JPH0499796A - New lipopeptide and antitumor agent - Google Patents

Abstract

NEW MATERIAL:The lipopeptide of formula I [R is -COrCH2)14CH3]. EXAMPLE:S-{2,3-bis(palmitoyloxy)-2R-propyl}-N-palmitoyl-(R)-cysteinyl- (S)- seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine. USE:An antitumor agent. PREPARATION:The objective lipopeptide of formula I can be produced e.g. by reacting (R)-(-)-3-iodo-1,2-propanediol of formula II with a protected cysteine, reacting with palmitoyl chloride to convert two hydroxyl groups into palmitoyl esters, removing the carboxyl-protecting group of amino acid, successively reacting with serine having protected alpha-amino group, etc., serine, asparagine and alanine to synthesize a peptide chain, removing the protecting groups and finally acylating the terminal amino group.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to novel vopeptides and antitumor agents.

Conventional techniques and their problems Bacterial cell wall peptidoglycan and ribopolysaccharide localized in the outer membrane of the cell wall can be used as immune enhancers (adjuvants) for mammals.
It is known to exhibit a variety of biological activities such as anti-inflammatory, pyrogenic, anti-ulcer, and non-specific infection-protective activities. These cell surface substances have a variety of functions and biological activities, such as regulating the body's immune function, host and cell recognition, receptor function, adhesion, and inducing cell differentiation in very small amounts. .

Recently, there has been a great deal of interest in basic research to elucidate the nature of these molecular structures and functions or activity expression at the molecular level, as well as in their effective utilization.

Bacteria are broadly classified into Durham-negative bacteria and Durham-positive bacteria.
The main difference between the two lies in the surface structure of the cells. The outer membrane of Escherichia coli, a representative strain of Durham-negative bacteria, contains several types of proteins in large amounts, including proteins OmpA, OmpF,
OmpC is a major riboprotein and is called a major outer membrane protein.

Major riboproteins interact with both the peptidoglycan layer and the outer membrane, and are thought to play an important role in the assembly of the outer membrane on the peptidoglycan layer. OmpF and OmpC are important proteins for the function and structure of the outer membrane. Furthermore, recently, ribopeptide, which exists between the cell membrane and cytoplasm of Durham-negative bacteria, has attracted attention as a substance that exhibits cell division-inducing activity in lymphocytes by stimulating B cells. Furthermore, five substances have been reported to have strong activity in the peptide portion of ribopeptides. Synthesis methods for this substance have also been reported, but only the racemic form has been synthesized. This synthesis method uses a racemic glycerol derivative as a starting material, and performs a peptide synthesis reaction after acylating the N-terminus of cysteine. However, peptide synthesis using this method may cause racemization of the cysteine moiety, and is therefore not a preferred method.

Under these circumstances, it is desired that a simple and reliable method for synthesizing optically active ribopeptides be developed.

Means for Solving the Problems Therefore, as a result of intensive research in order to solve the above problems, the present inventor used an optically active glycerol obtained by an enzymatic method as a starting material, and also converted the N-terminus of cysteine to trichloroethoxy. By protecting the cysteine moiety with a carbonyl group, racemization of the cysteine moiety was prevented, and we succeeded in synthesizing new natural and non-natural Noribopeptides, and also succeeded in synthesizing derivatives with enhanced activity. completed.

Lipopeptides present in Durham-negative bacteria are potent activators of B lymphocytes [V, BraunBioc
hem, Biop)+ys, ^cfa,,415.
335 (1975)]. In order to analyze the molecular structure that expresses the biological activity of B lymphocytes, we attempted to synthesize an analog of the N-terminal portion of this lipopeptide. The conclusion S
-(2,3-bis(palmitoyloxy)-(2R8)
-propyl)-N-valmitoyl-(R)-cysteinyl-(S)-seryl-(S)-seryl-(S)-asbarginyl-(S)-alanine activates B lymphocytes in vivo and in vitro. Found [W, G
, Be5sler.

Johnson, R.B., Wiesmul, K.H.
ler and G, Jung, HoppeSe7
er 2. Ph7siol, Chem,, 36
3. 767 (1982), Jobnson, R.B.
, S., Kohl K., H. Wiesmullet, G.
Jung, and W, G, Besslet I
monobiology, 135. 1900
(1985), W.G., Be5sler, M.
COI, A, Lex, B, 5uhr K,
HWiesmuller and G. Jung.
J, ImmunoloH, 135°1900 (1
985) Ko.

The present inventors have developed a novel S-
(2,3-bis(palmitoyloxy)-(2R and 2S)-propyl)N-valmitoyl-(R)-cysteinyl-(S)seryl-(S)-seryl-(S)-asparaginyl-(S)- Alanine [compounds of the following general formulas (1) and (3) and its derivative N-(2-trichloroethoxy-carbonyl) [compounds of the following general formulas (2) and (4)] were synthesized. In addition, compounds of the following general formulas (5) to (9) were also synthesized in the same manner as above.

CH20R R-C7s-Ser-3er-Asn-^1a-OH[
In the formula, R represents -Co(CH2)+, sCH3.

]H20R Troc-C7s-Ser-5er-^sn-^1a-
0) 1 [In the formula, Troc represents -COOCH2CC/3.

R is the same as above. ] R-C7s-3er-3er-Asn-Ala-OH0
-C-H CH20R [wherein R is the same as above. ] Troc-Cys-3er-3er-Asn-^Ia-
OHCH2OR [wherein R and Troc are the same as above. ]CF+2 OR OH2 H-C7s-3er-3er-Asn-Ala-OHT
roc-Cys Ser-3er-Asn-OH [wherein R is the same as above.

Ko [In the formula, R and Tr C is the same as above.

OH2 OR OH2 Troc-C7s-3er-OH -C7s Ser-3er-Asn [In the formula, R and Troc are the same as above.

[In the formula, R is the same as above.

OH2 OR Above - format %formula% Reaction process formula below It is manufactured according to the method shown in 1.

Troc-C7s-5er-3er H [In the formula, R and Troc are the same as above.

Reaction scheme-1 ()I-Cys-OBu')2→(T+oc-Cy
s-OBu')2->Troc-Cys-OBu' OH20R -)RO-C-H CH201(Cys-5er(Bu') Set(Bu') Asn-^1a-OBu OH-C-H C)I20 [( CI(2OR OH20R OH2I 0-C-H Troc -1CH2-Cys-OBu' 0-C-H Troc CI(2-Cys-OBu'->RO-C-)1 C7s-3e "(Bu' )-3e "(Bu' )A
sn-Ala-OBuOH20R)1-3et(Bu')-5et(Bu')Asn
-Ala-OBu [wherein R and Troc are the same as above. ]CH2-C7s
-OH For each of the above reactions, the reaction conditions used when synthesizing ordinary peptides can be applied as appropriate.

the To give a concrete example, It is as follows.

120R In the above reaction scheme, used as starting material 0-C-H Troc Formula (10) The compound is smu er etc. the method of [References can be found in the above Prepared by.

PiriC)12-C7s-3er(Bu')Ser(Bu
N-protection of the compound of formula (10) was carried out with trichloroethoxychloroformate (3 volumes) in )-^sn-Ala-OBu dine followed by triethylamine (
The compound of formula (12) is prepared by reduction with dithioerythritol (4 volumes) in chloroform in the presence of 3 volumes).

