day 7 peritoneal M0 was obtained and stimulated with LPS. A significant reduction in PGE2 release compared to the control was found.

In another test, the influence of the "procoagu-lant activity" (PCA) is examined. After stimulation with LPS, human endothelial cells synthesize PCA, measured as a reduction in the clotting time of plasma. Mo after LPS treatments and peritoneal leukocytes (obtained from rabbits) also shorten the clotting time of recalcified plasma after triggering the general Shwartzman reaction. To investigate the influence of PCA generated by LPs in vitro, thioglycolate-stimulated peritoneal Mo from B6D2FrMäu-sen was tested in an experimental design a overnight with LPS alone or

treated with LPS and test substance in DMEM medium without fetal calf serum (FCS). In experimental design b, such mouse Mo were treated with LPS or test substance for 24 hours and stimulated with LPS overnight after changing the medium. The clotting time of recalcified human control plasma after the addition of the multi-frozen and sonicated Mo suspension was measured using the checkmark method. Addition of test substance reduces the PCA triggered by LPS below that of the control value (extension of the clotting time). Pretreatment with test substance also leads to a reduction in PCA.

In vivo, the influence of LPS-induced PCA in mouse peritoneal leukocytes after pretreatment of the animals with test substance was shown to be wet: B6D2Fr mice were treated with LPS or test substance administered intraperitoneally on day 1, 2 and 3. In addition, all Tîere received 1.5 ml i.p. Peritoneal Mo was obtained on day 6, the sample of each animal was adjusted to 1 × 106 / ml cells and stimulated with LPS in DMEM medium without FCS for 24 hours. The PCA of these cells was determined as described above. Pretreatment with LPS or with test substance results in a significantly reduced PCA. A similar finding was found in preliminary experiments with rabbits. The PCA of peritoneal rabbit leukocytes after triggering the general Shwartzman reaction could also be reduced by pretreatment with LPS or with test substance.

In the inhibition of the local Shwartzman reaction, 3 chinchilla rabbits were treated on day 1, 2 and 3 with LPS or test substance, each i.v. or i.p., pretreated. On day 6, the intradermal administration of 40, 20, 10, 5.2, 5 and 1.25 µg LPS per skin site took place. On day 7, the reaction was carried out with 2 µg LPS / kg IV. triggered. The reaction was assessed semi-quantitatively by assessing skin necrosis. There was almost complete suppression of the Shwartzman reaction by LPS pretreatment or by pretreatment with test substance.

The compounds of the formulas Ia and III also have activity against tumors, as was demonstrated in the following model tests:

1. Investigation of the induction of tumor necrosis factor (INF)

To stimulate mouse bone marrow macrophages, bone marrow cells from BDFr mice (approx. 10-13 days old, induced with CSF) on 1 x 106 cells / ml in medium [RPMI 1640 + 1% pen / step (5000 U / ml) + 1% Glutamine] diluted and incubated in flat microtiter plates with the substance solutions in the concentration range 100 (ig to 0.1 µg / ml final concentration (1:10 dilution levels) for 4 hours at 37 ° C / 5% CO 2. After filtration through 0.45 µm The supernatants are frozen at -70 ° C until the TNF test is carried out.

L 929 cells are pre-cultured in microtiter plates to 3 × 104 cells / well / 100 jj.1 overnight (37 ° C./5% CO 2), 100 (j.1 of the respective sample is added and further dilution in series in 1: 2 steps After adding 100 μl of actinomycin D (8 μg / ml medium) to each well, the mixture is incubated for a further 18 hours at 37 ° C./5% CO. After removal of the supernatants, the remaining cell lawn is stained with Giemsa solution and the absorption at 620 nm measured on the Titertek Mulüskan autoreader (flow) A unit of TNF is defined as the activity which causes 50% lysis of the L 929 target cells.

2. B16F1 melanoma test

B16F1 melanoma cells are grown in vitro for 5 days. One day before the experiment, the cells are broken up into individual cells using EDTA Tiypsin and the total amount is returned to the same bottle.

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see synchronized with new medium. The cells are grown and adjusted to 106 / ml medium. 0.1 ml of cell suspension (105 cells) are injected intravenously into the tail vein. The mice are sacrificed 21 days later and the number of tumors in the lungs is determined. B6D2Fi mice are used for this test. The test substance is dissolved and injected intraperitoneally on days -6, -4 and - 1. It has shown,

that the substances of the formula Ia and III according to the invention have immunoprophylactic activity, the number of B16F1 melanoma metastases in the lungs has been reduced. To check the therapeutic activity, the mice were treated on the 3rd, 6th, 8th, 10th, 13th and 15th day after tumor cell inoculation. Here, too, there was a clear reduction in metastasis.

The compounds of the formulas Ia and III can therefore be used as modulators of non-specific antimicrobial resistance, for systemically increasing the immune response and for increasing non-specific immunity. Compounds of formulas Ia and III are thus indicated, e.g. for the treatment or supportive treatment (i.e. together with other specific or supportive forms of therapy) of conditions with a reduced immune response, in particular with a reduced humoral immune response and / or reduced hypersensitivity reaction of the delayed type and for the treatment of conditions in which there is otherwise a modulation of the immune response is desirable. In particular, compounds of the formulas Ia and III are indicated for the treatment or supportive treatment of disease states due to idiopathic immunodeficiencies of the type which occur in geriatric patients or in patients with severe burns or generalized infections. The compounds of the formulas Ia and III are also indicated for the treatment or supportive treatment of viral diseases (such as disseminated herpes, progressive pox diseases and disseminated varicella diseases) and also of Marbus Hodgkin and other malignant tumors. In addition, the compounds of the formulas Ia and III are suitable for the prophylaxis of endotoxin shock, e.g. for accidents, burns and surgical interventions as well as an anti-inflammatory agent.

For the applications mentioned, the indicated parenteral dose is between approximately 0.1 mg and approximately 70 mg, administered once to achieve an adjuvant effect, e.g. in the case of supportive treatment, or daily, in the latter case the dose being divided into 2 to 4 administrations per day or given in sustained release. Indicated dose units for administration therefore contain between approximately 0.025 and approximately 35 mg of the formula Ia or III substance if administered daily, or up to 70 mg of the formula Ia or III substance if a single, adjuvant treatment is desired.

Because of their immunomodulating activity, compounds of the formulas Ia and III are also suitable as adjuvants for vaccines. For this type of use, an indicated dose between 0.5 and 100 mg, preferably approximately 70 mg, is administered on the day of vaccination with a possible repetition at the same dose 2 to 4 weeks later.

Pharmaceutical preparations containing the compounds of formula I can be prepared according to standard pharmaceutical procedures, e.g. by mixing with conventional, pharmaceutically acceptable diluents or carriers. Such preparations can be made, for example, in the form of injectable solutions and also form part of the invention.

The invention relates to the compounds of the formula I or III as chemotherapeutic agents, in particular as immunomodulators, as antiviral agents and as anti-inflammatory agents.

In the following examples, which explain the invention in more detail, but in no way limit its scope

len, all temperatures are given in degrees Celsius. EDTA means ethylenediaminetetraacetic acid, DTE stands for 1,4-dit-hioerythritol, UDP for uridine phosphate and Tris for tris (hydroxy-methyl) aminomethane.

Example 1: 6-0- [2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3hydroxytetradecanoylamido] -ß-D-glucopyranosyI] -2,3-di-0 [(R) -3-hydroxytetradecanoyl] -1 -O-phosphono-aD-glucopy-ranose

2.03 mg UDP-2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3-hydroxytetradecanoylamido] -1 -O-phosphono-aD-glu-copyranose and 1, 42 mg of 2,3-di-0 - [(R) -3-hydroxytetradeca-noyl] -l-0-phosphona-aD-glucopyranose, both as Tris salts, are dissolved in 10 mM Tris buffer, pH 7 0.2 mM EDTA and 0.2 mM DTE dissolved and incubated with 60 ul enzyme extract at 30 °.

