CA2059272A1 - 4,6-0-hydroxyphosphoryl-glucosamine derivatives - Google Patents

Abstract

A B S T R A C T

4,6-O-hydroxyphosphoryl-glucosamine derivatives as shown in the following formula [I] and its pharmaceutically-acceptable salt:

[I]

wherein R1 and R2 indicate a hydrogen atom or a hydroxy group; one of R3 and R4 indicates -OCO(CH2)nCH3, -CH2(CH2)nCH3 or -O-CH2(CH2)nCH3, and the other indi-cates a hydrogen atom; 1 is an integer of 4-16; m is an integer of 4-16; and n is an integer of 6-18.

Description

2~9272 S P E C I F I C A T I O N
"4,6-O-HYDROXYP~OSPHORYL-GLUCOSAMINE DERIVATIVES"
[Technical Field]
This invention relates to novel 4,6-O-hydroxyphosphoryl-glucosamine derivatives and pharmaceutically-acceptable salts thereof.
The compcunds of the present invention show lipid A-like activity, and are useful as pharmaceutical drugs such as immunopotentiation agent and anti-tumour agent.
[Background Art]
Surface layers of Gram-negative bacteria are com-posed of a cell membranes, a call wall peptidoglycan, and an outer membrane. The outer membrane contains lipopolysaccharides (hereinafter abbreviated LPS).
LPS is a main ingredient of endotoxin which induces endotoxin shock, and consists of an acidic protein component, a high-molecular polysaccharide component, and a phospholipid component.
LPS induces various morbid conditions such as pyrogenesis, bleeding, arthritis, and encephalomyelitis.
LPS is also known to show a host protection effect of immune-activating mechanism such as macrophage-activation, B-cell blastogenesis activity, and cell-mediated immunity-activation, as well as antitumour effect such as IFN(interferon) induction and TNF(tumour necrosis factor) induction.
A main part of LPS which shows these activities 2~9272 among said three parts is a phospholipid part, which is called lipid A. The lipid A comprises fatty acid resi-due and phosphoric acid both of which are combined with disaccharide amine, and has the following formula [Japanese Bacteriology Journa]. 40(1), 57(1985) and Proc.Natl.Acad.Sci.USA 80, 4624(1983)]:

o o o_ ~, o ., o o ~
,1 .~

IY

o W ~ s~
/\ ~ ~ .
~o o~ o ~V5~272 A recent study has revealed that either a non-reducing subunit or a reducing subunit as shown above alone can show the lipid A-like activity, and various analogues have been synthesized based on this finding.
Examples of the analogues are disclosed in European Patent Application Disclosure No.224260, Japanese Patent Application Disclosure No. 62888/90, and Japanese Patent Application Disclosure No. 25494/90, etc..
As described above, extensive studies have been conducted in order to obtain lipid A analogues, specifi-cally by modifying them with various substituents and by changing substituent sites introduced. However, no lipid A analogue has been developed which can be pharmaceutically applicable, mainly because the same substituent shows the different activities depending on its introduced site, thus making the study on pharma-ceutical application of the lipid A-like analogues difficult. Therefore, lipid A analogue of higher activ-ity and lower toxicity is expected to develop.
[Disclosure of Invention]
The object of the invention is to produce novel compounds which show a more effective lipid A-like activity and low toxicity.
Inventors of the present invention have been ener-getically studied on lipid A derivatives in order toattain said object. As the result, the inventors have found novel compounds which show strong lipid A-like 20~27~

activity such as mytogenic activity of varying strengths depending on analogues, TNF-inducing activity, and IFN-inducing activity, and whi.ch nevertheless show low toxicity, and have completed the present invention based s on this findings.
Novel 4,6-O-hydroxyphosphoryl-glucosamine deriva-tives according to the present invention have the fol-lowing general fOormula [I]:

ll 6 HO-P

\O_ ~ O

~ ~ R
I NH

fH-R3 C=O [I]

(CH2)m (ICH2)1 wherein Rl and R2 indicate a hydrogen atom or a hydroxy group; one of R3 and R4 is -C(CH2)nCH3~ -CH2(CH2)nCH3 or -O-CH2(CH2)nCH3 and the other is a hydrogen atom; 1 is an integer of 4-16; m is an integer of 4-16; and n is an integer of 6-18.
This invention also relates not only to said com-pounds but also to their pharmaceutically-acceptable salts. Examples o~ these salts are inorganic alkali metal salts, alkali-earth metal salts, and organic amine ~V~9272 salts. Specifically, salts of the compounds with sodium, potassium, lithium, calcium, triethanolamine, diethanolamine, monoethanolamine, triethylamine, etc.
are exemplified.
4,6-_-hydroxyphosphoryl-glucosamine derivatives [I]
according to the present invention have two structural characteristics as follows. First, the pyranose ring is acylated at the 3-position with a- or ~-alkylated fatty acids, a- or ~-alkoxylated fatty acids, or a- or ~-acyloxylated fatty acids. Second, hydroxyphosphoryl groups [>P(0)OH] are introduced to the 4~ and 6-positions of the pyranose ring. The present compounds [I] are expected to be useful through these characteris-tics as pharmaceutical drug such as immune-activating agent. The present invention also includes all stereoisomers of the compounds ~I] and a mixture thereof.
These compounds [I] can be produced according to the following reaction steps:

20~272 ~0~
\ \~ \
HO Rl l R~ H or -OSE
[ 1 ] SE=-CH2CH2si ( CH3 ~ 3 R2 ' FIRST STEP CH3 ( CH2 ) 1CHCH2C2H

R2 ' =-OSEM
or -H

SEM=-CH20CH2CH2si ( CH3 ) 3 _~~0 '' __ ~ O\

~ R ' [2] C=O

fH-R2 ' ( ICH2) 2~272 SECOND STEP CH3(CH2)mCHCHC02H

one of R3, R4 iS -C(CH2)nCH3~
-CH2(CH2)nCH3 or -ocH2(cH2)ncH3;
the other of them is Hydrogen ~0--O~

R50 Rl' NH
C=O

[3] CH-R2' l l ¦ R5=-COCHCH(CH2)nCH3 ( ICH2) THIRD STEP

205~272 HO
Ho /~ o\

R50 ~ Rl' NH
C=O

[4] CH-R2' ( ICH2)1 FOURTH
STEP

O~

R50 ~ ~ Rl' NH
C=O

[5] CH-R2' ( ICH2)1 20~272 FIFTH STEP
(Rl'=-H and R2'=-H) , 1l ~ \
\0 ~ ~ \
RsO~
NH
F=

[6] fH2 Rl=H,OH
fH-R2 R2=H,OH
------ ( ICH2 ) 1 SIXTH STEP

1l \O

` ~ Rl ' NH
C~I-R3 C=O
CH-R4 1 [I]

(CH2)m t ( fH2 ) 1 2~59272 Description of said flow 1 is in detail as follows:
<the first step>
The known compound [1] derived from D-glucosamine tsee Japanese Patent Disclosurle No. 197582/86) is amidated to form an amide compound [2]. This procedure is performed by making the com,pound [1] to react with a fatty acid compounds whose hydroxyl group at 3-position is protected with 2-~trimethylsilyl)ethoxymethyl group (-SEM group) or with straight-chain fatty acid compound having no hydroxy group, in an inert solvent such as dichloromethane, in the presence of a condensation agent such as dicyclohexylcarbodiimide (DCC) and l-ethyl-3(3-dimethylami.nopropyl)-carbodiimide hydrochloride (WSC-HCl).
<the second step>
The compound [2] obtained in the first step is caused to react with an acylating agent thereby acylating the hydroxyl group at the 3-position of the ring in order to obtain a compound [3]. Examples of the acylating compound ara a- or ~-alkyl fatty acid, a- or ~-acyloxy fatty acid and a- or ~-alkoxy fatty acid (R50H). This step is performed in a solvent such as dichloromethane in the presence of dimethyiaminopyridine (DMAP) of catalytic amount, and a condensation agent such as DCC and WSC-HCl.
<the third step>
The compound [3] obtained in the second step is 2~9272 hydrolyzed with an acid such as acetic acid solution, in order to eliminate the protection groups at the 4- and 6-position, yielding a compound [4].
<the fourth step>
.S The compound [4] is made to react with phenyl dichlo~ophosphate in an inert solvent such as dichloromethane in the presence of a base such as pyri-dine and DMAP in order to obtain a compound [5].
<the fifth step>
This step is performed in order to eliminate pro-tection groups at hydroxy groups when Rl' and/or R2' are protected hydroxy groups. Therefore, when both Rl' and R2' are hydrogen atoms, this step is not required. When both Rl' and R2' are protected hydroxy groups, both protection groups may be simultaneously eliminated, or they may be separately eliminated in a stepwise manner.
The elimination step can be performed in various known manners. For example, when the Rl' and~or R2' of the compound [5] are -OSE, the compound [5] is dissolved in an inert solvent such as dichloromethane, and an acid such as boron trifluoride etherate (BE3.OEt2) or a fluo-ride ion generating agent such as tetrabutylammonium fluoride is added to the solution in order to easily eliminate the protection groups.
It is noted that the protection groups in the Rl' and/or R2' are not restricted to -OSE described above, and they may be, for example, benzyl groups (-Bn group).

, 20~2~2 When they are benzyl groups, they are easily eliminated through catalytic hydrogenation in the presence of a catalyst such as platinum and palladium.
<the sixth step~
The compound [6] is hydrogenated over platinum dioxide (Pt32), etc. in a solvent such as ethanol, methanol, and acetic acid to afford an objective com-pound [I].
This objective compound [I] can be also prepared according to the following reaction from a lipid A ana-logue obtained in a known manner.

2~9272 OH
HO \ ¦
HO / \ O

R50 ~- ~ ~ R
H
C=O
CH2 Rl=H,OH
¦ R2=H,OH

[7] CH-R2 ( ICH2 ) 1 IR3 IR4 CH3 R5=-COCHCH(CH2)mCH3 SEVENTH
STEP

\o ~ O\

~ Rl NH [I]
C=O
CH2 Rl=H,OH
R2=H,OH
ICH_R2 CH2) ~5~272 ~ 15 -This flow 2 comprises the following seventh step.
<the seventh step>
A compound [7] (for example, see Japanese Patent Disclosure No . 62888/90) is made to react wi-th a conden-satlon agent such as DCC and WSC.HCl in a solvent such as tetrahydrofuran~THF), dichloromethane, and chloro-form. By this reaction, the compound [7] is cyclized by intramolecular condensation to afford the objective compound [I].
Among a- or ~3- alkylated fatty acid, a- or ~-acyloxylated fatty acid, and a- or ~-alkoxylated fatty acid, some are known, and others are easily prepared from known compounds. Examples of methods for producing these substituents are as follows:
~Method for Producing a-alkyllated fatty acid]
Flow 3 CH3(CH2)nCH2 CH3(CH2)nCH2x CH3(CH2)mCH2CH2COOH CH3(CH2)mCH2CHCOOH

wherein X indicates halogen.
This reaction i5 performed in aprotic solvents such as tetrahydrofuran (THF) containing hexamethylphosphoric triamide (HMPA), etc.. First, a straight chain carbox-ylic acid having the corresponding number of carbons is added with two equivalents of strong base such as lith-ium diisopropylamide (LDA) in order to form dianion of the carboxylic acid. Next, the dianion is made to react with straight chain alkylhalide having the corresponding 205~272 number of carbons to obtain the a-alkylated fatty acid.
[Method for Producing ~-alkylated fatty acid]
Flow 4 CH3(CH2)nCH2 CH3tcH2)ncH2Mgx CH3(CH2)mCHO ~ > CH3(CH2)mCHOH -- >

CH3(CH2)nfH2 CH3(CH2)nfH2 CH3(CH2)mC=0 CH3(CH2)mC=CHCOOEt CH3~CH2)nfH2 CH3(CH2)nfH2 CH3(CH2)mCHCH2COOEt CH3(CH2)mCHCH2COOH

Straight chain alkyl halide having the correspond-ing number of carbons is made to react T~ith metal magnesium in an aprotic solvent such as THF in order to form Grignard reagent. The straight chain aldehyde hav-ing the corresponding number of carbons are made to react with the Grignard reagent to yield a secondary alcohol. This alcohol is oxidized with an oxidizing agent such as pyridinium chlorochromate (PCC) and Jones reagent in an inert solvent such as dichloromethane to form a ketone.
Separately, triethyl phosphonoacetate is added with 2s base such as sodium hydride in order to form carboanion.
The Wittig reaction of the carboanion and the aforementioned ketone yields a, ~-unsaturated ester.