The compound of formula (14) is produced by reacting the compound of formula (12) with the compound of formula (13) in dimethylformamide in the presence of N,N-diisopropylethylamine (4 volumes).

A compound of formula (15) can be prepared using balmitoyl chloride (2 volumes) and N,N-diisopropylethylamine (4 volumes) in methylene chloride in the presence of a catalytic amount of 4-dimethylaminopyridine. ) is produced by esterifying the compound.

The compound of formula (16) can be prepared by formula (16) using trifluoroacetic acid.
It is produced by deprotecting the jerl-butyl group of the compound 15).

The compound of formula (18)<7) is K, HoWiesmul
[The literature follows the above-mentioned method, using dicyclohexylcarbodiimide (1 volume) and 1-hydroxybenzotriazole (2 volumes) as coupling reagents, and combining the compound of formula (16) with the formula ( 17
) is produced by coupling with the compound of

The compound of formula (2) is produced by treating all the protecting groups of the fert-butyl groups of the compound of formula (18) obtained above with trifluoroacetic acid.

Moreover, the compound of formula (19) is produced by treating the trichloroethoxycarbonyl group of the compound of formula (18) with zinc in acetic acid to remove it.

The compound of formula (20) is prepared by acylating the compound of formula (19) with balmitoyl chloride and N,N-diisopropylethylamine in methylene chloride.

The compound of formula (1) is produced by treating all the protecting groups of the +er+-butyl groups of the compound of formula (20) obtained above with trifluoroacetic acid.

The compound of formula (3) and the compound of formula (4) can be obtained by using the formula H21 110-C-H C)I20H in place of the compound in the above reaction scheme and carrying out each reaction shown in the above reaction scheme. , manufactured.

Moreover, the above-mentioned polypeptides of types (5) to (9) are produced by the following reaction schemes -2 to 6.

Reaction scheme % Formula % Reaction scheme -3 X-5e+-0H-+Z-3er(Bu')-0Bu
'→H-5er(Bu')-0Bu') IC!
(2,3) H20R 0-C-H Troc CI (20R CH2-C7s-OH 0-C-H Troc Ct12-Cys-3er(Bu')-0Bu'
H20R RO-C-1 (Troc CH2-Cys-3er-OH [In the formula, R and Troc are the same as above. Z represents an N-benzyloxycarbonyl group.] [In the formula, R is the same as above.] Reaction step Formula-4 Reaction scheme-5 H-Asn-OBu')IC7・I/28)O-1→2-3er(Bu')-Asn-OBu(28)
(29).

C)+2 OR 0-C-H roc RO-C-)1 T+oc CH2-C7s-3er(Bu')-Set(Bu
')C)+2OR0-C-Hroc [wherein R, Troc and Z are the same as above. koBu H-3et(Bu')-Ser(Bu')-Asn
-OBuCH20R 0-C-H roc C)+2-Cys-OHCH20R 0-C-H roc C)12-Cys-Set(Bu')-3et(B
u' )-85n-OBuCH20R 0-C-H roc CH2-Cys-3er-3er-OH [in the formula R, Tr
oc and Z are the same as above. ] Reaction process formula % Formula % [In the formula, R is the same as above. ] For each of the above reactions, the reaction conditions used when synthesizing ordinary peptides can be applied as appropriate.

For example, the compound of formula (5) is produced by deprotecting the Ierl-butyl group of the compound of formula (19) using trifluoroacetic acid.

The compound of formula (23) is obtained by converting the compound of formula (21) into
-butylation and then N of the resulting compound of formula (22)
-Produced by deprotecting the benzyloxycarbonyl group.

The compound of formula (24) can be used with DCC as a coupling reagent.
It is produced by coupling the compound of formula (23) and the compound of formula (16) in dimethylformamide using

The compound of formula (6) can be prepared by formula (2) using trifluoroacetic acid.
It is produced by deprotecting the 1erl-butyl group of the compound 4).

The compound of formula (26) is a compound of formula (23) with HOBt
and DCC, followed by deprotection of the N-benzyloxycarbonyl group of the resulting compound of formula (25).

The compound of formula (27) can be used with DCC as a coupling reagent.
It is produced by coupling the compound of formula (26) and the compound of formula (16) in dimethylformamide using

The compound of formula-(7) can be prepared using trifluoroacetic acid by formula (
It is produced by deprotecting the 1erl-butyl group of the compound 27).

The compound of formula (32) is a compound of formula (28) with z-3e
t(Bu')-OH is reacted with DCC, then the N-benzyloxycarbonyl group of the obtained compound of formula (29) is deprotected, and the obtained compound of formula (30) is further treated with z-3er. It is produced by reacting (Bu')-OH with DCC and then deprotecting the N-benzyloxycarbonyl group of the resulting compound of formula (31).

The compound of formula (33) can be used with DCC as a coupling reagent.
It is produced by coupling the compound of formula (32) and the compound of formula (16) in dimethylformamide using

The compound of formula (8) can be synthesized by formula (3) using trifluoroacetic acid.
It is produced by deprotecting the tert-butyl group of the compound 3).

The compound of formula (34) is produced by removing the trichloroethoxycarbonyl group of the compound of formula (33) by treating it with zinc in acetic acid.

The compound of formula (9) can be prepared by formula (3) using trifluoroacetic acid.
It is produced by deprotecting the 1ert-butyl group of the compound 4).

The target compound obtained by the method shown in each of the above reaction schemes can be separated from the reaction system by conventional separation means and further purified.

As this separation and purification means, for example, distillation method, recrystallization method, column chromatography, ion exchange chromatography, gel chromatography, affinity chromatography, preparative thin layer chromatography, solvent extraction method, etc. can be adopted.

The active ingredient compound thus obtained is effective as an anti-ulcer agent, and the drug is used in the form of a parenteral pharmaceutical preparation. The formulation contains commonly used fillers, extenders, binders,
It is prepared using diluents or excipients such as wetting agents, disintegrants, surfactants, and lubricants. Various forms of this pharmaceutical preparation can be selected depending on the therapeutic purpose, and representative examples include tablets, gunpowders, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, and injections ( solutions, suspensions, etc.). When forming into a tablet, a wide variety of carriers that are well known in this field can be used. Examples include lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, excipients such as silicic acid, water, ethanol, propatool, simple syrup, glucose solution, starch solution. , gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, binder such as polyvinylpyrrolidone, dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, lauryl Sodium sulfate, stearic acid monoglyceride, starch, disintegrants such as lactose, white sugar, stearin, cocoa butter,
Disintegration inhibitor for hydrogenated oil etc., quaternary ammonium base,
Absorption enhancers such as sodium lauryl sulfate, glycerin,
Moisturizing agents such as starch, adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, purified talc,
Lubricants such as stearate, boric acid powder, polyethylene glycol, etc. can be used. Furthermore, tablets may be coated with conventional coatings, such as sugar-coated tablets, gelatin-coated tablets,
They can be enteric-coated tablets, film-coated tablets, double tablets, or multilayer tablets. When forming the powder into a gunpowder, a wide variety of carriers conventionally known in this field can be used. Examples include excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc, binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol, and disintegrants such as laminaran and agar. can be used.