This enzyme extract can be obtained as follows:

E. coli JB1104 is grown in L-Broth (preculture +10 µg ampidllin / ml at 30 ° overnight; main culture at 30 ° in a shaking incubator, starting from OD55o nm = 0> 1 to OD550nm = 1). The cells are harvested by centrifugation at 10,800 g (10 minutes / 40), the 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 pellet is then resuspended in the above buffer and disrupted with ultrasound (5 x 20 seconds / 30 seconds pause with 120 watts). After centrifugation (12,000 g, 10 minutes / 4 °) to separate the cell residues, ultracentrifugation (150,000 g, 90 minutes / 70). The top 2A of the supernatant are obtained. This fraction is subjected to a fractional ammonium sulfate precipitation. The range of 10-40% ammonium fat is used as the enzyme preparation. The pellet obtained in this way is taken up in the above buffer.

After the reaction has ended (reaction control via thin layer chromatography), the mixture is acidified to pH 2 with citric acid and centrifuged. The pellet is taken up in chloroform / methanol / acetic acid / water (65/15/5/2) and chromatographed on silica gel using the same eluent. The fractions containing the product are collected and evaporated until there are no more organic solvents and the rest lyophilized. The lyophilisate is dissolved in tetrahydrofuran / water (8/2) and converted into the Tris salt in this solvent via a cation exchanger in the Tris form, the tetrahydrofuran is spun off and the residue is lyophilized. All Rf values are determined on silica gel plates.

Rf (in chloroform / methanol / glacial acid / water = 65 / 25/5/5) = 0.48

Example 2: 6-0- [2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3-hydroxytetradecanoylamido] -ß-D-glucopyranosyl] -2-deoxy-3 -0 - [(S) -3-hydroxytetradecanoyl] -2 - [(S) -3-hydroxytetrad-canoylamido] -1 -O-phosphono-aD-glucopyranose

20 parts by volume of a 5 mM solution of 2-deoxy-3-0 - [(S) -3-hydroxytetradecanoyl] -2 - [(S) -3-hydroxytetradecanoyIa-mido] -l-'0-phosphono-aD-glucopyranose in 10 mM Tris / HCl buffer (pH = 7) with 0.2 mM EDTA and 0.2 mM DTE, 20 parts by volume of a 5 mM solution of UDP-2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3hydroxytetradecanoylamido] -1-O-phosphono-aD-glucopyranose in the same buffer and 20 parts by volume of a 100 mM Tris / HCl buffer (pH = 7) with 2 mM EDTA and 0.2 mM DTE reacted with 40 parts by volume of an enzyme preparation (preparation as described in Example 1) in 10 mM Tris / HCl buffer (pH = 7) with 2 mM EDTA, 2 mM DTE and 0.1% Triton X-100 at 30 ° for 8 hours, the final concentration of Triton X-100 is adjusted to 0.1%. The reaction mixture is lyophilized, approximately the starting volume is dissolved in the eluent for subsequent chromatography on molecular weight separation (Sephadex LH 20) (eluent:

9

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

672 789

Pyridine / acetic acid / water = 98/1/1) and filtered. The volume of the column used is about 20 times the sample application volume. The pure product fractions are collected, concentrated in vacuo, suspended in pyrogen-free water and lyophilized. After lyophilization, Triton X-100 is removed by digesting with diethyl ether. After centrifugation, the pellet is dissolved in a mixture of chloroform / methanol (1/1), filtered and the solvents removed in vacuo. The stoichiometrically calculated amount of L-lysine (free base) is added to the aqueous suspension of this pellet to obtain the mono-L-lysine salt in the form of an aqueous 100 mM solution and lyophilized again.

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

Example 3: 6-0- [2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 [(R) -3-hydroxytetradecanoylamido] -ß-D-glucopyranosyl] -2-deoxy-3- 0 - [(R) -3-hydroxytetradecanoyl] -l-0-phosphono-2 - [(R) 3-tetradecanoyloxytetradecanoylamido] -aD-glucopyranose

20 volume parts of a 5 mM solution of 2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -1 -0-phosphono-2 [(R) -3-tetradeca-noyloxyteradecanoylamido] -aD-glucopyranose in 10 mMTris / HCl buffer (pH = 7) with 0.2 mM EDTA and 0.2 mM DIE, 20 parts by volume of a 5 mM solution of UDP-2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] - 2 - [(R) -3hydroxytetradecanoyla-mido] -l-0-phosphono-ad-glucopyranose in the same buffer and 20 parts by volume of a 100 mM Tris / HCl buffer (pH = 7) with 2 mM EDTA and 0.2 mM DTE are reacted with 40 parts by volume of enzyme extract (preparation as described in Example 1) in 10 mM Tris / HCl buffer (pH = 7) with 2 mM EDTA, 2 mM DTE and 0.1% Triton X-100 at 30 ° for 48 hours, the final concentration of Triton X-100 is adjusted to 0.1%. The reaction mixture is lyophilized, treated with pyridine and filtered, the filtrate is concentrated in vacuo and chromatographed on silica gel with the following eluent (chloroform / methanol / water / 2-phenethylpicolonium bromide = 800/175 / 22.5 / 2.5). The pure product fractions are collected and, by shaking with 20 mM H3P04 in H20, the 2-phenethylpicolonium bromide serving as ion pair former is removed. The organic phase is concentrated in vacuo, the stoichiometrically calculated amount of L-lysine (free base) is added to obtain the mono-L-lysine salt in the form of an aqueous 100 mM solution and lyophilized.

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

Analogously to that described in Examples 1 to 3, the following compounds of the formula I can also be obtained, the amount of enzyme being selected depending on the compounds of the formula II and III used so that the reaction preferably proceeds in about 24 to 48 hours:

Table rj = r2 = r3 = r, = (R) -3-hydroxytetradecanoyl

example

W

e.g.

Y

X

Rf

4th

O

NH

NH

NH

0.45

5

NH

NH

NH

NH

0.40

6

O

O

O

NH

0.49

7

O

NH

■ NH

O

0.39

8th

NH

NH

O

NH

0.41

9

O

O

O

O

0.48

Example 10: 6-0- [2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(Rj-3-hydroxytetradecanoylamido] -ß-D-glucopyranosyl] -2-deoxy-1 - 0-phosphono-3-0-tetradecanoyl-2-tetradecanoylamido-a-dglucopyranose

10th

The procedure is analogous to that described in Examples 1 to 3 and the Utelverbindung is obtained.

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

5

Example 11: 6-0- [2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3-hydroxytetradecanoylamido] -ß-D-glucopyrenosyl] -2-ace-tamido -2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -l-0-phosphono-aD-glucopyranose io The procedure described in Examples 1 to 3 is followed and the ute compound is obtained.

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

15 Example 12: 6-0- [2-Deoxy-2 - [(R) -3-hydroxytetradecanoyla-mido] -ß-D-glucopyranosyl] -l-0-phosphono-a-D-glucopyranose

5 mg 6-0- [2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3-hydroxytetradecanoylamido] -ß-D-glucopyranosyl] -2,3-di- 0 - [(R) -3hydroxytetradecanoyl] -1 -O-phosphono-aD-glucopy-20 ranose are dissolved in chloroform / methanol (1/1) and treated with aqueous 25% ammonia ('/ 3 by volume) and poured over Left at night. After the end of the reaction, the mixture is evaporated, the residue is digested with diethyl ether to remove the fatty acids which have been split off, the precipitate is filtered off with suction and washed with ether. The residue is dissolved with water and the calculated amount of Tris to prepare the di-tris salt and lyophilized.