2~5~27~

Next, this ester is subjected to hydrogenation in a solvent such as ethyl acetate in the presence of palladium carbon to form saturated ester. Finally, this ester is hydrolyzed in a solvent such as aqueous ethanol in the presence of base such as potassium hydroxide to obtain ~-alkylated fatty acid.
[Method for Producing a- or ~-acyloxylated fatty acid]
Flow 5 OH OH
CH3(CH2)mCHCH2COOH >, CH3(CH2)mCHCH2cOOcH2cOc6H5 CH3(CH2)nclO

CH3(cH2)ncox ~ CH3(CH2)mcHcH2cOocH2coc6H5 or CH3(CH2)nCOOH

f CH3(CH2)nclO

> CH3(CH2)mCHCH2CH

2- or 3-hydroxycaroboxlyic acid of straight chain having the corresponding number of carbons (for example, 3-hydroxycarboxylic acid is shown in the flow 5) is acylated as follows. First, the hydroxycarboxylic acid is reacted with phenacyl bromide in a solvent such as ethyl acetate in the presence of a base such as triethylamine to form phenacyl ester. The hydroxy group at the 2- or 3-position of the phenacyl ester is 2~5~272 acylated by being reacted with acid chloride having the corresponding number of carbons in the presence of a base such as pyridine, or with straight chain carboxylic acid having the corresponding number of carbons in the presence of a condensation agent such as DCC and WSC HCl in an inert solvent such as dichloromethane. Next, the acylated phenacyl ester is treated with zinc powder and acetic acid in order to eliminate a phenacyl group. As the result, a- or ~-acyloxylated fatty acids are obtained.
[Method for Producing a- or !3-alkoxylated fatty acid]
Flow 6 OH OH
CH3(CH2)mCH2CHCOOH CH3(CH2)mCH2CHCOOAl OH OH
CH3(CH2)mCH2CHCH2OH > CH3(CH2)mCH2CHCH2OTr CH3(CH2)ncH2Ol CH3(CH2)ncH2OMs ~ CH3(CH2)mCH2CHcH20Tr >
CH3(CH2)ncH2lO CH3(CH2)ncH2lO
CH3(CH2)mCH2CHCH2OH > CH3(CH2)mCH2CHCOOH
wherein A~ indicates an alkyl group such as methyl group and ethyl group, Tr indicates a protPction group such as trityl group for a hydroxyl group, and Ms indicates a mesyl group or tosyl group, etc.................................... ;`
2- or 3-hydroxycaroboxylic acid having the 20~27~

corresponding number of carbons (for example, 2-hydroxycarboxylic acid is shown in the flow 6) is esterified with methyl iodider ethyl iodide or the like in an aprotic solvent such as benzene in the presence of a base such as 1,8-diazabicyclo[5,4,0]7-undecene (DBU).
The obtained ester is reduced with a reducing agent such as lithium aluminium hydride in a solvent such as THF to yield diol. Next, of OH groups in the obtained diols, only primary OH group is selectively protected with a protection group such as trityl group. The protected alcohol is reacted with straight chain alcohol which has the corresponding number of carbons and which is mesylated or tosylated, in an aprotic solvent such as THF, in the presence of a base such as potassium hydride or sodium hydride and a phase transfer catalyst such as tetra-n-butylammonium iodide, to introduce an alkoxy substituent. Next, the protection group (trityl group) at the primary hydroxy group is eliminated by using an acid such as p-toluensulfonic acid. Finally, the obtained alcohol is oxidized with an oxidizing agent such as Jones reagent and PCC in order to obtain ~-alkoxy substituted fatty acid.
Here is the description of pharmaceutical applica-tions of the compounds according to the present invention.
The compound of the general formula [I] is generally administered systemically or topically, and 20~272 orally or parenterally.
Although administered dose varys with age, weight, and symptom of a patient in question, therapeutic effect desired, administration route~ treatment period, etc., O.O1-lOOmg of the compound is generally administered orally or parenterally to an adult once to several times a day.
Solid compositions prepared to be orally admini-stered according to this invention include tablets, powder, granules, etc.. These solid compositions are obtalned by mixing at least one active substance with at least one inert diluent or dispersing agent. Examples of the diluents or dispersing agents include lactose, mannitol, glucose, hydroxypropylcellulose, crystalline cellulose, starch, polyvinylpyrrolidon, magnesium alumlno-metasilicate, etc.. Other than these diluents or dispersing agents, absorbents such as anhydrous sil-ica powder, etc. may be mixed with the compound [I].
Further, the solid compositions may contain additives other than inactive diluents, according to a general method.
The tablets or pills stated above may be coated, if desired, with acid soluble films or enteric coating films such as saccharose, gelatin, hydroxypro-pylcellulose and hydroxypropylmethylcellulose phthalate.Some tablets or pills may be coated, if desired, with two or more these films. Also powder or granules may 20~272 be encapsulated within capsules made of gelatin, ethylcellulose, etc..
Examples of liquid compositions for oral admini-stration include pharmaceutically acceptable emulsion, solution, suspension, syrup, erixil, etc.. These liquid compositions may contain inert diluents generally utilized, e.g., purified water, ethanol, vegetable oils, emulsifying agent. Further, auxiliary agents such as moisturing agents or suspending agents, edulcorants, flavouring agents, perfumes, and antiseptics may be con-tained in the compositions.
Injectable preparations for parenteral administra-tion may contain sterilized aqueous or non-aqueous solvents, solubilizing agents, suspending agents, and emulsifying agents. Examples of the aqueous solvents, solubilizing agents, and suspending agents include dis-tilled water for injection, saline solution, cyclo-dextrin and its derivatives, organic amines such as triethanolamine, diethanolamine, monoethanolamine, and triethylamine, and inorganic alkalines.
Examples of the non-aqueous solvent include propy-leneglycol, polyethyleneglycol, vegetable oils such as olive oil, and alcohols such as ethanol. Examples of non-aqueous solubilizing agents include surfactants (which forms mixed miscells) such as polyoxyethylene hydrogenated castor oil, and sucrose fatty acid ester, lecithin, and hydrogenated lecithin twhich forms ,;

20~27~

liposomes), etc.. Emulsion preparations are also included in the non-aqueous solution preparation, which are obtained by using non-aqueous solvent such as vegetable oils with emulsifying agents such as lecithin, polyoxyethylene hydrogenated castor oils, and polyoxyethylenepolyoxypropyleneglycol.
Examples of other composit,ons which are adminstered via any route other than per os are topical solutions, liniments such as ointments, suppositories, pessaries, etc., each of which contains at least one active substance and is prepared according to the disclosed method.
Hereinafter are described pharmacological actions of the compounds according to this invention by way of experimental examples. The compounds according to this invention have showed significant effects for various tests such as IL-l-producing activity, and also showed low toxicities for tests such as local Schwartzman reaction, and pyrogenicity. Some accivities are stated as follows.
Experimental Example 1 (2- production stimulating activity in neutrophils) 2- production stimulating activity in neutrophils was evaluated utilizing the following experimental system [see J. Exp. Med., 160, 1656-1671. (1984)].
To the peritoneal cavity of C3H/HeN mouse (male~

8-9 week-aged), physiological saline containing 2~5~27~

0.2% (w/v) casein was administered. Three hours later, peritoneal exudate cells (90% or more of which are neutrophils) were collected. These cells (1.7 x 1o6 cells~m~tube) were incubated in the presence of the compound (10 ~g/m~) according to this invention at 37C for 60 minutes. After addition of 80 ~M of cytochrome C and 0.1 ~M of formyl-methionyl-leucyl-phenylalanine (FMLP), the mixture was incubated in the presence of or in the absence of superoxide dismutase (SOD) at 37C for 10 minutes. Then, SOD-inhibitable cytochrome C reduction was estimated from the differ-ence between absorbances at 550 nm and 541.7 nm, and from molar absorption coefficient (16.5 x 103).
2- production-stimulating activity was shown in Stimulation % in the following formula.

the amount Of 2- produced in the presence of the compound Stimulation = accordi~L~o-f 2- produced x 100-100 ( ) in the absence of the compound according to the invention The compound according to the present invention showed the activity in the followiny Table 1.
Control compound in the Table 1 is 2-deoxy-2[(3R)-3-hydroxytetradecanamide]-4-O-phosphono-3-0-[(3R)-3-tetradecanoyloxytetradecanoyl]-D-glucopyranose (GLA-60).

2~5~2~2 Table 1 .. . ..... _ CompoundStimulat ion _ ~
_Experiment 1 Experiment 2 No compound 0 0 Control 60 60 Example 155 __ Example 4 __ Experimental Example 2 (TNF-producing activity) TNF-producing activity was evaluated utilizing the following experiment system.
The first stimulating agent, 5% Corynebacterium parvum suspension (0.2 m~ physiological saline solution was intravenously administered to ICR mouse (female, 6-7 week-aged). Nine days later, the second stimulating `
agent, the compound of this invention was intravenously administered to the same mouse at 10 ~g~mouse. In 90 minutes, 0.5 - 1 m~ of blood was taken from the retro orbital plexus. The obtained blood was allowed to clot at room temperature for five to six hours, and centri-fuged at 7200 x g for five minutes to separate serum.
The obtained serum was incubated at 56C for 30 minutes for inactivation before use in the following experiment.
TNF activity in the serum was measured with cyto-toxicity assay using L929 cells. L929 cells were pre-pared in concentration of 6 x 104 cells/well (0.1 m~) RPMI 1640 medium containing 10% FBS (fetal bovine serum) 20~272 and 2 ~g/m~ actinomycin D in 96-well plates. Serial dilution of obtained serum in RPMI 1640 medium contain-ing 10% FsS was added to each well in the plate (0.1 m~/well). After a 48 hr incubation at 37C, the viable cells were fixed with methanol. These cells were then s~ained with 0.2% crystal violet, and the dye was extracted with 1% SDS (sodium dodecyl sulphate).
Next, absorbance at 550 nm was measured. Finally, cytotoxicity ratio (%) was calculated according to the following formula, and the reciprocal of dilution of the serum showing 50% cytotoxicity was determined for TNF
titer in serum (U/m~).
Cytotoxicity (%) = [ODs50 (medium alone) - OD550 (serum obtained by administering compounds of the invention)] x 100/ODsso (medium alone) The compounds of this invention revealed activities shown in the following Table 2.
Table 2 ~ __ _ Compound The Amount of TNF in the Serum (U/m~) Experiment 1 Experiment 2 No compound < 10 < 10 Control 158000 80000 Example 1133000 __ Example 2 __ 102000 20~27~

Experimental Example 3 (Mitogen Activity) Mitogen activity of the compounds according to the present invention was evaluated by utilizing the following experimental system [see Eur.J.Immunol., 14, 109-114. (1984)].
The spleen of C3H/HeN mice (male, 6-10-week age) were isolated in an aseptic manner. The spleen tissue was loosened in Dulbecco's modified Eagle medium (DMEM) and then subjected to a stainless mesh in order to fil-ter the spleen cells. Next, erythrocytes contained in the collected cells were made to hemolyzed, and the i obtained cells were suspended in RPMI 1640 medium con-taining 5% FsS for use.
Evaluation of mitogen activity of the compounds according to the present invention was made by measuring the amount of 3H-thymidine in~orporated into the cells during the culture of the cells which were treated with the compounds according to the present invention.
First, the spleen cells were transferred to a 96-well plate at 5 x 105twell (100 ~). To each well, the com-pounds according to the present invention of a given concentration (100 ~) was added, and the obtained solution in each well was cultured under 5% CO2 at 37C
for 48 hours. After that, 3H-thymidine was added at 1 ~Ci/well (50 ~), followed by a culture for four hours. The obtained cells were washed with phosphate buffered saline (PBS), and the amount of 3H-thymidine 20~272 incorporated into the cells (the amount of radio-activity) was determined by a lic~uid scintillation counter. The result was shown by calculating the fol-lowing stimulation index.