When molding into a half-open form, a wide variety of conventionally known carriers can be used. Examples include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like. Capsules are usually prepared by mixing the active ingredient compound with the various carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules, etc. according to conventional methods. When prepared as injections, solutions, emulsions and suspensions are preferably sterile and isotonic with blood, and when formulated into these forms, diluents commonly used in this field may be used. For example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. can be used.

In this case, a sufficient amount of salt, glucose, or glycerin may be included in the pharmaceutical preparation to prepare an isotonic solution, and usual solubilizing agents, buffers, soothing agents, etc. may be added. You can. Furthermore, coloring agents, preservatives, perfumes, flavoring agents, sweeteners, etc., and other pharmaceuticals can also be included in the pharmaceutical preparation, if necessary.

The amount of the active ingredient compound to be contained in the anti-ulcer agent of the present invention is not particularly limited and can be appropriately selected from a wide range, but is usually about 1 to 70% by weight, preferably about 5 to 70% by weight in the pharmaceutical composition. The content is preferably 50% by weight.

The method of administering the anti-ulcer agent of the present invention is not particularly limited, and is administered in a manner depending on various formulation forms, age, sex and other conditions of the patient, degree of disease, etc. For example, tablets, powders, solutions, suspensions, emulsions, granules, and capsules are administered orally. In the case of injections, it may be used alone or with glucose,
It is administered intravenously in a mixture with a conventional replacement fluid such as amino acids, and further administered alone as needed intramuscularly, intradermally, subcutaneously, or intraperitoneally. If it is partially opened, it is administered rectally.

The dosage of the anti-ulcer agent of the present invention is appropriately selected depending on the usage, the age, sex and other conditions of the patient, the severity of the disease, etc., but usually the amount of the active ingredient compound is about 0.001 kg per kg of body weight per day. The amount is preferably about 6 to 50 mg. In addition, in dosage unit formulations, the active ingredient compound may contain about 10 to 1000
It is desirable that the content be in the range of mg.

EXAMPLES Below, reference examples and examples are given to further clarify the present invention.

Reference example 1 (S)-(+) 1-0-acetyl-2-0 pyridine 6011 in 13.44 g of hensylglycerol
and p-toluenesulfonyl chloride 19 under water cooling.
．． 45 g was added and stirred overnight. The reaction solution was poured into ice water, extracted with dichloromethane, and the organic layer was extracted with IN hydrochloric acid (15
The solution was washed with saturated brine, dried over magnesium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent: isopropyl ether:chloroform-1:10). , 21.75 g (yield: 98%) of the target compound was obtained.

Colorless Oil Reference Example 2 21.68 g of (R) -(+) -10-acetyl-2-0-benzyl-3-0-1 silglycerol obtained in Reference Example 1 above was mixed with 25% ammonia water 10/2/methanol. 150. The mixture was added to a mixed solvent of n and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, dichloromethane was added to the residue,
Washed with purified water and saturated brine, dried over magnesium sulfate, filtered, concentrated the filtrate under reduced pressure, and purified the residue with silica gel chromatography (developing solvent: chloroform:ethanol = 20:1) to obtain the target compound 18. .43 g (yield: 96%) was obtained.

Colorless oil reference example 3 (R) obtained in reference example 2 above in 100 y/ethanol
Add 18.43 g of -(+)-2-0-benzyl-1-0-tosylglycerol and add 5% Pd-C (50% water content).
2g of was added, and catalytic reduction was carried out in the presence of a hydrogen stream for 15 hours. The reaction solution was filtered, and the filtrate solution was concentrated under reduced pressure to obtain 13.69 g (yield: 98%) of the target compound.

Colorless Oil Reference Example 4 A mixture of 1.9 g of (R)-(-)-3 tosyloxy-1,2-propanediol obtained in Reference Example 3 above, 5 xi of acetone, and 3.63 g of sodium iodide was placed in a sealed tube. The mixture was stirred at 90°C for 9 hours.

The reaction solution was filtered through a glass filter, the filtrate was concentrated under reduced pressure, ether was added to the residue, stirred, extracted, and filtered again.
After adding 0.1N sodium thiosulfate to the furnace solution until the color disappeared, the organic layer was dried with magnesium sulfate and filtered.
The furnace solution was concentrated, n-hexane was added to the residue and stirred, and the crystalline portion was filtered through a glass filter and dried to obtain 1.05 g (yield: 65%) of the target compound.

Yellow crystal mp: 35-37°C [α]o--6.0° (C=1.15, chloroform) IR (KBr, νmax): 3334 (OH)c+
n-' Reference Example 5 15 g of (S) -(+)-1-0-acetyl-2-O-benzylglycerol and 13 g of diisopropylethylamine were dissolved in dichloromethane, and while stirring under water cooling, 6.5 g of methoxymethyl chloride was added. It was dissolved in dichloromethane and added dropwise. The mixture was stirred at room temperature overnight, washed with water and saturated brine, and the organic layer was dried over magnesium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent: hexane: ethyl acetate = 5: 1). 33 g (yield: 97%) of the target compound was obtained.

Reference Example 6 Synthesis of tylglycerol (S)-1-〇-acetyl-2 obtained in Reference Example 5 above
18 g of -0-benzy-3-0-methoxydimethylglycerol was dissolved in an ethanol solution of 2.7 g of sodium hydroxide, and the mixture was stirred for 1 hour under water cooling. After neutralizing with 6N hydrochloric acid, ethanol was distilled off and the residue was extracted with dichloromethane. The dichloromethane layer was dried over magnesium sulfate and concentrated under reduced pressure to obtain 15 g (yield: approximately 100%) of the target compound.

Reference Example 7 (R)-2-0-benzyl-3 obtained in Reference Example 6 above
-0-Methoxymethylglycerol (15 g) and triethylamine (10 g) were dissolved in dichloromethane. Under water cooling,
While stirring, p-toluenesulfonyl chloride was dissolved in dichloromethane and added dropwise. - After stirring overnight, the mixture was washed with water and saturated brine. Dry the organic layer with magnesium sulfate,
After filtration, the filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel chromatography (developing solvent: hexane: ethyl acetate = 5:1).
) to obtain 25 g (yield: 98%) of the target compound.

Reference Example 8 (S)-1-0-tosyl-2- obtained in Reference Example 7 above
0-benzyl-3-0-methoxymethylglycerol 2
5 g was added to a methanol solution containing 152 A' of 6N hydrochloric acid, and the mixture was stirred at 60° C. for 8 hours.

After neutralizing with 2N sodium hydroxide solution, methanol was distilled off, the residue was extracted with dichloromethane, the organic layer was washed with water and saturated brine, dried over magnesium sulfate, and concentrated under reduced pressure to obtain 21 g of the target compound ( A yield of 98% was obtained.

White amorphous Reference Example 9 Synthesis of (S)-1-0-1 silglycerol 9.05 g of (S)-1-0-tosyl-2-0-benzyl-glycerol obtained in Reference Example 8 above 1 Pd-C (50 % water content)
1.0 g and ethanol 5011 were treated in the same manner as in Reference Example 3 to obtain 6.54 g (yield: 99%) of the target compound.