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

30th

Example 13: 2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 -] (R) -3-hydroxytetradecanoylamido] -1 -O-phosphono-aD-ghico-pyranose a) 4,6- 0-Benzylidene-3-0 - [(R) -3-benzyloxytetradecanoyl] -35 2 - [(R) -3-benzyloxytetradecanoylamido] -2-deoxy-1 -O-dibenzyl-phosphono-a-D-glucopyranose:

To a solution of 3.9 g of 4,6-O-benzylidene-2 - [(R) -3-benzyloxytetradecanoylamido] -2-deoxy-1 -O-diben-zylphosphono-aD-glucopyranose cooled to -10 ° , 2 g of (R) -3-benzyloxytetrade-40 canoic acid and 50 mg of 4-dimethylaminopyridine in 20 ml of methylene chloride, 2 g of dicyclohexylcarbodiimide are added and the reaction mixture is kept at 4 ° overnight. It is then filtered, the filtrate evaporated, the residue taken up in a little toluene / ethyl acetate (8/2) and chromatographed with the same solvent mixture. Mp: 96-98 ° [a] o ° = + 33.3 ° (c = 1, chloroform)

Rf (toluene / ethyl acetate = 2/1) = 0.5

b) 2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3-hydroxyte-5o tradecanoylamido] - 1-O-phosphono-aD-glucopyranose 4.2 g 4, 6-0-Benzylidene-3-0 - [(R) -3-benzyloxytetradeca-noyl] -2 - [(R) -3-benzyloxytetradecanoylamido] -2-deoxy-1 -O-diben-zylphosphono-aD-glucopyranose dissolved in 900 ml of tetrahydrofuran / water (85/15) and hydrogenated with 1.5 g of 10% palladium on 55 activated carbon for 2 hours at 10 bar and 40 °. The catalyst is then filtered off, the tetrahydrofuran is evaporated off and the aqueous suspension is lyophilized.

[cx] d = + 28.5 ° (c = 0.2, chloroform + 1 drop of methanol)

60 Rf (chloroform / methanol / acetic acid / water = 125 / 75/10/20) = 0.56

Further cleaning can be carried out as follows:

10 g 2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3-65 hydroxytetradecanoylamido] -l-0-phosphono-aD-glucopyranose and 1.7 g Tris in 150 ml of methanol are sonicated in an ultrasonic bath at 45 ° for 12 minutes. The suspension is centrifuged and then the clear supernatant becomes dean from the pellet

animals. 1.7 g of Tris (dissolved in 20 ml of methanol) are added to this solution, the solution is inoculated and allowed to crystallize at room temperature. The di-tris salt of the title compound is obtained.

The mother liquor is evaporated. The residue and the pellet are dissolved together in a mixture of 300 ml of chloroform, 600 ml of methanol and 240 ml of water (pyrogen-free). A further 300 ml of chloroform and 300 ml of 0.1 N hydrochloric acid are added to this solution, the mixture is shaken gently and the phases are separated. The chloroform phase is separated off and evaporated. The residue consists of contaminated 2-deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(R) -3-hydroxytetradecanoylamido] -1 -0-phosphono-a-I> glucopyranose.

The 2-deoxy-3-0 - [(R) -3-hydroxytetradeca-noyl] -2 - [(R) -3-hydroxytetradecanoylamido] -l-0-phosphono-aD-glucopyranose thus obtained is, as described above, treated with Tris and methanol in an ultrasonic bath and centrifuged. The solution is applied to a Sephadex LH 20 column and eluted with methanol. The appropriate fractions are pooled and evaporated. The mono-tris salt of the Utel compound is obtained.

5.1 g of this mono-Tris salt are dissolved in 40 ml of methanol at 45 ° in an ultrasonic bath, a solution of 0.74 g of Tris in 10 ml of methanol is added, inoculated and left to crystallize first at room temperature and then at 4 °. Further crystalline di-tris salt of the title compound is obtained.

The residue of the mother liquor and the pellet from the centrifugation • can be returned to a cleaning cycle.

Mp: 183-185 ° (decomposition)

[<x] d = + 15 ° (c = 1, water)

Example 14: 3-Deoxy-2-0 - [(R) -3-hydroxytetradecanoyl] -3 - [(R) -3-hydroxytetradecanoylamido] -1 -O-phosphono-aD-gluco-pyranose a) 2-0- [(R) -3-Benzyloxytetradecanoyl] -3 - [(R) -3-benzyloxyte-tradecanoylamido] -3-deoxy-l-0-dibenzylphosphono-4,6-0-iso-propylidene-aD-glucopyranose

To a solution of 4.85 g of 2-0 - [(R) -3-benzyloxytetradecanoyl] -3 - [(R) -3-benzyloxytetradeca-noylamido] -3-deoxy-4,6-0 cooled to - 70 ° -isopropylidene-D-glucopyranose in 40 ml of absolute tetrahydrofuran, 8 ml of 1.6 M butyllithium in hexane are added dropwise. After 5 minutes, a solution of 3.8 g of dibenzylphosphorochloridate in 10 ml of benzene is added dropwise at this temperature. The mixture is stirred for a further 10 minutes at -70 °, 0.3 ml of acetic acid is added and the solution is concentrated to a quarter of its 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 (sodium sulfate) and evaporated. The residue is chromatographed (toluene / ethyl acetate = 7/3). Rf (toluene / ethyl acetate = 2/1) = 0.58

b) 3-Deoxy-2-0 - [(R) -3-hydroxytetradecanoyl] -3 - [(R) -3-hydroxytetradecanoylamido] -1 -O-phosphono-a-D-glucopyranose

A solution of 980 mg of 2-0 - [(R) -3-benzyloxytetradeca-noyl] -3 - [(R) -3-benzyloxytetradecanoylamido] -3-deoxy-l-0-diben-zylphosphono-4,6-0 -isopropylidene-aD-glucopyranose in 100 ml of tetrahydrofuran is hydrogenated for about 1 hour with 300 mg of 10% palladium on activated carbon at normal pressure. The catalyst is then filtered off, water (10 ml) and Dowex AG50WX8 H + are added, and the mixture is stirred at room temperature until the isopropylidene group is completely split off. The ion exchanger is filtered off, the solution is neutralized with Tris and tetrahydrofuran and water are evaporated off on a Rotavapor. The residue is dissolved in methanol and chromatographed on Sephadex LH 20.

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

11 672 789

Example 15: 2,3-di-0 - [(R) -3-hydroxytetradecanoyl] -l-0-phosphono-aD-glucopyranose a) 4,6-0-benzylidene-2,3-di-0 - [( R) -3-benzyloxytetradeca-noyl] -1-odibenzylphosphono-aD-glucopyranose

5 To a solution of 1 g of 4,6-O-benzylidene-2,3-di-0 - [(R) -3-benzyloxytetradecanoyl] -D-glucopyranose in 10 ml of absolute tetrahydrofuran, cooled to -70 °, become 0. 9 ml of 1.6 M butyl lithium in hexane were added dropwise. After 5 minutes, a solution of 420 mg of dibenzyl phosphorochloridate in 4 ml of benzene is added dropwise at this temperature. The mixture is stirred for a further 10 minutes at -70 °, neutralized with acetic acid and the solution is evaporated. The residue is chromatographed (silica gel, hexane / toluene / ethyl acetate = 4/4/1).

Rf (toluene / ethyl acetate = 6/1) = 0.65 15 b) 2,3-di-0 - [(R) -3-hydroxytetradecanoyl] -l-0-phosphono-a-D-glucopyranose

230 mg of 4,6-0-ben2ylidene-2,3-di-0 - [(R) -3-benzyloxytetrade-canoylJ-IO-dibenzylphosphono-aD-glucopyranose are dissolved in tetrahydrofuran / water (9/1) and mixed with 80 mg 10% Palla-20 dium hydrogenated on activated carbon for about 5 hours at normal pressure. The catalyst is then filtered off, the solution is neutralized with Tris and evaporated. The residue is chromatographed with methanol on Sephadex LH 20.

Rf (chloroform / methanol / acetic acid / water = 125/25 75/10/20) = 0.65

Example 16: 2-Deoxy-3-0 - [(S) -3-hydroxytetradecanoyl] -2 - [(S) -3-hydroxytetradecanoylamido] -1 -O-phosphono-a-D-glucopyranose

30 a) 4,6-0-Benzylidene-3-0 - [(S) -3-benzyloxytetradecanoyl] -2 - [(S) -3-benzyloxytetradecanoylamido] -2-deoxy-l-0-dibenzylphosphono-aD -glucopyranose

The procedure is analogous to that described in Example 13a) and the title compound is obtained.