The Radioactive Amount when the The Radioactive compound is added - Amount when the to the meclium medium only is Stimulation Index = ( The Radioactive Amourlt when the medium only is added (cpm) The compounds according to the present invention 0 showed the following activities.
Table 3 Compound Stimulati on Index Experiment 1 Experiment 2 No compound 0 0 Control 15.8 8.4 Example 1 32.1 __ Example 4 __ 20.7 Experimental Example 4 (Colony Stimulating Factor-Inducing Activity) By utilizing the following experimental example, in vivo colony stimulating factor (CSF) inducing activity of the compounds according to the present invention was evaluated [see Immunology, 21, 427-436. (1971)].

5 ~g of the present compounds according to the pre-sent invention were administered to the caudal vein of C57BL mice (male~ 8-10-week age). After six hours, the 2~9272 blood was sampled from the plexus venous orbitalis of said mice. This blood was allowed to stand at 4C for two hours for sufficient coaggulation, and then centri-fuged at 1500 x g. The resultant supernatant solution was collected for use as a CSF-containing serum sample.
Separately, from the femora of mice which were the same series as the mice used for sampling the serum, the bone marrow cells were sampled. Specifically, both ends of the femora isolated in an aseptic manner were cut, and an injection needle was inserted to one end to aspirate the bone marrow cells into a culture solution ( DMEM ) . The obtained cell suspension was sufficiently stirred, washed with the culture solution several times, and suspended in the culture solution again.
Next, the cell culture prepared as said was adjusted to a final concentration of 105/m~ by utilizing a medium (DMEM) containing 0.3% agar, 25% horse serum, and 50 ~M 2-mercaptoethanol. To this solution, O.lm~ of said serum sample which had been diluted to 1/3 with said DMEM medium was added, and the obtained solution was transferred to a culture plate of 35mm diameter.
Then, the resultant culture solution was cultured under 7% C2 at 37C for seven days to form colonies. Such colonies as containing at least 20 cells which are not separated so far were counted, and the numbers thus obtained were taken CSF inducing activity of the com-pounds according to the present invention.

205927~

This activity was shown in the following Table 4.

Table 4 The Number of Colony Formed/
Compound The number of Bone Marrow Cells (105) _ ._ ExPeriment 1~xperiment 2 .--No compound 0 0 Control 89 53 Example 1 153 __ Example 4 __ 66 0 Experimental Example 5 (lethal toxicity in galactosamine-sensitized mice) Lethal toxicity in galactosamin-sensitized mice was evaluated by utilizing the following experiment system [see J.Biochem., 98, 395-406. (1985)].
To C57BL mouse (male, 7-week aged), 10 mg/mouse of D-galactosamine/HC~ was intraperitoneally admini-stered. Immediately after that, the compound of this invention was intravenously administered. After these administrations, general conditions of the mouse were observed every one hour for seven hours, and every day from the following day to the seventh day.
The compound of this invention showed lethal toxic-ity as in the following Table 5;

2 ~

Table 5 CompoundLD50 (~alactosamine-load)~ k~-Lipid A 0.3 :
Control 3.0 Example 1 31.3 Example 4 71.1 * ~ynthetic lipid A (LA-15-PP, 506, manufactured by Daiichi Xagaku Yakuhin) [Best Mode of Carrying Out the Invention]
Hereinafter is the detailed description of methods for producing the final objective compound [I] and its intermediates [1] to [7] by way of examples. However, it should be understood that the present invention is not restricted to these examples. For example, the fol-lowing compounds are also included in the present invention.
1,5-anhydro-2-deoxy-2-dodecanamido-3~ (2RS~-2-hexadecyloxydodecanoyl}-4,6-0-hydroxyphosphoryl-D-glucitol 1,5-anhydro-2-deoxy-2-dodecanamido-3-_-{(3RS)-3-hexadecyloxydodecanoyl)-4,6-0-hydroxyphosphoryl-D-glucitol 1,5-anhydro-2-deoxy-3-0-~(3RS)-3-dodecylhexadecanoyl}-2-hexadecanamido-4,6-0-hydroxyphosphoryl-D-glucitol 1,5-anhydro-2-deoxy-3-O-{(2RS)-2-dodecyloxyoctadecanoyl}-2-~(3R)-3-hydroxydodecanamido}-4,6-O-hydroxyphosphoryl-D-glucitol 1,5-anhydro-3-0-{(3RS)-3-decyloctadecanoyl~-2-deoxy-2-{(3RS)-3-hydroxyhexadecanamido}-4,6-O-hydroxyphosphoryl-D-glucitol 1,5-anhydro-2-deoxy-2-dodecanamido-3-O-~(2RS)-2 dodecyloxyoctadecanoyl}-4,6-O-hydroxyphosphoryl-D-glucitol 1,5-anhydro-3-0-{(3RS)-3-decyloctadecanoyl}-2-deoxy-2-hexadecanamido}-4,6-O-hydroxyphosphoryl-D-glucitol 2-deoxy-3-O-{(2RS)-2-dodecylhexadecanoyl}-4,6-O-hydroxyphosphoryl-2-tetradecanamido-D-glucopyranose 2-deoxy-4,6-O-hydroxyphosphoryl-2-tetradecanamido-3-0-{(2RS)-2tetradecanoyloxytetradecanoyl}-D-glucopyranose 2-deoxy-3-O-{(2RS)-2-dodecyloxyhexadecanoyl}-4,6-O-hydroxyphosphoryl-2-tetradecanamido-D-glucopyranose 2-deoxy-3-0-{(3RS)-3-dodecylhexadecanoyl}-4,6-O-hydroxyphosphoryl-2-tetradecanamido-D-glucopyranose 2~27~

2-deoxy-4~6-o-hydroxyphosphoryl-2-tetradecanamido-3-0-{(3RS)-3-tetradecanoyloxyte-tradecanoyl}-D-glucopyranose 2-deoxy-3-0-{(3RS)-3-dodecyloxyhexadecanoyl)-4,6-0-hydroxyphosphoryl-2-tetradecanamido-D-glucopyranose 2-deoxy-3-0-{(3RS)-3-dodecyloxyhexadecanoyl)-2-~(3RS)-3-hydroxyoctadecanamido}-4,6-0-hydroxyphosphoryl-D-glucopyranose Relations of the compound [I] according to the pre-sent invention, intermediates [1] to [7] for producing the compound [I], and compound numbers are shown in the following Table 6.

2~9~7~

X ___------ r r o _ , _ _ H ~ m v ~ ~ ~ ~ ~: H )~ ~ ~1 ~ ~
r-- r / U // / /--/ .r /--/--~ ~D ~D ~D ~D ~D ~D ~D ~D / ~D /, /, / /
~ ~ ~ Q U ~ a) ~ i~ ~ -,~ r /
O _ Lt) In U~ In Ln U) Ln Lr~ Lr) Ln / / / /
o ~ ~a ~ u ~ ~ q~ t~ ~ ,~ r / /
~ ~ ~ ~ ~ d' ~ ~ ~ ~ ~ ~ / / /
~ ~ ~a u ~ a) 4~ ~ ~ ~ ~r / / /
~ ~ ~- ~ ~ ~ ~ ~ ~ ~ /, / / /
~ ~ n u ~ Q U ~ ~ ,~ ~a / / /
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ / / / /
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ / / /
~D ~1 r-l ~1 r-l ,_1 ~1 r-l ~1 ~1 ~1 / / / /
,~2 E~ o ~ 00 o 0 o o o o o o o ,_~
E~ ~ :~ 5~: ~a~
~ ~ :~ ~ ~ :r: ~ ~ ~C~ ~: ~ ~ ~ _ ~
8 x~ o _ ~ _ __ _ o V ~ _ o V
~ ~ ~ ~ ~ ~ ~7 ~ r~ ~
5: ~ :: ~: ~ ~ ~ V ~: :~
o ~ ~ ~ ~ o .
,, ~ ~ ~ ~ ,, ~)~ ~ ~ ~1 ~ ~ ~ ~ N ~
~; :~ ~: 5: ~ 5~ :~ X ~ c~ V I~ 1: ~ ~
_ U O O ~i ~S r _ ~g O O ~ _ ~ o 0 ~ o ~ ~ o o o o o o o o .- ~ ~ _ ~ ~1 ~1 ~1 ~1 ~1 ~1 ~
- ~ ~ ~ ~ ~ ~ ~ x ~ - ~ ~ ~: ~ ~:
P~ ~ ~ ~ ~ ~ ~ I ~ ~ ~ O O O :~:
- - - - - - o 2~27~

Example 1 1~5-Anhydro-2-deoxy-4~6-o-hydroxyphosphoryl-2-{(3R)-3-hydroxytetradecanamido}-3-O-{(2R)-2-tetradecanoyloxytetradecanoyl}-D-glucitol; (Compound A) (the first step) 1,5-Anhydro-2-deoxy-4,6-O-isopropyriden-2-[(3R)-3-{2-(trimethylsilyl)ethoxymethoxy}tetradeCanamido]-D-glucitol; (compound 2a) 2-Amino-1,5-anhydro-2-deoxy-4,6-0-isopropyriden-D-glucitol(la) (5~8g)~ (R)-3-{2-(trimethylsilyl)-ethoxymethoxy}tetradecanoic acid (10.7g), and WSC-HCl (llg) were dissolved in dichloromethane (44m~)~ and the resultant solution was stirred under ice-cooling for reaction. The reaction was monitored utilizing a silica gel thin layer chromatography (chloroform:methanol =
20:1). After the reaction went to completion, the mix-ture was diluted with dichloromethane, washed with water, and dried with anhydrous magnesium sulfat~. The obtained solution was evaporated to remove the solvent, and the resultant residue was purified by a silica gel column chromatography (chloroform:methanol = 100:1). A
colourless crystal compound (2a) (14g, yield: 88~) was obtained.
[a]D: -6.90 (c = 1.10, CH2C~2) m. p.: 61.0 - 62.0C

.