Colorless Oil Reference Example 10 1.9 g of (S)-1-0-tosyl-glycerol obtained in Reference Example 9, 5 zl of acetone, and 3.63 g of sodium iodide were treated in the same manner as in Reference Example 4 to obtain the target compound. 1
．． 05g (yield: 65%) was obtained.

Yellow crystal mp: 35-37°C [α]D-+6.0° (C=1.35, chloroform) IR (KBr, νmax): 3334 (OH) cm
-'Reference Example 11 45 g of 60% perchloric acid was added to 15 g of cystine to dissolve it, 350 zl of 1erl-butyl acetate was added, and the mixture was stirred for 60 hours. 2M NaOH and 1M NaHCO3 were added to the reaction solution to adjust the pH to 7.8, followed by extraction with diethyl ether, washing with saturated brine, drying over magnesium sulfate, and filtration. The filtrate solution was concentrated under reduced pressure to obtain the target compound 14. 8 g (yield: 67%) was obtained.

Dilute yellow oil Reference example 12 Cystine di-1e "1-butyl ester [Formula (11)
Cystine di-1eft butyl ester obtained in Reference Example 11 0.53g, dichloromethane and pyridine 0
．． 50 g was added thereto, and while stirring under water cooling, 1.02 g of trichloroethyl chloroformate dissolved in dichloromethane was added dropwise, and the mixture was stirred for 3 hours.

Dichloromethane was added to the reaction solution, washed with IN hydrochloric acid and saturated brine, and the organic layer was dried over magnesium sulfate and filtered.
The furnace liquid was concentrated. The residue was purified by silica gel chromatography (developing solvent: n-hexane:ethyl acetate = 10:1) to obtain 0.47 g (yield: 60%) of the target compound.

Yellow oil Reference Example 13 N-2,2,2-trichloroethoxycarbonylcystine di-tert-butyl ester cysteine obtained in Reference Example 12 0.85 g, dithioerythritol 1
．． 0 g of triethylamine, 0.48 g of triethylamine, and chloroform were added, and the mixture was stirred for 2 hours under argon gas substitution. Chloroform was added to the reaction solution, and after washing with 5% citric acid and purified water under argon gas substitution, the organic layer was dried over magnesium sulfate, filtered, and the filtrate solution was concentrated under reduced pressure. Because it is susceptible to air oxidation, 1.01 g of the target compound was obtained without purification.
I got it.

Reference Example 14 After dissolving 2.4 g of N-carbobenzoxy-L-alanine in dichloromethane, a catalytic amount of sulfuric acid was added, and while cooling with water, isobutylene gas was blown into the solution for 4 to 5 minutes, followed by stirring for 3 days. The reaction solution was neutralized by adding triethylamine, concentrated under reduced pressure, and the residue was dissolved by adding ethyl acetate to give 4% NaHCO3.
The organic layer was washed with saturated brine, dried over magnesium sulfate, and then concentrated. The residue was purified by silica gel chromatography (developing solvent: n-hexane:ethyl acetate = 10:1) to obtain 2.02 g (yield: 2%) of the target compound.

Reference Example 15 Synthesis of L-alanine +erl-butyl ester hydrochloride Carbobenzoxy-alanine-1er obtained in Reference Example 14 above! Ethanol and 400 mg of Pd-C (50% water content) were added to 3.76 g of -butyl ester, and catalytic reduction was performed in the presence of a hydrogen stream for 3 hours. The reaction solution was filtered, a hydrochloric acid-ethanol solution containing an equivalent amount of 0.47 g of hydrochloric acid was added to the furnace solution, and the mixture was concentrated under reduced pressure to obtain 2.12 g of the target compound (
Yield: 87%) was obtained.

White crystal reference example 16 0.72 g of L-alanine Iert butyl ester hydrochloride obtained in reference example 15 above, 0.41 g of N-methylmorpholine and dimethylformamide were added, and while stirring,
1.06 g of carbobenzoxy-asparagine, 0.91 g of dicyclohexylcarbodiimide (rDCC) and 0 1-hydroxybenzotriazole
．． 61 g was added and stirred for 15 hours. A small amount of ethyl acetate was added to the reaction solution, filtered with suction, the filtrate was concentrated, dichloromethane was added to the residue, and this was mixed with 5% citric acid, purified water,
The organic layer was washed with %N a HC03 and saturated brine, dried over magnesium sulfate, filtered, and the filtrate was concentrated. To the residue, add the minimum amount of chloroform to dissolve it, add n-hexane to this until no precipitate occurs, filter the precipitate with suction through a glass filter, dry and re-precipitate (re-precipitation method), 1.07 g of the target compound. (Yield: 68%) White powder Reference Example 17 Carbobenzoxy-mono-asparaginyl-L-alanine 1erl-butyl ester obtained in Reference Example 16 above 0
．． 20g of ethanol and 5% Pd-C (containing 50%)
was added, and catalytic reduction was carried out in the presence of a hydrogen stream for 3 hours.

Filter the reaction solution, concentrate the reaction solution, and obtain 0.13 g of the target compound.
(yield: 97%).

White powder reference example 18 Carbobenzoxy-〇-1crt-butyl-shi-ceryl-shi-asparaginyl-L-alanine 1ert-
Synthesis of butyl ester Asparaginyl-L-alanine obtained in Reference Example 17 1erl-butyl ester 0.20g1 Carbobenzoxy-0-terl-butyl-serine 0.2
A mixture of 3 g of dimethylformamide, 0.12 g of 1-hydroxybenzotriazole, and 16 g of DCCo was stirred at room temperature for 15 hours. Add a small amount of ethyl acetate to the reaction solution, pass suction through a glass filter, concentrate the furnace solution under reduced pressure, add ethyl acetate to the residue, filter again with suction, concentrate the furnace solution, and dissolve the residue in dichloromethane. and 4% Na
After washing with HCO3 and saturated saline, the organic layer was dried and filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by the same reprecipitation method as in Reference Example 16 to obtain 0.31 g of the target compound (yield near 6%).

Reference Example 19 Synthesis of 0-ferf-butyl-L-seryl-L-asparaginyl-L-alanine terl-butyl ester Carbobenzoxy-0-te obtained in Reference Example 18 above
rt-butyl-ceryl-earth paraginyl L
A mixture of 0.80 g of -alanine 1ef-butyl ester, ethanol and 0.1 g of 5% Pd-C (50% water content) was subjected to catalytic reduction in the presence of a hydrogen stream for 3 hours.

The reaction solution was filtered, and the reaction solution was concentrated under reduced pressure to obtain 0.0% of the target compound.
58 g (yield: 97%) was obtained.

Synthesis of Reference Example 20 O -terl-butyl L- obtained in Reference Example 19 above
seryl-1-asparaginy-L-alanine-1erl-
Butyl ester 1.32g, carbobenzoxy-0-1
erl-butyl-thi-serine 0.97gXDCC0.7
A mixture of 4 g, HOBto, 50 g and dimethylformamide was stirred for 15 hours. Processing and purification of the reaction solution:
In the same manner as in Reference Example 18, 1.05 g (yield, 47%) of the target compound was obtained.