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

b) 2-Deoxy-3-0 - [(S) -3-hydroxytetradecanoyl] -2 - [(S) -3-hydroxytetradecanoylamido] -1 -O-phosphono-a-D-glucopyranose

The procedure is analogous to that described in Example 13b) and the title compound is obtained with an mp: 150-200 ° (dec.) 40 Rf (methylene chloride / methanol / ammonia = 1/1/1; lower phase) = 0.5

Example 17: 2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -l-0-45 phosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamido] -aD-glucopyranose a) 4.6-0 -Benzyliden-3-0 - [(R) -3-benzyloxytetradecanoyl] -2-deoxy-l-0-dibenzylphosphono-2 - [(R) -3-tetradecanoyloxytetra decanoylamido] -aD-glucopyranose

50 The procedure is analogous to that described in Example 13a) and the title compound is obtained with an mp: 82-95 °.

[a® = + 24.1 ° (c = 1, chloroform)

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

b) 2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] - l-O-

55 phosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamido] -a-D-glucopyranose

The procedure is analogous to that described in Example 21b) and the title compound is obtained.

[a] o = + 14.3 ° (c = 1, tetrahydrofuran / pyridine) 60 Rf (chloroform / methanol / acetic acid / water = 80/25/5/5) = 0.35

Example 18: 2-deoxy-l-0-phosphono-3-0-tetradecanoyl-2-tetra-decanoylamido-aD-glucopyranose 65 a) 4,6-0-benzylidene-2-deoxy-1-O-dibenzylphosphono-3 -O-tetradecanoyl-2-tetradecanoylamido-aD-glucopyranose

The procedure is analogous to that described in Example 13a), using tetradecanoic acid and with toluene / ethyl acetate

672 789

(4/1) as a solvent mixture, and receives the Utelverbindung of mp: 123-129 °

Rf (toluene / ethyl acetate = 2/1) = 0.6 b) 2-deoxy-l-0-phosphono-3-0-tetradecanoyl-2-tetradeca-noylamido-a-D-glucopyranose:

The procedure is analogous to that described in Example 21b) and the title compound is obtained.

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

Example 19: 2-Deoxy-2 - [(R) -3-hydroxytetradecanoylamido] -l-0-phosphono-3-0 - [(R) -3-tetradecanoyloxytetradecanoyl] -a-D-glucopyranose:

a) 4,6-0-Benzylidene-2 - [(R) -3-benzyloxytetradecanoyl-amido] -2-deoxy-l-0-dibenzylphosphono-3-0 - [(R) -3-tetra-decanoyloxytetradecanoyl] - AD glucopyranose:

The procedure is analogous to that described in Example 13a), using (R) -3-tetradecanoyloxytetradecanoic acid and with toluene / ethyl acetate (4/1) as the solvent mixture, and the title compound is obtained with an mp: 89-90 °.

Md = + 30.9 ° (c = 1, chloroform / methanol = 1/1) Rf (toluene / ethyl acetate = 4/1) = 0.5

b) 2-Deoxy-2 - [(R) -3-hydroxytetradecanoylamido] -l-0-phosphono-3-0 - [(R) -3-tetradecanoyloxytetradecanoyl] -a-D-glu-copyranose:

The procedure is analogous to that described in Example 21b). The lyophilisate is dissolved in methanol and chromatographed on Sephadex LH 20 with the same solvent.

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

Example 20: 2-Deoxy-2 - [(R) -3-hydroxytetradecanoylamido] -

1-O-phosphono-3-O-tetradecanoyl-cc-D-glucopyranose a) 4,6-0-benzylidene-2 - [(R) -3-benzyloxytetradecanoyl-amido] -2-deoxy-l-0-dibenzylphosphono -3-0-tetradecanoyl-aD-glucopyranose

The procedure is analogous to that described in Example 13a), using tetradecanoic acid and toluene / ethyl acetate (3/1) as the solvent mixture, and the title compound is obtained with an mp: 125.5-126.5 °.

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

b) 2-Deoxy-2 - [(R) -3-hydroxytetradecanoylamido] - l-O-phosphono-3-O-tetradecanoyl-a-D-glucopyranose:

354 mg of 4,6-0-benzylidene-2 - [(R) -3-benzyloxytetradecanoyl-amido] -2-deoxy-1-O-dibenzylphosphono-3-O-tetradecanoyl-aD-glucopyranose are dissolved in 30 ml of tetrahydrofuran / water (9/1) dissolved and hydrogenated with 200 mg palladium on activated carbon for 10 hours at normal pressure. The catalyst is then filtered off and the solution is brought to pH 7.5 with tris (hydroxymethyl) aminomethane. The tetrahydrofuran is evaporated off on a Rotavapor and the aqueous solution is lyophilized. The lyophilisate is digested twice with ether and then dried.

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

Example 21: 2-Acetamido-2-deoxy-3-0 [(R) -3-hydroxytetradeca-noyl] -l-0-phosphono-a-D-glucopyranose:

a) 2-Acetamido-4,6-0-benzylidene-3-0 - [(R) -3-benzyloxytetra-decanoyl] -2-deoxy-1 -O-dibenzylphosphono-aD-glucopyranose A solution of 3.55 g 2-acetamido-4,6-0-benzylidene-

2-deoxy-l-O-dibenzylphosphono-a-D-glucopyranose and 2.1 g of (R) -3-benzyloxytetradecanoic acid in 15 ml of methylene chloride are cooled to 0 ° and 1.32 g of dicyclohexylcarbodiimide and p-dimethylaminopyridine are added. The reaction is complete after 2 hours at this temperature. The reaction mixture is filtered, evaporated and chromatographed on silica gel once with methylene chloride / methanol (50/1) and a second time with toluene / ethyl acetate (2/1).

12

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

b) 2-Acetamido-2-deoxy-3-0 - [(R) -3-hydroxytetradeca-noyl] -l-0-phosphono-a-D-glucopyranose:

2.08 g of 2-acetamido-4,6-0-benzylidene-3-0 - [(R) -3-benzyl-5 oxytetradecanoyl] -2-deoxyl-1 -O-dibenzylphosphono-aD-glucopyranose are in Dissolved tetrahydrofuran / water (9/1) and hydrogenated for 3 hours with 1 g of 10% palladium on activated carbon at normal pressure. The catalyst is filtered off and the ritrate is first evaporated and then lyophilized. The lyophilisate is dissolved in methanol, neutralized with tris (hydroxymethyl) aminomethane and chromatographed with methanol over Sephadex LH 20.

[a] o = + 43.3 ° (c = 1, methanol)

Rf (chloroform / methanol / acetic acid / water = 125 / 75/10/20) = 0.4

15

Example 22: l-0-phosphono-2,3-di-0 - [(R) -3-tetradecanoyloxyte-tradecanoyl] -aD-glucopyranose a) 4,6-O-benzylidene-1 -0-dibenzylphosphono-2, 3-di-0 - [(R) -3-tetradecanoyloxytetradecanoyl] -aD-glucopyranose

20 The procedure is analogous to that described in Example 15a). Chromatographic purification on silica gel with ether / hexane (1/1) as the eluent mixture gives the ute compound.