7 ~

IR(nujol)cm~l:
3450, 3280, 1640, 1550, 1460, 1380, 860 - 835 H-NMR(30OMHz)~TMS CDC~3:
0.03(9H, s, Me3Si), 0.85 - 0.97(5H, m, CH2TMS, -Me), 1.20 - 1.60(20H, m, -CH2-), 1.43, 1.52(6H, each s, -CMe2)~
2,38, 2,48(2H, AB part of ABX, JAB = 14,9 Hz, JAX = 6.6 Hz, JBX = 4-0 Hz, -CH2CO-), 3.22(2H, m, H-l, H-5), 3.44(1H, brs, -OH), 3.54 - 3.65(4H, m, H-l, H-~, -CH2CH2TMS), 3.72(1H, t, J = 10.5 Hz, H-6), 3.87 - 3.92(2H, m, H-6, CH-OSEM), 4.01 - 4.09(2H, m, H-2, H-3), 4.67, 4.75(2H, AB, JAB = 6.6 Hz, -OCH2O-), 6.47(1H, d, J = 7.0Hz, NH) (the second step) 1,5-Anhydro-2-deoxy-4,6-O-isopropyriden-3-O-{(2R)-2-tetradecanoyloxytetradecanoyl)-2-[(3_)-3-t2-(trimethylsilyl)ethoxymethoxy}-tetradecanamido]-D-glucitol; (Compound 3a) The compound 2a (1.73g), (R)-2-tetradecanoyloxytetradecanoic acid (1.4g), WSC-HCl (1.19g), and DMAP (189mg) were dissolved in dichloro-methane (14.7m~), and the obtained solution was stirred 2 7 ~

for three hours for reaction. The reacted solution was diluted with dichloromethane, washed with water, dried with anhydrous magnesium sulfate, and concentrated under a reduced pressure. The obtained residue was purified by a silica gel column chromatography (n-hexane: ethyl acetate = 3:1) to obtain an amorphous compound (3a) (2.53g, yield: 82.2%).
[~]D: +9.3 (c = 1.1, CHC~3) IR(film)cm~l:
3386, 2928, 2858, 1746, 1657, 1543, 1466, 1379 H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, Me3Si), 0.79 - 0.96(11H, m, -Me, CH2TMS), 1.12 - 1.83(64H, m, -CH2-)~
1.35, 1.45(6H, each s, >CMe~), 2.18 - 2.47(4H, m, -COCH2-)~
3.09 - 3.29(2H, m, H-l, H-5), 3.48 - 3.98~6H, m, H-l, H-4, H2-6, -CX2CH2TMS), 4.04 - 4.26(2H, m, H-2, CHOSEM), 4.62 - 4.68(2H, AB, JAB = 6.8 Hz, -OCH2O-), 4.86(1H, t, J = 6.3 Hz, >CHOCO-), 4.93(1X, t, H-3), 5.99(1H, d, J = 7.3 Hz NH) (the third step) 1,5-Anhydro-2-deoxy-3-O-((2R)--2-tetradecanoyloxytetrade-canoyl)-2-[(3R)-3-~2-(trimethylsilyl)ethoxyemthoxy)-tetradecanamido]-D-glucitol; ~Compound 4a) The compound 3a (2.5g) was dissolved in 95% acetic acid solution (32m~), and the obtained solution was stirred in a water bath at 50C for five hours to be reacted. The reacted solution was then diluted with toluene and concentrated under reduced pressure. The obtained residue was purified by a silica gel column chromatography (chloroform: methanol = 100:1) to obtain an amorphous compound (4a) (1.6g, yield: 67.7%).
[a]D: +12.6~ (c = 1.65, CHC~3) IR(film)cm~l:
3550 - 3150, 2926, 2858, 1742, 1655, 1543, 1460, 1365 H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, Me3Si), 0.80 - 0.99(11H, -Me, CH2TMS), 1.17 - 1.89(64H, m, -CH2-)~
2.20 - 2.44(4H, m, -COCH2-~
2.85(lH, brs, -OH), 3.14(1H, t, J = 12.3 Hz, H-l), 3.25 - 3.37(1H, m, H-5), 3.'18 - 3.68(3H, m, H-4, CH2CH2T~S), 3.70 - 3.81(1H, m, H-6~, 3.82 - 3.96(2H, m, H-l, H-6) ~A`

.;
.

2~2~

4.01 - 4.20(2H, m, H-2, CHOSEM), 4.64 - 4.70(2H, AB, JAB = 6.9 Hz, -OCH2O-), 4.82(1H, t, J = 6.5 Hz, >CHOCO-), 4.89(1H, t, J = 10.1 Hz, H-3), 6.13(1H, d, J=7,4 Hz, NH) (the fourth step) 1,5-Anhydro-2-deoxy-4,6-o-phenoxyphosphoryl-3-O-~(2R)-2-tetradecanoyloxytetradecanoyl~-2-[l3R)-3-~2-(trimethylsilyl)ethoxymethoxy} tetradecanamido]-D-glucitol; (Compound 5a) The compound 4a (1.6g) was dissolved in pyridine (1.6m~) and dichloromethane (3.3m~). To the resultant solution, phenyl dichlorophosphate (0.41m~) was dropwise added under ice-cooling, followed by stirring for reaction. After four hours, the reacted solution was diluted with chloroform, washed with water, dried with anhydrous magnesium sulfate, and evaporated under a reduced pressure to remove the solvent. The resultant residue was purified by a silica gel column chromatogra-phy (chloroform) to obtain an amorphous compound 5a (437mg, yield: 23.2%).
IR(film)cm~l:
3306, 2926, 2858, 1744, 1655, 1595, 1460, 1379 1207, 944, 690 H-NMR(300MHz)~TMS CDC~3:
0.01, 0.02(9H, each s, SiMe3) 20~2~

0.80 - 1.00(1lH, m, -Me, -CH2TMS), 1.14 - 1.69(64H, m, -CH2-), 2.20 - 2.50(4H, m, -COCH2-)~
3.18 - 3.32(1H, m, H-l), 3.49 - 3.79(3H, m, H-5, -CH2CH2TMS), 3.88 - 4.00(1H, m, >CH-OSEM), 4.06 - 4.56(5H, m, H-l, H-2, H-4, H2-6), 4.63 - 4.70(2H, AB, JAB = 11.5 Hz, -OCH2O-), 4.72 - 4.88(1H, m, >CHOCO-), 5.12, 5.16(1H, each t, J = 9.6 Hz, J = 7.1 Hz, H-3), 6.00, 6.03(1H, each d, J = 6.9 Hz, J = 7.1 Hz, NH), 7.11 - 7.44(5H, m, Ph) (the fifth step) 1,5-Anhydro-2-deoxy-2-{(3R)-3-hydroxytetradecanamido)-4,6-_-phenoxyphosphoryl-3-O-{(2R)-2-tetradecanoyloxytetradecanoyl}-D-glucitol;
(Compound 6a) The compound 5a (437mg) was dissolved in dried dichloromethane (8.7m~). To the solution, boron trifluoride etherate (0.44m~) was dropwise added under ice-cooling, followed by stirring for thirty minutes for reaction. The reacted solution was diluted with dichloromethane, and washed with water, aqueous sodium bicarbonate solution, and water in this order. - .

The obtained solution was then dried with anhydrous magnesium sulfate, and evaporated under a reduced pres-sure to remove the solvent. The obtained residual was purified by a silica gel column chromatography (chloroform: methanol = 100:1) to afford an amorphous compound (6a) (249mg, yield: 64.7%).
IR(film)cm~l:
3320, 2924, 2858, 1742, 1657, 1524, 1207 lH-NMR(30OMHz)~TMS CDC~3:
0.88(9H, t, J = 6.3 Hz, Me) 1.13 - 1.76(64H, m, -CH2-), 2.21 - 2.49(4H, m, -COCH2-), 3.21 - 3.42(2H, H-l, OH) 3.53 - 3.63, 3.69 - 3.80(1H, each m, H-53, 3.85 - 3.96(lH, m, >C_-OH), 4.01 - 4.56(5H, m, H-l, H-2, H-4, H2-6), 4.70 - 4.85(1H, m, >CHOCO-), 5.19 - 5.32(1H, m, H-3), 6.50, 6.60(1H, each d, J = 8.5 Hz, J = 7.9 Hz, NH), 7.11 - 7.43(5H, m, Ph) (the sixth step) The compound 6a (50mg) was dissolved in acetic acid (5m~). The solution was added with platinum dioxide (20mg), and stirred in H2 atmosphere under pressure (1.5kg/cm2) for two hours for reaction. The 20~92~2 reacted solution was then filtered to remove the catalyst, and the filtrate was concentrated under a reduced pressure. The obtained residue was suspended in 1,4-dioxane, and the obtained suspension was lyophilized to obtain a white powder compound (A) (45mg, yield: 97.7%).
H-NMR: Hydrogen signals on the benzene ring completely disappeared.
m. p.: 103.6 - 104.5C (decomp.) 10IR(nujol)cm~l: 3350, 1738, 1657, 1540 SI-MS: 887(M-H)-Example 2 1,5-Anhydro-2-deoxy-3-O-{(2RS)-2-dodecanoyloxyhex-15adecanoyl}-2-~(3_)-3-hydroxydodecanamido}-4,6-O-hydroxyphosphoryl-D-gluCitol; (Compound ~) (the first step) 1,5-Anhydro-2-deoxy-4,6-O-isopropyriden-2-[(3_)-3-~2-(trimethylsilyl)ethoxymethoxy}-dodecanamido]-D-glucitol; (Compound 2b) A compound 2b was formed (1.7g, yield: 57.3%) according to the same manner as that for the compound 2a, except that (R)-3-{2-(trimethylsilyl) Pthoxymethoxy}-dodecanoic acid (2.lg) was used.
[a]D: -5.17 (c = 0.97, CHC~3) m. p.: 91 - 94C

20~2~2 lR(KBr)cm~1: 3488, 2860, 1466, 1251, 1203, 1104 H-NMR: the same as that for the compound 2a except for a -CH2- integration value (the second step) 1~5-Anhydro-2-deoxy-3-o-{(2Rs-2-dodecanoyloxyhe adecanoyl}-4,6-O-isopropyriden-2-[(3R)-3-{2-(trimethylsilyl)ethoxymethoxy}dodecanamido]-D-glucitol;
(Compound 3b) A compound 3b was formed (1.6g, yield: 89.0%) according to the same manner as for the compound 3a, except that the compound 2b (l.Og) and (RS)-2-dodecanoyloxyhexadecanoic acid (800mg) were used.
IR: the same as that for the compound 3a 1H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, -SiMe3), 0.86 - 0.94(1lH, m, -Me, -CH2TMS), 1.19 - 1.35(60H, m, -CH2-), 1.35, 1.37, 1.45, 1.47(6H, each s, >CMe2), 2.23 - 2.46(4H, m, -COCH2-), 3.11 - 3.29(2H, m, H-l, H-5), 3.52 - 3.99(6H, m, ~-2, X-4, H2-6, -OCH2CH2TMS), 4.06 - 4.23(2H, m, H-2, >CHOCO), 4.95(1H, t, H-3), 6.01 - 6.05(1H, m, NH)~

2~272 (the third step) 1,5-Anhydro-2-deoxy-3-0-~(2RS)-2-dodecanoyloxyhexade-canoyl}-2-[(3R)-3-~2-(trimethylSilyl)ethoxymethoxy}-dodecanamido]-D-glucitol; (Compound 4b) Compound 4b was obtained (l.Og, yield: 52.8%) according to the same manner as that for the compound 4a, except that the compound 3b (2.0g) was used.
IR: the same as that for the compound 3a lH-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, -SiMe3), 0.80 - 0.98(11H, m, -Me, -CH2TMS), 1.10 - 1.92(60H, m, -CH2-), 2.23 - 2.45(4H, m, -COCH2-), 3.15(1H, t, J = 9.8 Hz, H-l), 3.31 - 3.38(lH, m, H-5), 3.53 - 3.83(4H, m, H-4, H-6, OCH2CH2TMS), 3.83 - 3.95(2H, m, H-l, H-6), 4.07 - 4.20(2H, m, H-2, >CHOSEM), 4.64 - 4.73(2H, m, -OCH2O-), 4.73 - 4.97(2H, m, H-3, >CHOCO-), 6.16 - 6.20(lH, m, NH) (the fourth step) 1,5-Anhydro-2-deoxy-3-_-~(2RS)-2-dodecanoyloxyhexade-canoyl}-4,6-O-phenoxyphosphoryl-2-[(3R)-3-~2-(trimethylsilyl)ethoxymethoxy)dodecanamido]-D-glucitol;
(Compound 5b) 2~272 Compound 5b was obtained (82omg~ yield: 71.4%) according to the same manner as that for the compound 5a, except that the compound 4b (1.0g) was used.
IR: the same as that for the compound 5a 1H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, -SiMe3), 0.84 - 0.98(11H, m, -Me, -CH2TMS), 1.17 - 1.77(60H, m, -CH2-)~
2.23 - 2.50(4H, m, -COCH2-), 3.20 - 3.32(1H, m, H-l), 3.52 - 3.78(3H, m, H-5, -OCH2CH2TMS), 3.87 - 3.98(1H, m, >CHOSEM), 4.08 - 4.60(5H, m, H-l, H-2, H-4, H2-6), 4.61 - 4.72(2H, m, -OCH2O-), 4.72 - 4.99(1H, m, >CHOCO), 5.09 - 5.23(1H, m, H-3), 6.08 - 6.41(1H, m, NH), 7.15 - 7.40(5H, m, Ph) (the fifth step) 1,5-Anhydro-2-deoxy-3-0-{(2RS)-2-dodecanoyloxyhexade-canoyl~-2-{(3R)-3-hydroxydodecanamido}-4,6-O-phenoxyphosphoryl-D-glucitol; (Compound 6b) The compound 6b was obtained (690mg, yield: 94.7%) according to the same manner for the compound 6a, except that the compound 5b (830mg) was used.
IR: the same as that for the compound 5a 205~272 H-NMR(30OMHz)6 TMS CDC~3:
0.89(9H, each t, J = 6.9 Hz, Me)~
1.23 - 1.96(60H, m, -CH2-), 2.20 - 2.46(4H, m, -COCH2-), 3.32 - 3.43(1H, m, H-l), 3.57 - 4.57(7H, m, H-l, H-2, H-4, H-5, H2-6, >CHOH), 4.73 - 4.90~lH, m, ~CHOCO), 5.21 - 5.33(1H, m, H-3), 6.29 - 6.64(1H, m, NH), 7.14 - 7.42(5H, m, Ph) (the sixth step) Compound B was obtained (80mg, yield: 57.8~) according to the same manner for the compound A, except that the compound 6b (150mg) was used.
H-NMR: Hydrogen signal on the benzene ring com-pletely disappeared.
m. p.: 122-126C (decomp.) IR(film)cm~l: 3586, 2928, 1738, 1649, 1261 Example 3 1,5-Anhydro-2-deoxy-3-0-{(2RS)-2-hexadecanoyloxydodecanoyl~-2-~(3RS)-3-hydroxyhexadecanamido}-4,6-O-hydroxyphosphcryl-D-glucitol; (Compound C) 2~2~