Reference Example 21 Carbobenzoxy-〇-f obtained in Reference Example 19 above
erf-butyl-ceryl-0-terl-butyl-
Shi-seryl-shi-asparaginyl-L-alanine-te
0.78 g rl-butyl ester, ethanol and 5%
A mixture of 0.1 g of Pd-C (50% water content) was subjected to catalytic reduction for 3 hours in the presence of a hydrogen stream. Filter the reaction solution,
The furnace liquid was concentrated under reduced pressure to obtain 0.55 g of the target compound (yield: 8
8%).

Reference Example 22 S-(2,3-dihydroxy-2R-propyl) (R)-(-)-3 to iodo-1,2 obtained in Reference Example 4 above
- 0.43 g of propanediol.

A mixture of 0.68 g of N-2,2,24-lichloroethoxycarponylcysteine di-1erI-butyl ester obtained in Reference Example 13, 1.0 g of N,N-diisopropylethylamine, and dimethylformamide was stirred at room temperature for 15 hours. . Dichloromethane was added to the reaction solution, and after washing with an aqueous solution containing an equivalent amount of 0.8yI of hydrochloric acid and saturated brine, the organic layer was dried over magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the residue. Purified by silica gel chromatography (developing solvent: chloroform, methanol = 15:1) to obtain 0.49 g of the target compound (yield:
59%).

Yellow-brown oil [α o = -4,5°IR (KBr, νmax): 3405 (OH, N
H) 1731 (COO) cm → S-(2,3-dihydroxy-2S-propyl)-N-2,2,2-trichloroethoxycarbonyl-(R)-cysteine tert- in the same manner as each side of the above reference example Butyl ester was obtained.

[α]D=+9.16°IR (KBr, νmax): 3414 (OH, NH
)17.23 (COO) cm' Reference Example Each side S-(2,3-dihydroxy-2R-propyl)-N-2,2,2 trichloroethoxycarbonyl-(R)- obtained in Reference Example 22 above Cysteine tert-butyl ester (0164 g) A mixture of dichloromethane, 0.046 g of 4-dimethylaminopyridine, and 0.78 g of diisopropylethylamine was cooled with water and while stirring, a dichloromethane solution of 0.88 g of valmitoyl chloride was added dropwise, and the mixture was stirred for 5 hours. Add dichloromethane to the reaction solution,
After washing with % citric acid, purified water, 5% NaHCO3, and saturated brine, the organic layer was dried over magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Chloroform:methanol-1:3 was added to the residue, recrystallization was performed, and target compound 1 was obtained.
．． 0 g (yield near 3%) was obtained.

White crystal mp: 43 to 45°C S-(2,3-bis(palmitoyloxy)-23-propyl)N-2,2,24
Lichloroethoxylponyl(R)-cysteine 1e
I got rt.

mp: 45-46°C Reference Example 24 Butyl ester mp: 31-33°C S-(2,3-bis(palmitoyloxy)-28-propyl)N-2 in the same manner as in Reference Example 24 above ,2,2
4-lichloroethoxycarponyl (R)-cysteine was obtained.

mp: 31 to 32°C Reference Example 25 S-(2,3-bis(palmitoyloxy)-2R-propyl)-N2.2.2-4 obtained in Reference Example 23 above
Lichloroethoxycarbonyl(R)-cysteine 1e
Trifluoroacetic acid 2 to 0.41 g of rt-butyl ester
yl was added and stirred for 1 hour. Dichloromethane was added to the reaction solution, washed with purified water, dried over magnesium sulfate, filtered, and the filtrate solution was concentrated under reduced pressure to obtain 0.3 of the target compound.
7g was obtained.

White powder S-(2,3-bis(palmitoyloxy)-2R-propyl)-N2 obtained in Reference Example 24 above. 2.2-Trichloroethoxycarbonyl(R)-cysteine 0.
32 g-O-terl obtained in Reference Example 21 above
-Butyl-Seryl-○-1erf-Butyl-L-Seryl-L-Asparaginyl-L-Alanine-1e"1-
Butyl ester 0.21g, DCCo, 09g, HOB
06g of tO and dimethylformamide were added and stirred for 15 hours.

The reaction solution was treated in the same manner as in Reference Example 18, and the resulting residue was recrystallized from chloroform:methanol=1:3 to obtain 0.25 g (yield: 50%) of the target compound.

White crystal mp: 132-134°C [α]o=+3.8” (chloroform) IR (KB
r, νmax): 3288 (NH).

1737 (COO), 1641°1541 (CONH) cm” S-(2,3-bis(palmitoyloxy)-23-propyl)N-2,2,2-
1-lichloroethoxylponyl(R)-cysteinyl-
〇 -ferl-butyl(S)-seryl-0-ferl
-Butyl-(S)-seryl-(S)-asparaginyl-
(S)-alanine-tert-butyl ester was obtained.

mp: 146-148°C [α]D=-6.61° (chloroform) IR (KB
r, νmax): 3288 (NH).

1739 (COO), 1639゜1541 (CONH) cm''1 Reference Example Reference 26 S-(2,3-bis(palmitoyloxy)-2R-propyl) -N2.2.2- obtained in Example 25
1-dichloroethoxycarbonyl(R)-cysteinyl-〇-1crt-butyl-(S)seryl-0-fer
t-Butyl-(S)-seryl-(S)-asparaginyl-(S)-alanine fert-butyl ester 0.15
A mixture of 0.75 g of zinc powder, 0.75 g of zinc powder, and acetic acid was stirred at room temperature for 15 hours. Filter the reaction solution, add dichloromethane to the furnace solution, neutralize with saturated NaHCO3, wash the organic layer with purified water and saturated brine, dry over magnesium sulfate, filter, and concentrate the furnace solution. Target compound 0.13g (yield: 9
6%).

mp 2 118-121°C S-(2,3-bis(palmitoyloxy)-23-propyl)(R)-cysteinyl-〇-tert-butyl-(S)-seryl-0
-tert-butyl-(S)-seryl-(S)-asparaginyl-(S)-alanine-1e-butyl ester was obtained.

Reference Example 27 S-(2,3-bis(palmitoyloxy)-2R-propyl)(R)-cysteinyl-〇-1erl-butyl-(S)-seryl-0-f obtained in Reference Example 26 above
erl-butyl-(S)-seryl(S)-asparaginyl-(S)-alanine-1erl-butyl ester 0.
While stirring a mixture of 06 g, dichloromethane, 6.1 mg of DMAP, and 26 mg of diisopropylethylamine under water cooling, 14 mg of valmitoyl chloride was dissolved in dichloromethane, added dropwise, and stirred for 5 hours. Reference example 2 using the reaction solution
Treat in the same manner as Step 3, and dissolve the residue in chloroform:methanol=
Recrystallization was performed using 1=3 to obtain 54 mg (yield near 6%) of the target compound.