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

b) l-0-Phosphono-2,3-di-0 - [(R) -3-tetradecanoyloxytetrade-25 canoyl] -a-D-glucopyranose

470 mg of 4,6-O-benzylidene-1-O-dibenzylphosphono-2,3-di-0 - [(R) -3-tetradecanoyloxytetradecanoyl] -aD-glucopyranose are dissolved in 47 ml of tetrahydrofuran and 1 hour with 230 mg of palladium hydrogenated on activated carbon at normal pressure. The catalyst is then filtered off, the filtrate is evaporated and the residue is chromatographed on silica gel (chloroform / methanol / water / triethylamine = 15/10/2 / 0.2). The appropriate fractions are collected and evaporated; the ute compound is obtained as the di-triethylamine salt. 35 Rf (chloroform / methanol / acetic acid / water = 80 / 25/5/5) = 0.5

Example 23: 2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2-40 [(S) -3-hydroxytetradecanoylamido] -1 -O-phosphono-aD-glucopyranose a) 4.6 -0-Benzylidene-3-0 - [(R) -3-benzyloxytetradecanoyl] -2 - [(S) -3-benzyloxytetradecanoylamido] -2-deoxy-1 -O-dibenzylphosphono-aD-glucopyranose

45 The procedure is analogous to that described in Example 13a).

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

b) 2-Deoxy-3-0 - [(R) -3-hydroxytetradecanoyl] -2 - [(S) -3-hydroxytetradecanoylamido] -1 -O-phosphono-a-D-glucopyranose

The procedure is analogous to that described in Example 13b), so Rf (methylene chloride / methanol / ammonia = 1/1/1, lower phase) = 0.5

Fast atom bombardment (FAB) mass spectroscopy (negative mode) was carried out to characterize the compounds (Raetz, J. Biol. Chem. 259/4852 [1984]).

example

Dimensions

example

Dimensions

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1325

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1324

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1324

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1323

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1534

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1292

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1140

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647

NMR spectra

Example 1 (C5D5N)

9.82 (d, 9 Hz, 1H); 6.65 (dd, 8.8 and 3.5 Hz, 1H); 6.25 (t, 10 Hz,

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1F); 5.83 (t, 10H, 1H); 5.63 (d, 8.8 Hz, 1H); 5.35 (dd, 10 and 2.5 K, 1H); 4.0 (t, 10 Hz, 1H); 3.83 (m 1H).

Example 13 (CD3OD)

5.49 (dd, 3.5 and 7.5 Hz, 1H); 5.24 (dd, 9.5 u, 10.5 Hz, 1H); 4.17 (ddd, 2.5.3.5 and 10.5 Hz, 1H); 4.0 (m, 3H); 3.90 (dd, 2 and 12 Hz, 1H); 3.75 (dd, 5.5 and 12 Hz, 1H); 3.71 (s, Tris); 3.63 (t, 10 Hz, 1H).

Example 14 (CDC13 / CD30D)

5.77 (dd, 3.5 and 7.5 Hz, IH); 4.77 (ddd, 2.5, 3.5 and 10.5 Hz, 1H); 4.38 (dd, 9.5 and 10.5 Hz); 3.8 to 4.1 (m, 4H); 3.73 (dd, 5.5 and 12 Hz, 1H); 3.53 (t, 10 Hz, IH).

Example 15 (CD3OD)

5.71 (dd, 3.5 and 7.5 Hz, 1H); 5.48 (t, 10 Hz, 1H); (ddd, 2.4 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).

Example 16 (CDa3 / CD3OD)

5.58 (dd, 3 and 7 Hz, 1H); 5.22 (t, 10 Hz, 1H); 4.19 (dt, 3 and 10 Hz, 1H); 3.8 to 4.1 (m, 4H); 3.7 (s, Tris and dd, 1H); 3.46 (t, 10 Hz, 1H).

Example 17 (CDCl3 / CD3OD)

5.46 (dd, 3 and 7 Hz, 1H); 5.22 (t, 10 Hz, 1H); 5.17 (m, 1H); 4.19 (dt, 3 and 10 Hz, 1H); 3.9 to 4.1 (m, 3H); 3.7 (s, Tris); 3.65 (dd, IH); 3.51 (t, 10 Hz, 1H).

Example I8 / stage a) (protected) (CDC13)

7.38 (m, 15H); 5.70 (dd, 3.5 and 6 Hz, 1H); 5.63 (d, 9.5 Hz, 1H);

5.50 (s, 1H); 5.26 (t, 10 Hz, 1H); 5.08 (m, 4H); 4.38 (m, 1H);

4.08 (dd, 5 and 10 Hz, 1H); 3.95 (dt, 5 and 10 Hz, 1H); 3.73 (t, 10 Hz, 1H); 3.69 (t, 10 Hz, 1H); 2.29 (m, 2H); 1.89 (m, 2H).

Example 19 / Stage a) (protected) (CDC13)

7.2 to 7.45 (m, 20H); 6.34 (d, 9 Hz, 1H); 5.77 (dd, 3.5 and 6 Hz, 1H); 5.49 (s, 1H); 5.29 (t, 10 Hz, 1H); 5.14 (quintet, 6 Hz, 1H); 5.0 (m, 4H); 4.51 u. 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 u. 2.47 (ABX, 5.5, 7.5 and 15.5 Hz, 2H).

Example 20 / Stage a) (protected) (CDC13)

7.2 to 7.45 (m, 20H); 6.29 (d, 9 Hz, 1H); 5.74 (dd, 3.5 and 6 Hz, 1H); 5.49 (s, 1H); 5.30 (t, 10 Hz, 1H); 5.0 (m, 4H); 4.52 u. 4.42 (AB, 11 Hz, 2H); 4.44 (m, 1H); 4.03 (dd, 5 and 10 Hz, 1H); 3.94 (dt, 5 and 10 Hz, 1H); 3.7 (m. 3H); 2.15 to 2.35 (m, 4H).

Example 21 / Stage a) (protected) (CD3OD)

5.47 (dd, 3.5 and 7.5 Hz, 1H); 5.23 (dd, 9.5 and 10.5 Hz, 1H); 4.18 (ddd, 2.5.3.5 and 10.5 Hz, 1H); 3.90 (m, 3H); 3.85 (dd, 2 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).

Example 22 / Stage a) (protected) (CDC13)

7.3 to 7.45 (m, 15H); 5.92 (dd, 3.5 and 6.7 Hz, 1H); 5.60 (t, 9.5 and 10 Hz, 1H); 5.48 (s, 1H); 5.02 to 5.22 (m, 6H); 4.95 (ddd, 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).

Example 23 / Stage a) (protected) (CDC13)

7.2 to 7.42 (m, 25H); 6.49 (d, 9 Hz, 1H); 5.77 (dd, 3.5 and 6 Hz, 1H); 5.45 (s, 1H); 5.35 (t, 10 Hz, 1H); 5.0 (m, 4H); 4.54 u. 4.40 (AB, 11 Hz, 2H); 4.47 u. 4.36 (AB, 11 Hz, 2H); 4.45 (m, 1H);

3.9 to 4.1 (m, 2H); 3.6 to 3.8 (m, 4H); 2.58 (dd, 6.5 and 15.5 Hz, 1H); 2.34 (dd, 6 and 15.5 Hz, 1H); 2.2 (m, 2H).

The compounds required as starting products can be obtained as follows:

A) 4,6-0-Benzylidene-2 - [(R) -3-benzyloxytetradecanoyl-amido] -2-deoxy-l-0-dibenzylphosphono-a-D-glucopyranose (for Examples 13, 19 and 20)

a) 2 - [(R) -3-Benzyloxytetradecanoylamido] -2-deoxy-D-glucopyranose

A mixture of 5.4 g of D-glucosamine hydrochloride, 10.8 g of N - [(R) -3-benzyloxytetradecanoyloxy] succinimide and 5 ml of diisopropylethylamine in 25 ml of absolute dimethylformamide is stirred at room temperature for 24 hours. The solvent is largely evaporated off and the residue is dissolved in a mixture of 150 ml of chloroform, 300 ml of methanol and 120 ml of water. After adding a further 150 ml of chloroform and 150 ml of water, the lower phase is separated off and washed twice with an upper phase each made of 100 ml of chloroform, 100 ml of methanol and 90 ml of water. The solution is evaporated and dried in a high vacuum. The product thus obtained is used in the next stage without further purification.

For analytical purposes, a small amount of crude product is purified by chromatography (toluene / ethanol = 9/1). Mp: 150-158 °.

[a] r? = + 53 ° (c = 1, dimethylformamide) Rf (chloroform / methanol = 9/1) = 0.3

b) 4,6-0-Benzylidene-2 - [(R) -3-benzyloxytetradecanoyl-amido] -2-deoxy-D-glucopyranose

A solution of 5.83 g of 2 - [(R) -3-benzyloxytetradecanoyl-amido] -2-deoxy-D-glucopyranose, 3 g of benzaldehyde dimethyl acetal and 500 mg of p-toluenesulfonic acid. Monohydrate in 200 ml of absolute dimethylformamide is held at 55-60 ° and 30-40 mbar on a Rotavapor for about 3 hours. After that there is no more raw material. The majority of the dimethylformamide is evaporated, 500 ml of methylene chloride are added and the solution is extracted twice with 200 ml of dilute sodium bicarbonate solution and 200 ml of water. It is dried with sodium sulfate, evaporated and the residue is chromatographed (toluene / ethyl acetate = 6/4). Mp: 162-165 °.