(the first step) 1,5,-Anhydro-2-deoxy-4,6-O-isopropyriden-2-[(3RS)-3-~2-(trimethylsilyl)ethoxymethoxy} hexadecanamido]-D-glucitol; (Compound 2c) Compound 2c was obtained (2.6g, yield: 81.7%) according to the same manner as that for the compound 2a, except that (RS)-3-{2-(trimethylsilyl)ethoxymethoxy}-hexadecanoic acid (2.5g) was used.
lR(film)cm~l: 3612, 1926, 1460, 1251, 1199, 1102 1H-NMR(300MHz)~TMS CDC~3:
0.03(9H, s, -SiMe3), 0.85 - 0.98(5H, m, -CH2TMS, -Me), 1.22 - 1.33(?-4H, m, -CH2-)~
1.44, 1.52(6H, each s, >CMe2)~
2.31 - 2.56(2H, m, -CH2CO-), 3.16 - 3.25(2H, m, H-l, H-5), 3.54 - 3.65(4H, m, H-l, H-4, -OCH2CH2TMS), 3.72(1H, t, J = 10.5 Hz, H-6), 3.86 - 3.94(2H, m, H-6, >CHOSEM), 3.g8 - 4.13(2H, m, H-2, H 3), - 4.66 - 4.77(2H, m, -OCH2O-), 6.28 - 6.34(1H, m, NH) .

20~272 (the second step) 1,5-Anhydro-2-deoxy-3-_-{(2RS)-2-hexadecanoyloxydodecanoyl}-4,6-_-isopropyriden-2-[(3RS)-3-{2-(trimethylsilyl)ethoxymethoxy~hexadecanamido]-D-glucitol; (Compound 3c) Compound 3c was formed (2.8g, yield: 81.6~) accord-ing to the same manner as that for the compound 3a, except that the compound 2c (2.0g) and (RS)-2-hexadecanoyloxydodecanoic acid (1.5g) were used.
IR: the same as that for the compound 3a H-NMR(300MHz)~TMS CDC~3:
0.03(9H, s, -SiMe3), 0.87 - 0.93(11H, m, -Me, -CH2TMS), 1.15 - 1.35(68H, -CH2-), 1.35, 1.36, 1.45, 1,47(6H, each s, >CMe2), 2.28 - 2.61(4H, m, -COCH2O-), 3.15 - 3.30(2H, m, H-l, H-5), 3.48 - 3.95(6H, m, H-l, H-4, H2-6, -OCH2CH2TMS), 4.07 - 4.29(2H, m, H-2, >CHOSEM), 4.63 - 4.80(2H, m, -OCH2O-), 4.86 - 5.02(2H, m, >CHOCO, H-3), 6.00 - 6.57(lH, m, NH) 2~9272 (the third step) 1,5-Anhydro-2-deoxy-3-_-{(2RS)-2-hexadecanoyloxydodecanoyl}-2-[(3RS)-3-{2-(trimethylsilyl)ethoxymethoxy~hexadecanamido]-D-glucitol; (Compound 4c) Compound 4c was obtained (1.7g, yield: 70.2%) according to the same manner as that for the compound 4a, except that the compound 3c (2.5g) was used.
IR: the same as that for the compound 4a 1H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, -SiMe3), 0.85 - 0.98(11H, m, -Me, -CH2TMS), 1.19 - 1.90(68H, m, -CH2-)~
2.22 - 2.47(4H, m, -COCH2-), 3.10 - 3.26(lH, m, H-l), 3.26 - 3.39(lH, m, H-5), 3.50 - 3.94(6H, m, H-l, H-4, H2-6, -OCH2CH2TMS), 4.08 - 4.20(2H, m, H-2, >CHOSEM), 4.64 - 4.97(4H, m, H-3, -OCH2O-, >CHOCO-), 6.15 - 6.48(lH, m, NH) (the fourth step) 1,5-Anhydro-2-deoxy-3-_-~(2RS)-2-hexadecanoyloxydodecanoyl}-4,6-O-phenoxyphosphoryl-2-[(3RS)-3-{2-(trimethylsilyl)ethoxymethoxy}-hexadecanamiclo-D-glucitol; (Compound 5c) 20~272 Compound 5c was obtained ~1.3g, yield: 68.0%) according to the same manner as that for the compound 5a, except that the compound 4c (1.7g) was used.
IR: the same as that for the compound 5a 1H-NMR(300MHz)6TMS CDC~3:
0.03(9H, s, -SiMe3), 0.87 - 0.99(1lH, m, -Me, -CH2TMS), 1.19 - 1.88(68H, m, -CH2-)~
2.27 - 2.68(4H, m, -COCH2-), 3.20 - 3.37(lH, m, H-l), 3.51 - 3.82(3H, m, H-5, -OCH2CH2TMS), 3.82 - 3.99(1H, m, >CHOSEM), 4.11 - 4.60(5H, m, H-l, H-2, H-4, H2-6), 4.60 - 5.00(3H, m, -OCH2O-, >CHOCO-), 5.09 - 5.25(1H, m, H-3), 6.05 - 6.65(1H, m, NH), 7.16 - 7.41(5H, m, Ph) (the fifth step) 1,5-Anhydro-2-deoxy-3-0-~(2RS)-2-hexadecanoyloxydodecanoyl}-2-~(3RS)-3-hydroxyhexadecanamido}-4,6-O-phenoxyphosphoryl-D-glucitol; (Compound 6c) Compound 6c was obtained (1.2g, yield: 99.1%) according to the same manner as that for the compound 6a, except that the compound 5c (1.4g) was used.
IR: the same as that for the compound 6a ~0~27~

H-NMR(300MHz)~TMS CDC~3:
0.88(9H, each t, J = 6.3 Hz, -Me3), 1.19 - 1.92(68H, m, -CH2-), 2.20 - 2.46(4H, m, -COCH2-), 3.22 - 3.41(1H, m, ~-1), 3.53 - 4.56(7H, m, H-l, H-2, H-4, H-5, H2-6, >C_OH), 4.74 - 4.90(lH, m, >CHOCO), 5.18 - 5.31(lH, m, H-3), 6.22 - 6.83(lH, m, NH), 7.12 - 7.41(5H, m, Ph) (the sixth step) Compound C was obtained (140mg, yield: 72.6%) according to the same manner as that for the compound A, except that the compound 6c (210mg) was used.
H-NMR: Hydrogen signals on the benzene ring completely disappeared.
m. p.: 124-127C (decomp.) IR(film)cm~1: 3586, 2926, 2856, 1738, 1638, 1257 Example 4 1,5-Anhydro-2-deoxy-3-_-~(2RS)-2-dodecylhexadecanoyl)-4,6-_-hydroxyphosphoryl-2-~(3R)-3-hydroxytetradecan-amido}-~-glucitol; (compound D) 20~272 (the second step) 1,5-Anhydro~2-deoxy-3-O-{(2RS)-2-dodecylhexadecanoyl}-4,6-O-isopropyriden-2-[(3R)-3-{2-(trimethylsilyl)ethoxymethoxy}tetradecanamido]-D-glucitol; (Compound 3d) An amorphous compound 3d was prepared (2.5g, yield: 90%) according to the same manner as hat for the compound 3a, except that the compound 2a (1.6g) obtained in the first step of the exampl0 1 and (RS)-2-dodecylhexadscanoic acid (1.9g) were used.
IR(film)cm~l:
3~80, 2900, 1720, 1655, 1530, 1460, 1370 H-NMR(30OMHz)~TMS CDC~3:
0.03(9H, s, Me3Si), 0.83 - 0.94(11H, m, -CH2TMS, -Me), 1.12 - 1.75(68H, m, -CH2-)~
1.32 - 1.43(6H, each s, CMe2)~
2.15 - 2.40(3H, m, -COCH2-, -COCH<), 3.02 - 3.98(8H, m, H2-1, H-4, H-5, H2-6, -OCH2CH2TMS), 4.15 - 4.22(2H, m, H-2, >CH-OSEM), 4.65(2H, s, -O-CH2-O-), 4.92(1H, t, J = 9.6 Hz, H-3), 6.18(1H, d, J = 7.1 Hz, NH) 20~27~

(the third step) 1~5-Anhydro-2-deoxy-3-o-~(2Rs)-2-dodecylhexadecanoyl}
2-[(3R)-3-{2-(trimethylsilyl)ethoxymethoxy~-tetradecanamido]-D-glucitol; (Compound 4d) An amorphous compound 4d was formed (l.2g~
yield: 64.8%) according to the same manner as that for the compound 4a, except that the compound 3d (1.9g) was used.
IR(nujol)cm~l: 3500 - 3300, 1740, 1640, 1545 1H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, Me3Si), 0.80 - 1.00(1lH, m, -CH2TMS, -Me), 1.10 - 1.71(68H, m, -CH2-), 2.12 - 2.50(3H, m, -COCH2-, -COCH<), 3.13(1H, t, J = 10.3 Hz, H-l), 3.26 - 3.37(lH, m, H-5), 3.51 - 3.99(6H, m, H-l, H-4, H2-6, -CH2CH2TMS), 4.01 - 4.20(2H, m, >CH-OSEM, H-2), 4.67(2H, s, -O-CH2-O-), 4.84(1H, t, J = 10.2 Hz, H-3), 6.22(1H, d, J = 7.3 Hz, NH) (the fourth step) 1,5-Anhydro-2-deoxy-3-_-{(2RS)-2-dodecylhexadecanoyl}-4,6-O-phenoxyphosphoryl-2-[(3R)-3-{2-(trimethylsilyl)-ethoxymethoxy} tetradecanamido]-D-glucitol;
(Compound 5d) 2~27~