White crystal mp: 187-189°C S-(2,3-bis(palmitoyloxy)-28-propyl)N-balmitoyl-(R)-cysteinyl-〇-jcrt-butyl-( S
)-Ceryl-0-terf-butyl-(S)-Ceryl-
(S)-asparaginyl-(S)-alanine-1erl
-Butyl ester was obtained.

mp: 194-195°C Reference Example 28 N-carbobenzoxy-thi-serine 4.78g (20m
mol) was dissolved in dichloromethane, 2 to 3 drops of concentrated sulfuric acid were added, and isobutylene gas was blown into the solution for 4 to 5 minutes while cooling with water, followed by stirring for 3 days. Several drops of triethylamine were added to the reaction solution to neutralize it, and the mixture was concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate, washed with 4% sodium bicarbonate and then with saturated aqueous solution, and the organic layer was dried over sodium sulfate.
Concentrate and subject the residue to silica gel column chromatography (
Purification was performed using a developing solvent (n-hexane: ethyl acetate = 10:1) to obtain 3.97 g (yield: 57%) of the target compound.

Reference Example 29 N-carbobenzoxy-〇 obtained in Reference Example 28 above
-1erl-butyl-L-serine 1erl-butyl ester hydrochloride 1. Ethanol and Pd-C (50% water content) were added to 05 g (3 mmol), and catalytic reduction was performed for 3 hours in the presence of a steam stream. The reaction solution was filtered, and an equivalent amount of hydrochloric acid was added to the furnace solution. Add ethanol containing 11 g (3n+n+ol) and concentrate under reduced pressure to obtain 0 of the target compound.
．． 63g (yield: 97%) was obtained.

Reference Example 30 O -terl-butyl L- obtained in Reference Example 29 above
Serine 1eN-butyl ester hydrochloride 4 Q,
DMF and 16 mg (16n+n+ol) of N-methylmorpholine were added to Omg (0,16+nmol), and after stirring for several minutes, S-(2,3-bis(palmitoyloxy)
-2R-propyl)-N-2,2,2-trichloroethoxycarbonyl-(R)-cysteine 134 mg (0
, 16 mmol), DCC 36 mg (0,18 mmol)
1), 48 mg (0.32 mmol) of HOBt was added and stirred overnight. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, ethyl acetate was added to the residue, the mixture was filtered again, and the solution was concentrated. Dichloromethane was added to the residue, which was washed with 5% citric acid, 4% sodium bicarbonate and saturated brine, and the organic layer was dried over magnesium sulfate and filtered. The furnace solution was concentrated, and the resulting residue was subjected to silica gel chromatography (developing solvent: n-hexane: ethyl acetate = 7: 1).
95 mg (yield: 57%) of the target compound was obtained.

Oily [α]D−+9.69° (C=1.9, chloroform) FABMASS: m/z (M+H)” 1046 Reference Example 31 0 −1crt-Butyl-L obtained in Reference Example 29 above
-Serine tert-butyl ester hydrochloride 127a
DMF and 51 mg (o, 50 mmol) of N-methylmorpholine were added to +g (0,50 mmol), and after stirring for several minutes, 148 mg (0,50 mmol) of N-carbobenzoxy-0-ferj-butyl-serine, DC
C113mg (0,55mmol) and HOB tl
53 mg (1.0 mmol) was added and stirred overnight.

The reaction solution was treated in the same manner as in Reference Example 30, and the resulting residue was purified by silica gel chromatography (developing solvent: n-hexane:ethyl acetate = 7:1) to obtain the target compound 18.
0 mg (yield near 3%) was obtained.

Reference Example 32 N-carbobenzoxy-〇 obtained in Reference Example 31 above
-tert-butyl-shi-seryl-0-1 crt-butyl-shi-serine terj-butyl ester 99 mg (
Ethanol and Pd-C (50% water content) were added to 0.2 mmol), and catalytic reduction was performed for 3 hours in the presence of a steam stream. The reaction solution was filtered, the reaction solution was concentrated, and 72m of the target compound was obtained.
g (yield: 100%) was obtained.

Reference Example 33 0-1erl-butyl L- obtained in Reference Example 32 above
Seryl-0-1erj-butyl-serine terl-
Butyl ester85. 0 mg (0.24 mmol), DMF, S-(2,3-bis(palmitoyloxy)-2R-propyl)-N-2,2,2-trichloroethoxycarbonyl-(R)-cysteine 200 mg (
0,24 mmol), DCC54 mg (0,26 mm
ol), HOB t 80mg (0.52mmol)
was added and stirred overnight. The reaction liquid was filtered, the furnace liquid was concentrated, ethyl acetate was added to the residue, filtered again, and the furnace liquid was concentrated. Dichloromethane was added to the residue, and the mixture was washed with 4% sodium hydrogen carbonate and saturated brine. The organic layer was dried with magnesium sulfate and filtered. The reaction solution was concentrated, and the resulting residue was purified by silica gel chromatography (developing solvent: n-hexane:ethyl acetate-7=1) to obtain the target compound 19.
0 mg (yield: 67%) was obtained.

Oily [α D = +15.3° (C = 2, 14, chloroform) FABMASS: m/z (M + H)” 118
9 Reference Example 34 Synthesis of [compound of formula (29)] Asparagine Ietl-butyl ester hydrochloride 0,
59 g (2.5 mmol) of DMF and 0.5 g (2.5 mmol) of N-methylmorpholine. After adding 26 g (2.5 mmol) and stirring for several minutes, N-carbobenzoxy 0-ter
-butyl-mono-serine 0. 74 g (2.5 mmo
l), DCCo, 57 g (2,8 mmol) and HO
BtO, 77 g (5.0 mmol) was added and stirred overnight.

The reaction solution was treated in the same manner as in Reference Example 30, and the resulting residue was purified by reprecipitation to obtain 0.84 g (yield near 4%) of the target compound.

Reference Example 35 Synthesis N-carbobenzoxy-〇 obtained in Reference Example 34 above
-tert-butyl-seryl-asparagine fe
rt-butyl ester 0. 31g (0.7mmol)
Ethanol and Pd-C (50% water content) were added to the solution, and catalytic reduction was performed for 3 hours in the presence of a steam stream. The reaction solution was filtered and the reaction solution was concentrated to obtain 0.22 g of the target compound (yield: 10
0%) was obtained.

Reference Example 36 0-1ett-butyl-shi- obtained in Reference Example 35 above
Seryl-asparagine 1ert-butyl ester 0.
22 g (0.7 mmol), carbobenzoxy-0-te+4-butyl-thi-serine 0.21 g (0,
7 mmol), DCCo, 16 g (0.8 mmol)
), HOBtO, 21g (1.4mmol) and DM
F was added and stirred overnight. The reaction solution was treated in the same manner as in Reference Example 33, and the resulting residue was purified by reprecipitation to obtain 0.34 g (yield: 80%) of the target compound.

Reference Example 37 N-benzocarboxy-〇 obtained in Reference Example 36 above
-jert-butyl-(S)-seryl-terl-butyl-(S)-seryl-asparagine terl-butyl ester 0. Ethanol and Pd-C (50% water content) were added to 18 g (0.17 mmol), and catalytic reduction was performed for 3 hours in the presence of a steam stream. The reaction solution was filtered and the filtrate solution was concentrated to obtain 0.13 g (yield: 95%) of the target compound.