[cc] d = + 5.5 ° (c = 1, chloroform)

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

c) 4,6-0-Benzylidene-2 - [(R) -3-benzyloxytetradecanoyl-amido] -2-deoxy-1 -O-dibenzylphosphono-a-D-glucopyranose

A solution of 4.85 g of 4,6-O-benzylidene-2 - [(R) -3-benzyloxytetradecanoylamido] -2-deoxy-D-glucopyranose in 40 ml of absolute tetrahydrofuran is cooled to -70 ° 8 ml of 1.6 m butyllithium in hexane were added dropwise. After 5 minutes at this temperature a solution of 3.8 g of dibenzylphosphorochloridate in 10 ml of benzene is added dropwise. The mixture is stirred for a further 10 minutes at -70 °, 0.3 ml of acetic acid is added and the solution is concentrated to a quarter of its 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 (sodium sulfate) and evaporated. The residue is chromatographed (toluene / ethyl acetate = 7/3). Mp: 102-105 °

[a] o = + 31.7 ° (c = 1, chloroform)

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

B) 4,6-0-Benzylidene-2,3-di-0 - [(R) -3-benzyloxytetradeca-noyl] -D-glucopyranose (for example 15)

a) Trichloroethyl 2,3,4,6-tetra-O-acedtyl-ß-D-glucopyranoside To an ice-cooled solution of 2.34 g of penta-O-acetyl-

D-glucopyranose in 9 ml of trichloroethanol is added to 0.9 ml of boron trifluoride etherate and the reaction mixture is kept at this temperature for 4 hours. The solution is then poured onto 100 ml of ice water, extracted with 100 ml of methylene chloride, the organic phase is separated off, washed with 50 ml of sodium bicarbonate solution and 50 ml of water, dried with sodium sulfate and evaporated. After chromatographic purification (silica gel, toluene / ethyl acetate = 4/1), the product crystallizes from ethyl acetate / petroleum ether.

Mp: 138-140 °

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

b) trichloroethyl 4,6-O-benzylidene-β-D-glucopyranoside

7.13 g of trichloroethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside are dissolved in a mixture of methanol and methylene chloride, the solution is cooled with ice and with a sodium methanolate solution (60 mg sodium in 26 ml of methanol) was added. After 2 hours at 0 °, the mixture is neutralized with Dowex AG 50 WX8 H +, the ion exchanger is filtered off and the solvents on a Rotavapor

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away. The residue is dissolved in 60 ml of benzaldehyde, 4.1 g of zinc chloride are added and the mixture is stirred at room temperature for 10 hours. Then it is poured onto ice water, extracted with ether, the ether solution is dried (sodium sulfate) and evaporated. The residue is chromatographed (silica gel, toluene / ethyl acetate = 4/1).

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

c) Trichloroethyl 4,6-0-benzylidene-2,3-di-0 - [(R) -3-benzyl-oxytetradecanoyl] -ß-D-glucopyranoside

930 mg are added to a solution of 800 mg of trichloroethyl 4,6-O-benzylidene-β-D-glucopyranoside, 1.4 g of (R) -3-benzyloxyte-tradecanoic acid and 20 mg of dimethylaminopyridine in 10 ml of methylene chloride, cooled to 0 ° Dicyclohexylcarbodiimide added and the reaction mixture kept at 4 ° overnight. It is then filtered, the filtrate evaporated, the residue taken up 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-Ben2ylidene-2,3-di-0 - [(R) -3-benzyloxytetradeca-noyl] -D-glucopyranose

480 mg of trichloroethyl 4,6-0-benzylidene-2,3-di-0 - [(R) -3-ben-zyloxytetradecanoyl] -ß-D-giucopyranoside are dissolved in 50 ml of a mixture of dioxane / glacial acetic acid (1/1 ) dissolved and mixed with Zn dust in portions at room temperature until no more starting material is available. Then it is filtered, evaporated and the residue is chromatographed (silica gel, hexane / toluene / ethyl acetate = 2/5/1).

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

Q2-0 - [(R) -3-beiizyloxytetradecanoyl] -3 - [(R) -3-benzyloxytetra-decanoylamido] -3-deoxy-4,6-0-isopropylidene-D-glucopyranose (for example 14):

a) 3-Azido-3-deoxy-4,6-0-isopropylidene-D-glucopyranose

1.02 g of 3-azido-3-deoxy-D-glucopyranose are dissolved in 10 ml of absolute dimethylformamide, 940 ml 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 neutralized with sodium hydrogen carbonate, the solvent is evaporated off and the residue is chromatographed (silica gel, chloroform / methanol = 9/1).

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

b) tert-butyldimethylsilyl-3-azido-3-deoxy-4,6-0-isopropylidene-β-D-glucopyranoside:

345 mg of tert-butyldimethylsilyl chloride are added to a solution of 560 mg of 3-azido-3-deoxy-4,6-0-isopropylidene-D-glucopyranose and 310 mg of imidazole in 25 ml of dry methylene chloride and the mixture is stirred for 2 hours Room temperature. Then imidazole hydrochloride is filtered off and the filtrate is shaken twice with 10 ml of water each time, dried (sodium sulfate) and evaporated. The residue is chromatographed (silica gel, toluene / ethyl acetate = 12/1).

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

c) tert-Butyldimethylsilyl-2-Ó - [(R) -3-benzyloxytetradeca-noyl] -3 - [(R) -3-benzyloxytetradecanoylamido] -3-deoxy-4,6-0-isopropylidene-β-D -glucopyranoside

A solution of 3.1 g of tert-butyldimethylsilyl-3-azido-3-deoxy-4,6-0-isopropylidene-β-D-glucopyranoside in 100 ml of methanol is hydrogenated for 2 hours with 300 mg of 10% palladium on activated carbon. The catalyst is then filtered off and the ritrate is evaporated. The residue is dissolved together with 5.35 g of (R) -3-benzyloxymyristic acid and 100 mg of dimethylaminopyridine in 50 ml of dry methylene chloride, the solution is cooled with ice and 4.1 g of dicyclohexylcarbodiimide are added. After about 3 hours at room temperature, the mixture is filtered, the solvent is evaporated off and the residue is chromatographed (silica gel, toluene / ethyl acetate = 9/1).

Rf (toluene / ethyl acetate = 6/1) = 0.42 d) 2-0 - [(R) -3-benzyloxytetradecanoyl] -3 - [(R) -3-benzyloxyte-tradecanoylamido] -3-deoxy-4, 6-0-isopropylidene-D-glucopyra-nose

To a solution of 5 g of tert-butyldimethylsilyl-2-0 - cooled to - 60 ° - [(R) -3-benzyloxytetradecanoyl] -3 - [(R) -3-benzyl-oxytetradecanoylamido] -3-deoxy- 4,6-0-isopropylidene-ß-D-glucopyranoside in 100 ml of absolute tetrahydrofuran is added dropwise to 5.2 ml of a 1 m solution of tetrabutylammonium fluoride in tetrahydrofuran. The mixture is allowed to warm to - 40 ° and is kept at this temperature until there is no more starting material (about 30 minutes). Then add 5 ml of methanol, allow to come to room temperature and evaporate. The residue is partitioned between methylene chloride and water, the organic phase is dried (sodium sulfate) and the solvent is evaporated off. Chromatographic purification (silica gel, toluene / ethyl acetate = 3/1) gives the ute compound as an anomer mixture. Rf (toluene / ethyl acetate = 1/1) = 0.48 and 0.52

D) 4,6-0-benzylidene-2 - [(S) -3-benzyloxytetradecanoyl] -2-deoxy-l-O-dibenzylphosphono-a-D-glucopyranose (for example 16)

a) 2 - [(S) -3-Benzyloxytetradecanoylamido] -2-deoxy-D-glucopyranose

The procedure is analogous to that described under Aa) and the title compound is obtained.