An amorphous compound 5d was obtalned (893 mg, yield: 74.8%) according to the same manner as that for the compound 5, except that the compound 4d (l.lg) was used.
IR(film)cm~l:
3318, 2924, 2856, 1742, 1657, 1595, 1468, 1379, 1205, 963, 690 H-NMR(300MH~)~TMS CDC~3:
0.06(9H, s, -Me3Si), 0.71 - 1.04(1lH, -CH2TMS, -Me), 1.14 - 1.66(68H, m, -CH2-), 2.20 - 2.50(3H, m, -COCH2-, -COCH<), 3.11 - 3.30(lH, m, H-l), 3.54 - 3.80(3H, m, H-5, -CH2CH2TMS), 3.89 - 4.00(1H, m, >CH-OSEM), 4.11 - 4.58(5H, m, H-l, H-2, H-4, H2-6), 4.70(2H, s, -O-CH2--O-), 5.10, 5.16(1H, each t, J = 9.6 Hz, J = 9.6 Hz, H-3), 6.26, 6.33(1H, each d, J = 7.1 Hz, J = 7.2 Hz, NH), 7.13 - 7.46(5H, m, Ph) (the fifth step) 1,5-Anhydro-2-deoxy-3-0-{(2RS)-2-dodecylhexadecanoyl)-2-~(3R)-3-hyclroxytetradecanamido}-4,6-O-phenoxyphosphoryl-D-glucitol; (Compound 6d) , ~O~i~2rl12 An amorphous compound 6d was obtained (758mg, yield: 96.7%) according to the same manner as that for the compound 6a, except that the compound 5d (758mg) was used.
IR(nu~ol)cm~l:
3586 - 3366, 1736, 1640, 1539, 1164, 1048 H-NMR(300MHz)~TMS CDC~3:
0.88(9H, t, J = 6.2 Hz, -Me), 1.09 - 1.56(68H, m, -CH2-)~
2.10 - 2.50(3H, m, -COCH2-, -COCH<), 3.06 - 3.29(2H, m, H-l, OH~, 3.51 - 3.62, 3,67 - 3.79(1H, each m, H-5), 3.87 - 3.99(lH, m, >CH-OH), 4.07 - 4.56(5H, m, H-l, H-2, H-4, H2-6), 5.06, 5.13(1H, each t, J = 10.5 Hz, J = 9.9 Hz, H-3), 6.20, 6.28(1H, each d, J = 6.9 Hz, J = 5.7 Hz, NH), 7.10 - 7.44~5H, m, Ph) (the sixth step) A white powder compound D was obtained (38mg, yield: 83%) according to the same manner as that for the compound A with the exception that the compound 6d (50mg) was used.
lH-NMR: Hydrogen signals on the benzene ring completely disappeared.

20$9272 m. p.: 151.0-152.0C ~decomp.) IR(film)cm~l: 2924, 2856, 1736, 1649, 1543, 1247 Example 5 1,5-Anhydro-2-deoxy-3-0-{(2RS)-2-dodecyloctadecanoyl}-2-{(3R)-3-hydroxydodecanamido~-4,6-O-hydroxyphosphoryl-D-glucitol; Compound E) (the second step) 1,5-~hydro-2-deoxy-3-O-{(2RS)-2-dodecyloctadecanoyl}-4,6-O-isopropyriden-2-[(3R)-3-{2-(trimethylsilyl)-ethoxymethoxy}dodecanamido]-D-glucitol; (Compound 3e) A compound 3e was prepared (1.2g, yield: 67.1%) according to the same manner as that for the compound 3a, except that the compound 2b (l.Og) obtained in the first step of the example 2 and (RS~-2-dodecyloctadecanoic acid (853mg) were used.
IR: the same as that for the compound 3d lH-NMR: the same as that for the compound 3d (the third step) lr5-Anhydro-2-deoxy-3-o-{(2Rs)-2-dodecyloctadecanoyl}
2-[(3R)-3-{2-(trimethylsilyl)ethoxymethoxy}-dodecanamido]-D-glucitol; (Compound 4e) A compound 4e was obtained (838mg, yield: 71.7%) i according to the same manner as that for the compound 4a with the exception that the compound 3e (1.2g) was used.

2~5~27~

IR: the same as that for the compound 4d lH-NMR: the same as that for the compound 4d (the fourth step) 1~5-Anhydro-2-deoxy-3-o-{ (2Rs)-2-dodecyloctadecanoyl}-4,6-0-phenoxyphosphoryl-2-[(3R)-3-~2-(trimethylsilyl)-ethoxymethoxy}dodecanamido]-D-glucitol; (Compound 5e) A compound 5e was obtained (177mg, yield: 78.2%) according to the same manner as that for the compound Sa with the exception that the compound 4e (200mg) was used.
IR: the same as that for the compound 5d lH-NMR: the same as that for the compound 5d (the fifth step) 1,5-Anhydro-2-deoxy-3-0-~(2RS)-2-dodecyloctadecanoyl}-2-{(3R)-3-hydroxydodecanamido}-4,6-O-phenoxyphosphoryl-D-glucitol; (Compound 6e) Compound 6e was obtained (133mg, yield: 85.8%) according to the same manner as that for the compound 6a, except that the compound 5e (177mg) was used.
IR: the same as that for the compound 6d lH-NMR: the same as that for the compound 6d (the sixth step) Compound E was formed (36mg, yield: 78.5%) accord-ing to the same manner as that for the compound A, - . -. . .

~.

205~272 except that the compound 6e (50mg) was used.
H-NMR: Hydrogen signal on the benzene ring completely disappeared.
m. p.: 154.5-155.5C (decomp.) IR: the same as that for the compound D

Example 6 ,5-Anhydro-3-O-{(2RS)-2-decyloctadecanoyl~-2-deoxy-2-{(3RS~-3-hydroxyhexadecanamido)-4,6-O-hydroxyphoSphoryl-D-glucitol; (compound F) (the second step) 1,5-Anhydro-3-0-{(2RS)-2-decyloctadecanoyl~-2-deoxy-4,6-O-isopropyriden-2-[(3RS)-3-~2-(trimethylsilyl)-ethoxymethoxy~hexadecanamido]---glucitol; (Compound 3f) A compound 3f was prepared (822mg, yield: 48.6%) according to the same manner as that for the compound 3a, except that the compound 2c (l.Og) obtained in the first step of the example 3 and (RS)-2-decyloctadecanoic acid (724mg) were used.
IR: the same as that for the compound 3d lH-NMR: the same as that for the compound 3d, except for the -CH2- integration value.

(the third step) 1,5-Anhydro-3-O-{(2RS)-2-decyloctadecanoyl~-2-deoxy-4,6-0-phenoxyphosphoryl-2-[(3R',)-3-{2-(trimethylsilyl)-ethoxymethoxy~ hexadecanamido]-D-glucitol; (Compound 4f) Compound 4f was obtained (565mg, yield: 76.6%) according to the same manner as that for the compound 4a with the exception that the cornpound 3f (822mg) was used.
IR: the same as that for the compound 4d lH-NMR: the same as that for the compound 4d except for the -CH2- integration value.

(the fourth step) 1,5-Anhydro-3-O-{(2RS)-2-decyloctadecanoyl}-2-deoxy-2-~(3RS)-3-{2-(trimethylsilyl)ethoxymethoxy)-hexadecanamido]-D-glucitol; (Compound 5f) Compound 5f was obtained (184mg, yield: 81.6%) according to the same manner as that for the compound 5a with the exception that the compound 4f (200mg) was used.
IR: the same as that for the compound 5d H-NMR: the same as that for the compound 5d except for the -CH2- integration value.

(the fifth step) 1,5-Anhydro-3-0-~(2RS)-2-decyloctadecanoyl~-2-deoxy-2-((3RS)-3-hydroxyhexadecanamido~-4,6-O-phenoxyphosphoryl-D-glucitol; tCompound 6f) 2~2~

A compound 6f was obtained (145mg, yield: 89.7~) according to the same manner as that for the compound 6a, except for the compound 5f (184mg) was used.
IR: the same as that for the compound 6d lH-NMR: the same as that for the compound 6d except for the -CH2- integration value.

(the sixth step) A compound F was obtained (31mg, yield: 60.0%) according to the same manner as that for the compound A, except that the compound 6f (somg) was used.
H-NMR: Hydrogen signals on the benzene ring completely disappeared.
m. p.: 159.7-161.4C (decomp.) IR: the same as that for the compound D

Example 7 1,5-Anhydro-2-deoxy-4,6-O-hydroxyphosphoryl-2-{(3R)-3-hydroxytetradecanamido}-3-0-{(3R)-3-tetradecanoyloxytetradecanoyl}-D-glucitol;
(Compound G) (the second step) 1,5-Anhydro-2-deoxy-4,6-O-isopropyriden-3-O-{(3-)-3-tetradecanoyloxytetradecanoyl~-2-[(3R)-3-{2-(trimethylsilyl)ethoxymethoxy)-tetradecanamido]-_-glucitol; (compound 3g) An amorphous compound 3g was prepared (2.0g, yield: 79.6~) according to the same manner as that for the compound 3a with the exception that the compound 2a (l.38g) obtained in the first step of the example 1 and (R)-3-tetradecanoyloxytetradecanoic acid (1.12g) were used.
[a]D: +0.05 (c = 1.25, CHC~3) IR(film)cm~l:
3316, 2926, 2858, 1738, 1647, 1543, 851, 835 1H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, SiMe3), 0.78 - 0.98(1lH, m, -Me, -CH2TMS), 1.08 - 1.66(62H, m, -CH2-), 1.33, 1.46(6H, each s, >CMe2), 2.17 - 2.39(4H, m, -COCH2-), 2.47 - 2.65(2H, AB part of ABX, JAB = 32.2 Hz, JAX = 7-2 Hz, Jgx = 9-5 Hz, -NHCOCH2-), 3.10(1H, t, J = 9.9 Hz, H-l), 3.16 - 3.28(1H, m, H-5), 3.49 - 3.94(6H, m, H-1, H-4, H2-6, CH2CH2TMS), 4.02 - 4.20(2H, m, H-2, >CHOSEM), 4.62 - 4.68(2H, AB, JAB = 13.1 Hz, -OCH2O-), 4.89(1H, t, J = 10.4 Hz, H-3), 5.09 - 5.20(lH, m, -COCH2C_CO), 6.22(1H, d, J - 6.3 Hz, NH) . .

2~9272 (the third step) 1~5-Anhydro-2-deoxy-3-o-{(3R)-3-tetradecanoyloxytetrade canoyl}-2-[(3R)-3-{2-(trimethylsilyl)ethoxymethoxy3-tetradecanamido]-_-glucitol; (Compound 4g) An amorphous compound 4g was obtained (1.6g, yield: 84.2%) according to the same manner as that for the compound 4a with the exception that the compound 3g (2.0g) was used.
[a]D: +5.55 (c = 1.25, CHC~3) IR(film)cm~l:
3580 - 3190, 2922, 2856, 1736, 1651, 1551, 861, 835 H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, SiMe3), 0.80 - 1.00(11H, m, -Me, -CH2TMS), 1.12 - 1.71(62H, m, -CH2-), 2.24 - 2.41(4H, m, -COCH2-), ' 2.53(2H, d, J = 5.4 Hz, -NH, -COCH2-), 3.11(1H, t, J = 10.8 Hz, H-l), 3.26 - 3.38(1H, m, H-5), 3.43 - 4.21(8H, m, H-l, H-2, H-4, H2-6, -CH2CH2TMS, >CHOSEM), 4.70 - 4.63(2H, AB, JAB = 14.5 Hz, -OCH2O-), 4.81(1H, t, J = 10.3 Hæ, H-3), 5.05 - 5.17(1H, m, >CHCO-), 6.40(1H, d, J = 7~0 Hz, NH) (the fourth step) 1,5-Anhydro-2-deoxy-4,6-_-phenoxyphosphoryl-3-O-{(3R)-3-tetradecanoyloxytetradecanoyl)-2-[(3_)-3-~2-(trimethylsilyl)ethoxymethoxy~tetradecanamido]-D-glucitol; (Compound 5g) An amo~us compound 5g (0.821 g, yield: 70.3~ was obtained according to the same manner as that for the compound 5a with the exception that the compound 4g (1.029) hQS used.
IR(film)cm~l:
3586, 2926, 1744, 1667, 1539, 1466, 1379 H-NMR(300MHz)6TMS CDC~3:
0.02, 0.11 l9H~each s, -SiMe3), 0.81 - 1.00(11, m, -Me, -CH2TMS), 1.12 - 1.67(62H, m, -CH2-), 2.20 - 2.66(6H, m, -COCH2-), 3.08 - 3.28(1H, m, H-3), 3.50 - 3.79(3H, m, H-5, -CH2CH2TMS), 3.81 - 3.97(1H, m, >CHOSEM), 4.05 - 4.55(5H, m, H-l, H-2, H-4, H2-6), 4.61 - 4.79(2H, m, -OCH2O-)~
5.01 - 5.28(2H, m, H-3, >CHOCO), 6.40 - 6.51(1H, m, NH), 7.24 - 7.45(5H, m, Ph) 2~272 (the fifth step) 1,5-Anhydro-2-deoxy-2-{(3R)-3-hydroxytetradecanamido~-4,6-_-phenoxyphosphoryl-3-0-{(3R)-3-tetradecanoyloxytetradecanoyl~-D-glucitol; (Compound 6g) s An amorphous compound 6g (320mg, yield: 44.2%) was obtained according to the same manner as that for the compound 6a with the exception that the compount 5g (B21mg) was used.
IR(film)cm~l:
3586, 1742, 1651, 1543, 1168, 1052 H-NMR(300MHz)~TMS CDC~3:
0.88(9H, t, J = 5.0 Hz, Me), 1.18 - 1.70(62H, m, -CH2~
2.20 - 2.63(6H, m, -COCH2-), 3.18 - 3.42(2H, m, H-l, OH), 3.51 - 3.62, 3.70 - 3.80(1H, each m, H-5), 3.83 - 3.96(lH, m, >CHOH), 4.02 - 4.57(5H, m, H-l, H-2, H-4, H2-6), 5.04 - 5.21(2H, m, H-3, >CHOCO), 6.67, 6.77(1H, each d, J = 6.8 Hz, J = 4.9 Hz, NH), 7.14 - 7.43(5H, m, Ph) (the sixth step) A white powder of compound G was obtained (9omg~
yield: 97.7~ according to the same manner as that for the compound A, except that the compound 6g (100mg) was 2~27~