Reference Example 38 Leu(S)-seryl-asparagine (e"1-butyReference Example 39 0-1crt-butyl-(S) obtained in Reference Example 37 above
)-Ceryl-0-jett-butyl-(S)-Ceryl-
Asparagine 1ef-butyl ester 78, 0m
g (0.16 mmol), DMF, 5-(2,3-bis(palmitoyloxy)-2R-propyl)-1'
m-2,2,2-trichloroethoxycarbonyl-(R
)-cysteine 150mg (0.18mmol), D
CC40mg (0.20mmol), HOB t 5
4 mg (0.36 mmol) was added and stirred overnight. The reaction solution was treated in the same manner as in Reference Example 30, and the resulting residue was recrystallized from chloroform/ethanol-1=5 at -20°C.
The precipitated crystals were collected and 108 mg (yield: 52%) of the target compound was obtained.

[α p=+12. 7° (C=1.25, chloroform) FABMASS: m/z (M + H)” 130
5 S-(2,3-bis(palmitoyloxy)-2R-propyl) -N-2゜2.2- obtained in Reference Example 38
1-lichloroethoxycarbonyl(R)cysteinyl-
〇 -tert-butyl-(S)-seryl-0-ter
t-Butyl-(S)-seryl-asparagine jut-
650 mg of zinc powder and acetic acid were added to 130 mg (0.1 mmol) of butyl ester, and the mixture was stirred overnight. The reaction solution was treated in the same manner as in Reference Example 26 to obtain 94 mg (yield: 87%) of the target compound.

[α D=+3.26° (C=1, 84, chloroform) FABMASS: m/z (M+H)” 112
9 Synthesis of Example 1 S-(2,3-bis(palmitoyloxy)-2R-propyl)-N-valmitoyl-(R)-cysteinyl-〇-tert-butyl-(S
)-seryl-0-terl-butyl-(S)-seryl-
(S)-asparaginyl-(S)-alanine-1ert
- Add trifluoroacetic acid to 70 mg of butyl ester,
Stirred for 1 hour. The reaction solution was treated in the same manner as in Reference Example 24,
The residue was recrystallized from chloroform:methanol=1:1 to obtain 33 mg (yield: 53%) of the target compound.

white powder mp: 211-213℃ [α]D=+56.5° IR (KBr.

FABMASS Example 2 (C=1.02, chloroform) νmax): 3284 (OH, NH) (Coo)
, 1627° (CONH) cm" + m/z (M1H)"" 1270 Reference Example 25 above
S-(2,3-bis(palmitoyloxy) obtained in
-2R-propyl)-N-2,2,24lichloroethoxycarbonyl(R)-cysteinyl-〇-te'l
-Butyl-(S)-seryl-0-1erl-butyl-(
Trifluoroacetic acid was added to 90 [11 g of S)-seryl (S)-asparaginyl-(S)-alanine-terl-butyl ester and stirred for 1 hour. Reference example 24 using the reaction solution
The residue was treated in the same manner as chloroform:methanol=1
= 1 to obtain 32 mg of the target compound (yield: 4
5%).

White powder mp: 205-207°C [α] o = +9.20° (C = 1.00, chloroform) IR (KBr, νmax): 3300 (OH, N
H) 173'6 (COO), 1662°1537 (CONH) cm' FABMASS: m/z (M x H)" 120
5 Example 3 Synthesis of S-(2,3-bis(palmitoyloxy)-2S-propyl)-N-balmitoyl-(R)-S (R)-(-)-3 obtained in Reference Example 4 above -Iodine-
The target compound was obtained in the same manner as in Example 1, except that (S)-(+)-3-iodo-1,2-propanediol obtained in Reference Example 10 was used instead of 1,2-propanediol.

White powder mp: 210-212°C [αko o −28,3° (C=0.86,
IR (KBr.

FABMASS Chloroform) νmax): 3296 (OH, NH) (Coo)
, 1639° (CONH) cm': m/z (M+H)' 1270 Example 4 1538 (CONH) cm" FABMASS: m/z (M+H)" 120
5 Example 5 (S)-(+)-3-iodo obtained in Reference Example 10 instead of (R)-(-)-3-iodo-1,2-propanediol obtained in Reference Example 4 above The target compound was obtained in the same manner as in Example 2 except that 1,2-propanediol was used.

White powder mp: 204-207°C [α]D-+16.6° (C = 1.00° chloroform) IR (KBr, νmax): 3302 (OH, N
H) 1737 (COO), 1629°S-(2,3-bis(palmitoyloxy)-2R-propyl)(R)-cysteinyl-〇-1erl-butyl-(S)- obtained in Reference Example 26 above Serilu 0-t
erf-butyl-(S)-seryl(S)-asparaginyl-(S)-alanine jet-butyl ester 70m
Trifluoroacetic acid was added to g (0.06 mmol) and stirred for 1 hour. The reaction solution was treated in the same manner as in Reference Example 24, and the residue was recrystallized from chloroform:methanol-1,1 to obtain 33 mg (yield: 53%) of the target compound.

White powder mp: 198-200°C FABMASS: m/z (M+H)” 1032
Example 6 86%) was obtained.

White powder mp: 50-52°C FABMASS: m/z (~i+)()'''93
3 Example 7 S-(2,3-bis(palmitoyloxy)-2R-propyl)-N2.2.24lichloroethoxycarbonyl(R)-cysteinyl-〇 obtained in Reference Example 30 above
-feat-butyl-(S)-serine Trifluoroacetic acid was added to 130 mg (0,12n++nol) of 1ert-butyl ester and stirred for 1 hour. The reaction solution was treated in the same manner as in Reference Example 24, and the residue was recrystallized from chloroform:methanol=1:1 to yield 100 mg of the target compound (yield: 5-(2,3-bis( palmitoyloxy)-2R-propyl)-N-2,2,
24lichloroethoxycarbonyl(R)-cysteinyl-0-1eN-butyl-(S)seryl-0-1crt-
Butyl-(S)-serine jerl-butyl ester 19
Trifluoroacetic acid was added to 0 mg (0.1.6 mmol) and stirred for 1 hour. The reaction solution was treated in the same manner as in Reference Example 24, purified water was added to the residue, stirred, and one crystal was collected.
After drying, 130 mg (yield: 80%) of the target compound was obtained.

White powder mp: 102-105°C FABMASS: m/z (M+H)" 1 ml of trifluoroacetic acid was added to 1022 Example 8 (0.05 mn+ol) and stirred for 1 hour. The reaction solution was treated in the same manner as in Reference Example 24. Then, methanol:chloroform=1=1 was added to the residue, left overnight at -20°C, recrystallized, and the precipitated crystals were collected in an oven and dried to give 45 mg of the target compound (yield: 58
%) was obtained.

White powder mp: 183-185°C FABMASS: m/z (M+H)'''1135 Example 9 Synthesis S-(2,3-bis(palmitoyloxy)-2R-propyl)- obtained in Reference Example 38 above N2.2, 2.h
Lichloroethoxycarbonyl(R)-cysteinyl-〇-1erl-butyl-(S)-seryl-0-tent
-Butyl-(S)-seryl asparagine Iert-Butyl ester 60mg Synthesis 5-(2,3-bis(palmitoyloxy)-2R-propyl)(R)-cysteinyl-〇-1er obtained in Reference Example 39 above! -butyl-(S)ceryl-0-ten
t-Butyl-(S)-seryl asparagine tent-
1 ml of trifluoroacetic acid was added to 94 mg (0,087 mmol) of butyl ester and stirred for 1 hour. The reaction solution was treated in the same manner as in Reference Example 24 to obtain 45 mg of the target compound (
A yield of 58% was obtained.