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

b) 4,6-0-Benzylidene-2 - [(S) -3-benzyloxytetradecanoyl-amido] -2-deoxy-D-glucopyranose

The procedure is analogous to that described under Ab) and the title compound is obtained.

[a] i> = - 3.5 ° (c = 1, chloroform)

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

c) 4,6-0-Benzylidene-2 - [(S) -3-benzyloxytetradecanoyl-amido] -2-deoxy-1-O-dibenzylphosphono-a-D-glucopyranose

The procedure is analogous to that described under Ac) and the title compound is obtained.

[a] o = + 39.2 ° (c = 1, chloroform)

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

E) 4,6-0-Benzylidene-2-deoxy-1 -0-diben2ylphosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamido] -cc-D-glucopyranose (for example 17)

a) 2-Deoxy-2 - [(R) -3-tetradecanoyloxytetradecanoyla-mido] -D-glucose

A mixture of 650 mg of D-glucosamine hydrochloride, 1.9 g of N - [(R) -3-tetradecanoyloxytetradecanoyloxy] succinimide and 0.5 ml of diisopropylethylamine in 15 ml of dimethylformamide is stirred for 20 hours at room temperature. The solvent is then evaporated off and the residue is chromatographed on silica gel with ethyl acetate / methanol (8/1) as the solvent mixture.

[cc] d = + 25 ° (c = 1, methanol)

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

b) 4,6-0-Benzylidene-2-deoxy-2 - [(R) -3-tetradecanoyloxytetra-decanoylamido] -D-glucopyranose

A solution of 5.83 g of 2-deoxy-2 - [(R) -3-tetradecanoyloxyte-tradecanoylamido] dglucose, 3 g of benzaldehyde dimethyl acetal and 500 mg of p-toluenesulfonic acid monohydrate in 200 ml of absolute dimethylformamide is about 3 hours at 55- 60 ° and 30-40 mbar held on the Rotavapor. After that there is no more raw material. The majority of the dimethylformamide is evaporated, 500 ml of methylene chloride are added and the solution is extracted twice with 200 ml of dilute sodium hydrogen carbonate solution and 200 ml of water. It is dried with sodium sulfate, evaporated and the residue is chromatographed (toluene / ethyl acetate = 6/4). Mp: 162-165 °.

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[a] o - - 1.5 ° (c = 1, chloroform / methanol = 1/1) Rf (toluene / ethyl acetate = 1/1) = 0.32 c) 4,6-0-benzylidene-2-deoxy -l-0-dibenzylphosphono-2 - [(R) -3-tetradecanoyloxytetradecanoylamido] -aD-glucopyranose

1.25 ml of a solution of 950 mg of 4,6-0-benzylidene-2-deoxy-2 - [(R) -3-tetradecanoyloxytetradecano-ylamido] -D-glucopyranose in 40 ml of dry tetrahydrofuran are cooled to - 40 ° 1.6 m butyllithium added dropwise in hexane. After 5 minutes, a solution of dibenzyl phosphorochloridate in benzene is added dropwise and the mixture is stirred at this temperature for a further 5 minutes. The mixture is then neutralized with acetic acid, the solution is evaporated and the residue is partitioned between methylene chloride and water. The organic phase is dried with sodium sulfate, evaporated and chromatographed on silica gel (toluene / ethyl acetate = 3/1).

[cì] d = + 19.3 ° (c = 1, chloroform)

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

F) 4,6-0-benzylidene-2-deoxy-l-O-dibenzylphosphono-2-tetrade-canoylamido-a-D-glucopyranose (for example 18)

a) 2-Deoxy-2-tetradecanoylamido-D-glucose

A mixture of 3 g of D-glucosamine hydrochloride, 4.56 g of N-tetradecanoyloxysuccinimide and 2.8 ml of diisopropylethylamine in 40 ml of dry dimethylformamide is stirred for 24 hours at room temperature. The reaction product is then filtered off, washed with dimethylformamide and water, dried in vacuo and used in the next reaction without further purification.

b) 4,6-0-Benzylidene-2-deoxy-2-tetradecanoylamido-D-glu-cose

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-toluenesulfonic acid monohydrate are added and the mixture for 4 hours at 55-60 ° and 30-40 mbar on the Rotavapor. The solution is then evaporated and the residue is washed with dilute sodium hydrogen carbonate solution, water and ethanol, dried in vacuo and used directly in the next reaction.

c) 4,6-0-Benzylidene-3-0- (tert-butyldimethyIsilyl) -2-deoxy-2-tetradecanoylamido-D-glucopyranose

2.2 g of 4,6-0-benzylidene-2-deoxy-2-tetradecanoylamido-D-glucose are dissolved in 250 ml of dry dimethylformamide at 60 °, 960 mg of imidazole and 2.13 g of tert-butyldimethylchlorosilane are added and the reaction Maintained at this temperature for 24 hours. After adding 480 mg of imidazole and 1 g of tert-butyldimethylchlorosilane and a further 24 hours at 60 °, there is no longer any starting material. The solvent is evaporated, the residue is partitioned between methylene chloride and water, the organic phase is washed once with water, dried (sodium sulfate) and evaporated. The residue is chromatographed on silica gel using a gradient of toluene / ethyl acetate (20/1 to 2/1). Two main fractions are obtained: the utel compound (650 mg) and the corresponding 1,3-bis-silyl compound (1.82 g). The bis-silyl compound is dissolved in 25 ml of dry tetrahydrofuran, the solution is cooled to -40 ° and 2.5 ml of a 1 normal solution of tetrabutylammonium fluoride in tetrahydrofuran is added dropwise. The anomeric silyl protective group is split off after about 1 hour at -40 °. 3 ml of methanol are added, the mixture is allowed to warm to room temperature and the solvents are then removed in vacuo. The residue is partitioned between methylene chloride and water, the organic phase is extracted once with water, then dried and evaporated. Another 1.52 g of the title compound is obtained as an anomer mixture.

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

d) 4,6-0-benzylidene-3-0- (tert-butyldimethylsilyl) -2-deoxy-1-O-dibenzylphosphono-2-tetradecanoylamido-a-D-glucopyranose:

15 672 789

To a solution cooled to -60 ° of 2.46 g of 4,6-0-benzylidene-3-0- (tert-butyldimethylsilyl) -2-deoxy-2-tetrad-ecanoylamido-D-glucopyranose in 100 ml of dry tetrahydrofuran 3.12 ml of 1.6 M butyllithium in hexane are added dropwise. s After 5 minutes, a solution of dibenzylphosphorochloridate in benzene is added dropwise at this temperature. The mixture is stirred for a further 5 minutes at -60 °, neutralized with acetic acid and then evaporated. The residue is taken up in methylene chloride and extracted with water. Working up and chromatography under silica gel with toluene / ethyl acetate (3/1) as the eluent gives the title compound.

Rf (toluene / ethyl acetate = 7/4) = 0.75 e) 4,6-0-benzylidene-2-deoxy-10-dibenzylphosphono-2-tetradecanoylamido-aD-glucopyranose 15 To a solution of 2 cooled to -10 ° , 05 g of 4,6-O-benzylidene-3-0- (tert-butylimethylsilyl) -2-deoxy-l-0-dibenzyl-phosphono-2-tetradecanoylamido-aD-glucopyranose in 40 ml of dry tetrahydrofuran is added dropwise 2.5 ml of a 1 N solution of tetrabutylammonium fluoride in tetrahydrofuran and stirred for 2 hours at 0 °. Then 3 ml of methanol are added, the mixture is evaporated and partitioned between methylene chloride and water. It is worked up as usual and chromatographed on silica gel using toluene / ethyl acetate (3/2).