used.
H-NMR: Hydrogen signals on the benzene ring completely disap]peared.
[a]D: -1.55 (c = 1.1, CHC~3: MeOH = 1.1) m. p.: 274.1-277.9C (decomp.) IR(film)cm~1: 2858, 1719, 1651, 1462 Example 8 1,5-Anhydro-2-deoxy-4,6-_-hydroxyphosphoryl-2-{(3R)-3-hydroxytetradecanamido)-3-_-{(3RS)-3-undecylheptadecanoyl}-D-glucitol; (Compound H) (the second step) 1,5-Anhydro-2-deoxy-4,6-_-isopropyriden-2-[(3R)-3-{2-(trimethylsilyl)ethoxymethoxy}tetradecanamido]-3-_-{(3RS)-3-undecylheptadecanoyl}-D-glucitol; (Compound 3h) An amorphous compound 3h was prepared (4.12g, yield: 91%) according to the same manner as that for the compound 3h, except that the compound 2a (3.0g) obtained in the first step of the example 1 and (RS)-3-undecylheptadecanoic acid (2.0g) were used.
IR(film)cm~l:
3320, 2900, 1735, 1645, 1545, 1470, 1383 1H-NMR(300MHz)~TMS CDC~3:
0.03(9H, s, Me3Si), 0.86 - 0.97(11H, m, -CH2TMS, -Me), 1.18 - 1.60(66H, m, -CH2-), 205~272 1.36, 1.47(6H, each s, CMe2j, 1.85(1H, m, -CH<), 2.20 - 2.40(4H, m, -CH2CO-), 3.10 - 4.00(8H, m, H2-1, H-4, H-5, H2-6, -O-CH2CH2TMS), 4.16(2H, m, H-2, -CH-OSEM), 4.66, 4.68(2H, AB, JAB = 6.9 Hz, -OCH2O-), 4.94(1H, t, J = 9.6 Hz, H-3), 6.27(1H, d, J = 7.0 Hz, NH) "
(the third step) 1,5-Anhydro-2-deoxy-2-[(3R)-3-{2-(trimethylsilyl)-ethoxymethoxy)tetradecanmido]-3-O-{ ! 3RS)-3-undecylheptadecanoyl}-D-glucitol; (Compound 4h) An amorphous compound 4h was formed (1.84g, yield: 91%) according to the same manner as that for the compound 4a with the exception that the compound 3h (2.79g) was used.
IR(film)cm~l:
3600 - 3100, 2900, 1720, 1650, 1540, 1463, 1380 H-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, -SiMe3), 0.85 - 0.96(1lH, m, -CH2TMS, -Me), 1.10 - 1.65(66H, m, -CH2-)~
1.85(1H, m, -CH<), 2.20 - 2.40(4H, m, -COCH2-), 2.65(lH, brs, -OH), ' . ' ' 205~272 3.13(1H, t, J = 10.0 Hz, H-l), 3.32(lH, m, H-5), 3.52 - 3.95(ÇH, m, H-l, -CH2-CH2TMS, H-4, H2- 6 ) , 4.0 - 4.17(2H, m, H-2, -CH-OSEM), 4.63, 4.69(2H, AB, J = 7.0 Hz, -OCH2O-), 4.85(1H, t, J = 9.4 Hz, H-3), 6.29(1H, d, J = 7.3 Hz, NH) (the forth step) 1,5-Anhydro-2-deoxy-4,6-O-phenoxyphosphoryl-2-[(3R)-3-~2-(trimethylsilyl)ethoxymethoxy~-tetradecanamido]-3-0-{(3RS)-3-undecylheptadecanoyl}~
D-glucitol; (Compound 5h) An amorphous compound 5h was obtained (1.0g, yield: 80.5~) according to the same manner as that for the compound 5a with the exception that the compound 4h (l.lg) was used.
IR(film)cm-l:
3308, 2926, 1744, 1659, 1595, 1466, 1707, 1379, 944, 665 H-NMR(30OMHz)~TMS CDC~3:
0.02(9H, s, -SiMe3), 0.80 - 0.99(11H, m, -Me, -CH2TMS), 1.11 - 1.60(66H, m, -CH2-), 1.69 - 1.90(1H, m, >CH-), 2.14 - 2.41(4H, m, -COCH2-), 20~9272 3.08 - 3.27(1H, m, H-l), 3.50 - 3.77(3H, m, H--5, -CH2CH2TMS), 3.81 - 3.92(lH, m, >CHOSEM), 4.04 - 4.54(5H, m, H--l, H-2, H-4, H2-6), 4.61 - 4.72(2H, m, -t)CH2O-), 5.02 - 5.18(1H, m, H-3), 6.29, 6.36(1H, each d, J = 7.5 Hz, J , 4.9 Hz, NH), 7.10 - 7.40(5H, m, Ph) (the fifth step) 1,5-Anhydro-2-deoxy-2-{(3R)-3-hydroxytetradecanamido]-4,6-O-phenoxyphosphoryl-3-0-{(3RS)-3-undecylheptadecanoyl~-D-glucitol; (Compound 6h) A compound 6h was obtained (751mg, yield: 86.0%) according to the same manner as that for the compound 6a with the exception that the compound 5h (1.0g) was used.
IR(film)cm~l:
3586, 3296, 1742, 1651, 1543, 1168, 1052 1H-NMR(300MHz)6TMS CDC~3:
0.88(9H, t, J = 6.4 Hz, -Me), 1.14 - 1.52(66H, m, -CH2-)~
1.70 - 1.90(1H, m, >CH-), 2.14 - 2.45(4H, m, -COCH2), 3.13 - 3.31(1H, m, H-l), 3.52 - 3.64, 3.69 - 3.79(1H, each m, H-5), 3.85 - 3.99(1H, m, >CHOH), 2~272 4.08 - 4.57(5H, m, H-1, H-2, H-4, H2-6), 5.10 - 5.29t1H, m, H-3), 6.27, 6.39(1H, each d, J = 7.1 Hz, J = 7.3 Hz, NH), 7.11 - 7.42(5H, m, Ph) (the sixth step) A white power H was formed (38mg, yield: 82.7%) according to the same manner as that for the compound A with the exception that the compound 6h (lOOmg) was used.
H-NMR: Hydrogen signals on the benzene ring completely disappeared.
m. p.: 153.2-156.3C (decomp.) IR(film)cm~l: 2922, 1649, 1543, 1460 Example 9 1,5-Anhydro-2-deoxy-3-0-(2-dodecyltetradecanoyl)-4,6-0-hydroxyphosphoryl-2-tetradecanamido D-glucitol;
(Compound I) (the first step) 1,5-Anhydro-2-deoxy-4,6-0-isopropyriden-2-tetradecanamido-D-glucitol; (Compound 2i) A compound 2i was formed according to the same man-ner as that for the compound 2a with the exception that tetradecanoic acid (2.3g) was used.

20~272 [a]D: -9-4 (c = 1.01, CHC~3) m. p.: 108.0 - 109.0C
IR(film)cm~l: j;
3308, 2922, 2854, 1647, 1547, 1468 `
1H-NMR(30OMHz)6TMS CDC~3:
0.88(9H, t, J = 6.7 Hz, Me), 1.16 - 1.38(20H, m, -CH2-), 1.43, 1.52(6H, each s, >CMe2)~
1.57 - 1.69(2H, m, -COCH2cH2-)~
2.14 - 2.28(2H, m, -COCH2-), 3.18(1H, t, J = 10.8 Hz, H-l), 3.11 - 3.22(1H, m, H-5), 3.47 - 3.63(2H, m, H-l, H-4), 3.72(1H, t, J = 10.6 Hz, H-6), 3.90(1H, dd, J = 5.4 Hz, J = 10.8 Hz, H-6), 3.94 - 4.08(1H, m, H-2)~
4.15(1H, dd, J = 5.4 Hz, J = 10.9 Hz, H-3), 5.54(1H, d, J = 6.6 Hz, NH) (the second step) 1,5-Anhydro-2-deoxy-3-0-(2-dodecyltetradecanoyl)-4,6-0-isopropyriden-2-tetradecanamido-D-glucitol;
(Compound 3i) A compound 3i was obtained (1.3g, yield: 84.0%) according to the same manner as that for the compound 3a with the exception that the compound 2i (833mg) and 2-tetradecyldecanoic acid (776mg) were used.

~05~272 [a]D: -8.5 (c = 1.27, CHC~3) m. p.: 52.8 - 54.0C
IR(film)cm~l:
3298, 2922, 2854, 1734, 1649, 1545, 1468 1H-NMR(30OMHz)~TMS CDC~3:
0.88(9H, t, J = 6.7 Hz, Me), 1.10 - 1.65(66H, m, -CH2-), 1.35, 1.47(6H, each s, >CMe2)~
2.09(2H, t, J = 7.7 Hz, -COCH2-), 2.26 - 2.42(1H, m, COCH<), 3.12(1H, t, J = 10.0 Hz, H-l), 3.20 - 3.32(lH, m, H-5), 3.65 - 3.79(2H, m, H-1, H-6), 3.92 - (lH, dd, J = 5.2 Hz, J = 10.7 Hz, H-6), 4.06 - 4.27(2H, m, H-4, H-2), 4.92(1H, t, H = 9.7 Hz, H-3), 5.89(1H, d, J = 6.9 Hz, NH) (the third step) 1,5-Anhydro-2-deoxy-3-0-(2-dodecyltetradecanoyl~-2-tetradecanamido D-glucitol; (Compound 4i) A compound 4i was obtained (l.lg, yield: 84.0%) according to the same manner as that for the compound 4a with the exception that the compound 3i (1.2g) was used.
[a]D: -0.089 (c = 1.05, CHC~3) m. p.: 99.0C

~05~272 IR(film)cm~l:
3372, 3320, 2922, 2~56, 1736, 1647, 1537, 1466 H-NMR(30OMHz)~TMS CDC~3:
0.88(9H, t, J = 6.9 Hz, Me), 1.08 - 1.67(66H, m, -CH2-)~
2.09(2H, t, J = 7.8 Hz, -COCH2-), 2.27 - 2.46(1H, m, -COCH<), 3.13(1H, t, J = 10.5 Hz, H-1), 3.23 - 3.34(1H, m, H-5), 3.68 - 3.96(3H, m, H-l, H2-6), 3.98 - 4.19(2H, m, H-2, H-4), 4.89(1H, t, J = 9.7 Hz, H-3), 6.07(1H, d, J = 7.0 Hz, NH) (the fourth st~p) 1,5-Anhydro-2-deoxy-3-O-~2-dodecyltetradecanoyl)-4,6-O-phenoxyphosphoryl-2-tetradecanamido-D-glucitol;
(Compound 5i) Compound 5i was obtained (969mg, yield: 84.0%) according to the same manner as that for the compound 5a with the exception that the compound 4i (969mg) was used.
m. p.: 86.4 - 87.0C
IR(film)cm~l:
33~0, 2918, 2854, 1738, 1669, 1595, 1493, 1468, 1379, 1301, 1207, 768 , -2~27~