White powder mp: 216-218°C FABMASS: m/z (M+H)” 961 Next, pharmacological test examples are given.

■ Measurement of mitogen activity The spleens were removed from LPS-reactive C3H/He mice and LPS-unresponsive C3H/HeJ mice, spleen cells were isolated, and red blood cells were hemolyzed with 0.83% NH4Cl solution. The cells were removed to obtain a lymphocyte fraction.

In each well of a 96-well plate (manufactured by Falcon), 10
The above-mentioned lymphocytes suspended in RPMI-1640 culture medium containing % tallow serum were placed at 5 x 10 cells per well, and each well was filled with a test sample (compound of the present invention) and a positive control standard. Synthetic lipid A preparation (506, manufactured by Daiichi Kagaku Co., Ltd.) or S, LT derived from typhimu+ium
-2LPS was added and the lymphocytes in the plate were cultured at 37°C in a carbon dioxide cell incubator for 48 hours. after that,
0.25 μCi of 3H-thymidine per well was added to each well, and culture was continued at 37°C for 16 hours. After the culture was completed, the cultured cells were collected on paper using a Cellver Hester, and after drying, the The amount of radioactivity (3) (-thymine uptake, average count/min) of the cells was measured using a scintillation counter, and this was used as an index for determining cell juvenileization.

In addition, a control test was conducted in the same manner as above without adding any test sample, and based on the amount of radioactivity (average counts/min) obtained in the control test, this and each test sample were compared. The results of comparing the same amount of radioactivity (average counts/min) in the case of sample addition are obtained according to the following formula, and this is calculated as the stimulation index (S I).
ion 1 index).

Table 1 The results of the control test radioactivity are shown in Table 1 (1) (in the case of using C3H/He cells) and Table 1 (2) (in the case of using C3H/HeJ cells) below.

From Table 1 above, LPS or 506 is C3) 1/H
Although tile cell rejuvenation ability was observed for e cells, LPS
No reaction was observed in C3H/HeJ cells from non-responsive mice.

On the other hand, since each of the compounds of the present invention was found to have the ability to transform mice of both of the above-mentioned strains, it became clear that their effects were different from those of LPS. Among the compounds of the present invention, the compound obtained in Example 2 (KAB-2) having two valmitoyl groups had particularly strong activity.

(2) Lethal toxicity test using galactosamine-loaded mice 640 mg, 7 kg of galactosamine hydrochloride (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) was intraperitoneally administered to C57BL/6 mice, and immediately the compounds of the present invention (KAB-1 to KAB-4) were suspended. The solution was administered intravenously, and 24 hours later, the experimental mice were determined to be alive or dead, and the lethal toxicity of the test compound was determined.

The results obtained are shown in Table 2 below.

Table 2 also shows the results of the same test conducted on 506 as a comparative compound.

Table 2 Note that ND indicates that the experiment was not conducted.

From the table above, Lipid A synthetic product 506 kills test animals at a dose of 1 μg/mouse, but none of the compounds of the present invention kill test animals even at a dose of 50 μg/mouse. It is clear that the

■, Cell proliferation inhibitory activity test C3H/He mice were given 3% thioglycollate solution 2, Ow
1 was administered intraperitoneally, and peritoneal cells were collected on the fourth day.

Add 106 of the above cells per well to each well of a 24-well microplate (manufactured by Falcon), culture the cells for 2 hours at 37°C in a carbon dioxide cell incubator, and then wash the cells that do not adhere to the plate. A predetermined amount of the compound of the present invention was added to the macrophages obtained as a test sample, and the cells were further cultured at 37°C for 24 hours. After the culture was completed, the culture supernatant (containing TNF) was collected by centrifugation.

Next, using a 96-well microplate (manufactured by Falcon), a two-step dilution series (R
diluted with PMI-1640 medium) and added actinomycin to each well of L929 cells (4 x 104
cells/well) and cultured at 37° C. for 24 hours, then each well was washed with phosphate buffer, and then crystal violet was added to stain the cells. After drying, ethanol is added to liberate the pigment, and the amount of pigment is expressed as E
The absorbance was measured at 55 On'm, and the cell growth inhibiting activity (TNF production ability) of the test compound was determined from this.

The results obtained are shown in Table 3 below.

Table 3 From Table 3 above, all of the compounds of the present invention have clear cell proliferation inhibiting activity compared to LPS, especially KAB-
2 had strong activity.

■ Antitumor effect (tumor necrosis factor (TNF) effect) test B ALB/C? MethA tumor cells in mice 5
×105 mice were subcutaneously inoculated, and on the 7th and 9th day, 50 μg of the compound of the present invention was administered intravenously per mouse (test group, 1
(group of 5 animals). A control group (5 animals per group) was provided in which only physiological saline without the compound of the present invention was given.

200 days after tumor cell inoculation, tumors were excised, the average tumor weight (g+:SD) was determined, and the tumor cell proliferation inhibition rate was determined according to the following formula.

Inhibition rate (%) - (1-A/B)X100A: Average tumor weight of test group B: Average tumor weight of control group The results obtained are shown in Table 4 below.

From the above table, it is clear that among the compounds of the present invention, KAB-2 exhibits a significant tumor cell proliferation suppressive effect.

(2) Cytokine production ability test The cytokine production ability of the compounds of the present invention was examined. That is, a certain amount of the compound of the present invention was weighed and dissolved in 5% dimethyl sulfoxide-RPM11640 at a concentration of 10 μg/ill. LP S (Lipo
polysacchride) is LPS W
ECoI 055: 10ng/zI using B5 (manufactured by DIFCO)! RPM116 so that
40. R in a 24-well microplate,
PM11640 to 1000μ11 above DMSP'-RP
Compound of the present invention or target LP dissolved in M11640
100 μl of S solution and 100 μl of heparinized whole blood collected from a healthy person were added and cultured at 37° C. for 24 hours. After culturing, the supernatant was collected by centrifugation (3000 rpm, 10 minutes) and diluted 3 times with RPM11640. Supernatant (3
Each monoclonal antibody was used to measure the cytokine production amount of the diluted solution. Measured by ELISA method. The results are shown in Table 5 below. In addition, in Table 5, the production amount of each cytotonin is pg/zl.

Table 5 (that's all)

Claims (10)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R represents -CO(CH_2)_1_4CH_3. ] Lipopeptide represented by. (2) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, Troc represents -COOCH_2CCl_3. R is the same as above. ] Lipopeptide represented by. (3) General formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R is the same as above. ] lipopeptide represented by. (4) General formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R and Troc are the same as above. ] lipopeptide represented by. (5) General formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R is the same as above. ] lipopeptide represented by. (6) General formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R and Troc are the same as above. ] lipopeptide represented by. (7) General formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R and Troc are the same as above. ] lipopeptide represented by. (8) General formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R and Troc are the same as above. ] lipopeptide represented by. (9) General formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R is the same as above. ] lipopeptide represented by. (10) An antitumor agent containing the lipopeptide according to claims (1) to (9) as an active ingredient.