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

30 G) 2-acetamido-4,6-0-ben2ylidene-2-deoxy-l-0-dibenzyl-phosphono-a-D-glucopyranose (for example 21)

a) 2-Acetamido-4,6-0-benzylidene-3-0- (tert-butyldimethylsilyl) -2-deoxy-D-glucopyranose

A solution of 5.17 g2-acetamido-4,6-0-benzylidene-35 2-deoxy-D-glucopyranose, 3.4 g imidazole and 6.3 g tert-butyldimethylchlorosilane in 300 ml dimethylformamide is at 60- Held at 70 °. After 6 hours, another 500 mg of imidazole and 1 g of tert-butyldimethylchlorosilane are added and the mixture is kept at this temperature until the reaction has ended. The solvent is evaporated off and the residue is purified by methylene chloride / water extraction and subsequent chromatography with toluene / ethyl acetate (5/1). To split off the anomeric tert-butyldimethylsilyl group, the product is dissolved in 600 ml of dry tetrahydrofuran, the solution is cooled to -40 ° and 45-14 ml of a 1 M tetrabutylammonium fluoride solution are added. After 10 minutes, the reaction is stopped by adding 17 ml of methanol. The reaction mixture is evaporated, the residue is taken up in methylene chloride and extracted with water. The organic phase is dried and evaporated.

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

b) 2-Acetamido-4,6-0-benzylidene-3-0- (tert-butyldimethylsilyl) -2-deoxy-1-O-dibenzylphosphono-a-D-glucopyranose

The procedure is analogous to that described under Ec) and 55 is obtained after chromatography with toluene / ethyl acetate (1/1) as the solvent.

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

c) 2-Acetamido-4,6-0-benzylidene-2-deoxy-1-O-dibenzyl-phosphono-aD-glucopyranose eo To an ice-cooled solution of 4.4 g of 2-acetamido-4,6-0 -benzylidene-3-0- (tert-butyldimethylsilyl) -2-deoxy-l-0-dibenzylphosphono-aD-glucopyranose in 200 ml of dry tetrahydrofuran, 6.5 ml of a 1N solution of tetrabutylammonium fluoride in tetrahydrofuran are added dropwise and hold for 2 hours at 65 0 °. Then methanol (7 ml) is added and the solution is evaporated. A solution of the residue in methylene chloride is washed with water, dried and evaporated.

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

672 789

H) 4,6-0-Benzylidene-2,3-di-0 - [(R) -3-tetradecanoyIoxytetradeca-noyl] -D-ghicopyranose (for example 22)

a) Trichloroethyl-4,6-0-benzylidene-2,3-di-0 - [(R) -3tetradeca-noyloxytetradecanoyl] -ß-D-glucopyranoside

The procedure is analogous to that described under Bc), using (R) -3-tetradecanoyloxytetradecanoic acid as the acylating agent and toluene / ethyl acetate (98/2) as the eluent mixture. Rf (toluene / ethyl acetate = 9/1) = 0.75

b) 4,6-0-Ben2ylidene-2,3-di-0 - [(R) -3-tetradecanoyloxytetrade-canoyl] -D-glucopyranose

The procedure is analogous to that described under Bd). Chromatographic purification on silica gel with toluene / ethyl acetate (15/1) as the eluent mixture gives the title compound. Rf (toluene / ethyl acetate = 9/1) = 0.25

I) Synthesis of UDP-2,3-diacyl-hexoses

1.4 g of uridine-5'-phosphoromorpholidate-N, N'-dicyclohexyl-carboxamidine salt are dissolved in 25 ml of anhydrous pyridine and dried by evaporating twice and redissolving in 25 ml of dry pyridine each. One gives to this solution

16

a solution pretreated in the same way of 1 g of a 2,3-diacyl-hexose-1-phosphate in 25 ml of pyridine and left to stand at 37 ° for 24 hours. The reaction mixture is cooled with ice. Then 50 ml of chloroform and 12 ml of 90% formic acid are added. This solution is applied to a silica gel column and unreacted starting material is eluted with chloroform / pyridine / 90% formic acid (30/30/7). The column is washed free of pyridine with chloroform / methanol (9/1) and the XJDP derivative is then eluted with chloroform / methanol / 10% water (66/33/4). Chloroform and methanol are evaporated from the peak fraction in vacuo and the aqueous suspension is filtered. The precipitate is dissolved in 100 ml of tetrahydrofuran / water (2/1) and stirred for 2 hours with Dowex AG5QWX8 (tris-hydroxymethylaminomethane form). The ion exchanger 15 is filtered off, the majority of the tetrahydrofuran is evaporated off and the aqueous solution is lyophilized. The lyophilisate is used in the next reaction step without further purification.

Analogously to that described under I), the compounds of the formula II can be obtained starting from the corresponding compounds 2o of the formula III.

Claims (9)

672 789 PATENT CLAIMS 1. Saccharides of the formula HOHO— ' \ _ / \ - W R ' 3rd \ / R ' 4th • 0 - CH- HO- / \ Y I *1 -0 OH \ -0.P ^ / j OH "Î o X I * 2 wherein Ri ', R2', R3 'and R4' are the same or different and each represent hydrogen or an optionally substituted acyl group and W, X, Y and Z are the same or different and each represent oxygen or the imino group with the proviso that a) at least one of the substituents Rt ', R2', R3 'and R4' denotes an acyl group and b) if Z and X are imino, W and Y are oxygen, a) R / and R2 'are not simultaneous (R) -3-hydroxytetradecanoyl mean if R3 'and R4' are the same and for (R) -3-hydroxyte-tradecanoyl or R4 'for (R) -3-dodecanoyloxytetradecanoyl and R3' for tetradecanoyloxytetradecanoyl β) R2 'is not hydrogen or (R) -3-hydroxytetradecanoyl if R / and R3' are hydrogen and R4 'are (R) -3-hydroxytetradecanoyl, and y) R3' is not the - (co) 2. CH2 • (CHOH) 4 • CH2OH group means when the other substituents are hydrogen, in free form and in the form of their acid addition salts. 2. A compound of formula I according to claim 1, in free form or as a chemotherapeutically acceptable acid addition salt, as a pharmaceutical. 3. Saccharides of the formula HOHO— ' OH / ■ O.P. S OH III \ _0 / \ \ / 1 I Y? R »R" 1 2 • wherein R / 'and R2 "are identical or different and each represent hydrogen or an optionally substituted acyl group and a) Y represents O and X represents NH, but a) when R ^ 'is (R) -3-hydroxytetradecanoyl, R2 "is not (R) -3-hydroxytetradecanoyl or (R) -3-hexadecanoyloxyte-tradecanoyl, β) R ["does not represent hydrogen, and y) Ri" and R2 "do not simultaneously represent the acetyl or -CO • (CH2) 10 • CH3 group, b) Y is O and X is O and c) Y is NH and X is O, with the proviso that at least one of the two substituents R ^ 'and R2 "stands for acyl and, if X or Y stands for the imino group, the substituent Ri" or R2 "attached to the imino group does not mean hydrogen, in free form and in Form of their chemotherapy-tolerated acid addition salts. 4. A compound of formula III according to claim 3, in free form or as a chemotherapeutically acceptable acid addition salt, as a pharmaceutical. 5. The compound according to claim 3, in which 5 Y oxygen, X imino, R2 "3 (R) -hydroxytetradecanoyl, Ri "3 (R) -Tetradecanoyloxytetradecanoyl mean and the ring the configuration of the a-D-glucopyranose 10 owns. 6. A chemical therapeutic composition comprising a compound of formula I, defined in claim 1, in free form or as a chemotherapeutically acceptable acid addition salt, together with a chemotherapeutically acceptable diluent. 15 agents or carriers. 7. A chemical therapeutic composition comprising a compound of formula III, defined in claim 3, in free form or as a chemotherapeutically acceptable acid addition salt, together with a chemotherapeutically acceptable agent. Contains 20 fertilizers or carriers. 8. Use of a compound according to claim 1 in free form or as a chemotherapeutically acceptable acid addition salt, for the preparation of agents for modulating the non-specific antimicrobial resistance, for systemic increase in 25 Immune response and to increase non-specific immunity. 9. Use of a compound according to claim 3, in free form or as a chemotherapeutically acceptable acid addition salt, for the preparation of agents for modulating the non-specific antimicrobial resistance, for systemic increase in 30 immune response and to increase non-specific immunity. 35 DESCRIPTION