H-NMR(300MHz)~TMS CDC~3:
0.88(9H, t, J = 6.6 Hz, Me), 1.11 - 1.72(66H, m, -CH2-), 2.90(2H, t, J = 7.6 Hz, -COCH2-), 2.26 - 2.49(1H, m, -COCH<), 3.13, 3.19(1H, each t, J = 10.5 Hz, J = 10.2 Hz, H-l), 3.52 - 3.63, 3.67 - 3.79(1H, each m, H-5), ~.04 - 4.53t5H, m, H-l, H-2, H-4, H2-6), 5.03, 5.11(1H, each t, J = 9.9 Hz, J = 9.8 Hz, H-3), 5.86, 5.96(1H, each d, J = 6.8 Hz, J = 7.0 Hz, NH), 7.10 - 7.41(5H, m, Ph) (the sixth step) Compound I was formed (219mg, yield: 41.0%) according to the same manner as that for the compound A, with the exception that the compound (5i) (585mg) was used.
[a]D: -0.39 (c = 1.00, CHC~3) m. p.: 93.0 - 96.0C
IR(film)cm~l: 3296, 2924, 2856, 1736, 1651, 1543, 1468, 1379, 1257, 1178 2~272 Example 10 1,5-Anhydro-2-deoxy-4,6-O-hydroxyphosphoryl-2-{(3_)-3-hydroxytetradecanamido}-3-0-{(3RS)-3-tetradecyloxytetradecanoyl}-D-glucitol; (Compound J) (the second step) 1,5-Anhydro-2-deoxy-4,6-O-isopropyriden-3-O-~(3RS)-3-tetradecyloxytetradecanoyl}-2-[(3_-3-{2-(trimethylsilyl)ethoxymethoxy}tetradecanamido]-D-glucitol; (Compound 3j) An amorphous compound 3j was prepared (8.lg, yield: 87.5%) according to the same manner as that for the compound 3a, with the exception that the compound 2a (5~3g) obtained in the first step of the example 1 and (RS)-3-tetradecyloxytetradecanoic acid (4.1g) were used.
IR(XBr)cm~l:
3308, 2926, 2858, 1734, 1647, 1547, 1466, 1371, 1251, 1201, 1106, 1056 lH-NMR(300MHz)~TMS CDC~3:
0.02(9H, s, SiMe3), 0.75 - 1.00(llH, m, Me, -CH2TMS), 1.00 - 1.61(65H, m, -CH2`, >CH-), 1.34, 1.46(each s, Me2C<), 2.19 - 2.70(4H, m, -COCH2-), 3.04 - 3.96(H2-1, H-4, H-5, H2-6, -CH2CH2TMS, -OCH2CH2- ) ~
4.05 - 4.22(2H, m, H-2, >CHOSEM), ~5~272 4.57 - 4.72(2H, m, -OCH2O-)~
4.86 - 5.00(1H, m, H-3), 6.26(1H, d, J = 6.9 Hz, NH) _ (the third step) 1,5-Anhydro-2-deoxy-3-O-{(3RS)-3-tetradecyloxytetradecanoyl)-2-[(3R)-3-~2-(trimethylsiiyl)-ethoxymethoxy}tetradecanamido]-D-glucitol; ~Compound 4j) An amorphous compound 4j was obtained (6.lg, yield: 78.5%) according to the same manner as that for the compound 4a, with the exception that the compound 3j (8.0g) was used.
IR(~Br)cm~l:
3310, 2926, 2858, 1729, 1657, 1539, 1466, 1379, 1305, 1251, 1158, 1104 lH-NMR(300MHz)~TMS CDC~3:
0.02(9H, m, SiMe3), 0.78 - 1.00(11H, m, Me, -CH2TMS), 1.00 - 1.70(65H, m, -CH2-, >CH-), 2.21 - 2.78(4H, m, -COCH2-)~
3.12(1H, J = 10.5 Hz, H-l), 3.26 - 3.95(9H, m, H-l, H-4, H-5, H2-6, -CH2CH2TMS, -OCH2CH2-), 4.00 - 4.21(2H, m, H-2, ~CHOSEM), 4.65, 4.68(2H, AB, JAB = 6.8 Hz, -OCH2O-), 4.71 - 4.90(1H, m, H-3), 6.27 - 6.60(1H, m, NH) 2~27~

(the fourth step) 1,5-Anhydro-2-deoxy-4,6-O-phenoxyphosphoryl-3-_-{(3RS)-3-tetradecyloxytetradecanoyl}-2-[(3R)-3-~2-(trimethylsilyl)ethoxymethoxy} tetradecanamido]
D-glucitol; (Compound 5~) An amorphous compound 5~ was formed (87Sg, yield: 77.6%) according to the same manner as that for the compound 5a, with the exception that the compound 4;
(1.0y) was used.
IR(KBr)cm~l:
2926, 2858, 1744, 1657, 1543, 1493, 1466, 1305, 1251, 1104, 1054 H-NMR(300MHz)~TMS CDC~3:
0.02(9H, m, SiMe3), 0.77 - 1.00(11H, m, Me, -CH2TMS), 1.00 - 1.61(65H, m, -CH2-, >CH-), 2.15 - 2.72(4H, m, -COCH2-)~
3.09 - 4.55(12H, m, H2-1, H-2, H-4, H-5, H2-6, -CH2CH2TMS, -OCH2CH2, >CHOSEM), 4.55 - 4.72(2H, m, -OCH2O-), 5.01 - 5.20(1H, m, H-3), 6.22 - 6.40(1H, m, NH), 7.10 - 7.40(5H, m, Ph) 2~27~

(the fifth step) 1,5-Anhydro-2-deoxy-2-{(3R)-3-hydroxytetradecanamido}-4~6---phenoxyphosphoryl-3-o-(3Rs)-3-tetradecyloxytetradecanoyl~-D-gluCitol; (Compound 6;) An amorphous compound 6j was obtained (710g) according to the same manner as that for the compound 6a, with the exception that the compound 5j (845mg) was used.
IR(KBr)cm~l:
3296, 2922, 2856, 1742, 1655, 1595, 1547, 1491, 1468, 1309, 1207, 1174 H-NMR(30OMHz)~TMS CDC~3:
0.88(9H, t, J = 6.4 Hz, Me), 1.00 - 1.63(65H, m, -CH2-, >CH-), 2.10 - 2.71(4H, m, -COCH2-), 3.18 - 4.60(10H, m, H2-1, H-2, H-4, H-5, H2-6, >C_OH, -OCH2CH2-), 5.11 - 5.31(3H, m, H-3), 6.40 - 6.70(1H, m, NH), 7.11 - 7.45(5H, m, Ph) (the sixth step) A white powder of compound (J) was obtained (9lmg, yield: 93.4%) according to the same manner as that for the compound A, with the exception that the compound 6j (lOOmg) was used.
lH-NMR: Hydrogen signals on the benzene ring 20~2~2 completely disappeared.
m. p.: 161-164C
IR(KBr)cm~l: 3272, 2924, 2854, 1740, 1647, 1543, 1468, 1363, 1259, 1176 Example 11 2-deoxy-4,6-_-hydroxyphosphoryl-2-{(3R)-3-hydroxytetradecanamido)-3-O-~(2RS)-tetradecanoyloxytetradecanoyl}-D-glucopyranose;
(Compound K) (the seventh step) 30mg of 2-deoxy-2-{(3R)-3-hydroxytetradecanamido~-4-O-phosphono-3-_-{(2RS)-2-tetradecanoyloxytetradecanoyl}-D-glucopyranose(7k) which can be produced according to the known manner (Japanese Patent Disclosure No. 62888/90) was dissolved in a mixture solvent of tetrahydro~uran:
chloroform (1:1) (lom~). To the resultant solution, DCC
(5mg) was added, followed by stirring for three hours.
The reacted solution was subjected to a Sephadex column (LH-20, chloroform:methanol=l:l). Further, the solution was lyophilized by utilizing 1,4-dioxane to obtain a compound K.
m. p.: 158-160C
IR(Ksr)cm-l: 3300, 2950, 2860, 1740, 1680, 1590, C4gHggNO12P (903-21) 2~2~2 theoretical value: C = 63.83%, H = 9.93%, N = 1.55%
actual value: C = 64.04%, H = 9.76%, N = 1.49%

Example 12 2-Deoxy-3-_-{(2RS)-2-dodecylhexadecanoyl~-4,6-O-hydroxyphosphoryl-2-~(3R)-3-hydroxytetradecanamido}--glucopyranose; (Compound L) (the seventh step) Compound L was obtained according to the same man-ner as that for the compound K, with the exception that 2-deoxy-3-O-{(2RS)-2-dodecylhexadecanoyl)-2-{(3R)-3-hydroxytetradecanamido}-4-O-phosphono-D-glucopyranose (71) which can be produced in the known method (Japanese Patent Disclosure No. 25494/90) was used.
m. p.: 167-169C
IR(KBr)cm~l: 3400, 2930, 2850, 1720, 1640, 1550 C4gHg3NO11P (891-24) theoretical value: C = 64.69%, H = 10.52%, N = 1.57%
actual value: C = 64.65%, H = 10.29%, N = 1.82%

Example 13 2-Deoxy-4,6-O-hydroxyphosphoryl-2-{(3R)-3-hydroXytetra-decanamido~-3-O-~(3 )-3-tetradecanoyloxytetradecanoyl}-D-glucopyranose; (Compound M) (the seventh step) Compound M was obtained according to the same man-ner as that for the compound R, with the exception that 2-deoxy-2-~(3R)-3-hydroxytetradecanamido}-4-O-phosphono-3-0-((3R)-3-tetradecanoyloxytetradecanoyl}-D-glucopyranose (7m) which can be produced by the known method (Japanese Disclosure No. 62889/90) was used.
[a]D: -2.0 (c = 0.5, CHC~3:MeOH = 1.1) m. p.: 168-170C
C4gHggNO12P (903-32) theoretical value: C = 63.83%, H = 9.93%, N = 1.55%
actual value: C = 63.82%, H = 10.02%, N = 1.33%
Example 14 2-Deoxy-4,6-O-hydroxyphosphoryl-2-~(3R)-3-hydroxytetradecanamido}-3-O-~(3RS)-3-undecylheptadecanoyl}-D-glucopyranose; (Compound N) the seventh step) Compound N was obtained according to the same man-ner as that for the compound K, with the exception that 2-deoxy-2-(~3R)-3-hydroxytetradecanamido~-4-O-phosphono-3-0-~(3RS)-3-undecylheptadecanoyl)-D-glucopyranose (7n) which can be produced by the same known method ~Japanese Patent No. 241866/89) was used.
"`A~

2~27~

m. p.: 176-179C
C4gHglNOloP (873.23) theoretical value: C = 66.02%, H = 10.50%, N = 1.60%
actual value: C = 66.20%, H = 10.24%, N = 1.89~

Claims

C L A I M S

1. 4,6-O-hydroxyphosphoryl-glucosamine derivatives as shown in the following formula [I] and its pharmaceutically-acceptable salt:

[I]

wherein R1 and R2 indicate a hydrogen atom or a hydroxy group; one of R3 and R4 indicates -OCO(CH2)nCH3, -CH2(CH2)nCH3 or -O-CH2(CH2)nCH3, and the other indi-cates a hydrogen atom; 1 is an integer of 4-16; m is an integer of 4-16; and n is an integer of 6-18.