JPH049397A - New lipopeptide and antitumor agent - Google Patents

Abstract

NEW MATERIAL:A polypeptide shown by formula I [R is group shown by the formula -CO(CH2)14CH3]. EXAMPLE:S-{2,3-Bis(palmitoyloxy)-2R-propyl}-N-palmitoyl-(R)-cysteinyl- (S)- seryl-(S)-seryl-(S)-arpartyl-(S)-alanine. USE:An antitumor agent. PREPARATION:For example, (R)-(-)-3-iodo-1,2-propanediol shown by formula II is reacted with N-2,2,2-trichloroethoxycarbonylcysteineditert-butyl ester, then with palmitoyl chloride and hydrolyzed to give a compound shown by formula III (Troc is COOCH2CCl3). This compound is reacted with a protected peptide shown by formula IV (Bu<t> is tert-butyl) and deprotected to give a compound shown by formula I.

Description

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

Conventional techniques and their problems Lipopolysaccharide localized in bacterial cell wall peptidoglycan and cell wall outer membrane is an immune enhancer (adjuvant) 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 large amounts of several types of proteins, including proteins OmpA, OmpF,
OmpC and major lipoproteins are called major outer membrane proteins.

Major lipoproteins 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. Recently, lipopeptide, 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 lipopeptides. 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 lipopeptides 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 peptides, and also succeeded in synthesizing derivatives with enhanced activity. The invention has been completed.

Popeptides present in Durham-negative bacteria are potent activators of 8928 bulbs [V, BraunBioc
hem, Bioph7s, ^cta,,415,33
5 (1975)]. In order to analyze the molecular structure that expresses the biological activity of B lymphocytes, we investigated the N of this ribopeptide.
I tried analog synthesis of one end part. As a result, 5-(2
,3-bis(palmitoyloxy)-(2R8)-propyl) -N-valmitoyl-(R)-cysteinyl-
(S)-Seryl(S)-seryl-(S)-asparginyl-(S)alanine in vivo and in vitro 8928
It was found that activating the sphere [W, G, Be
5slerRB, Jobnson, K, H, Wi
esmuller and G, Jung, Hop
peSe7er 2. Physiol, Chem,,
363. 767 (1982), R.B., Joh.
Inson, S., Kohl, and H.

Wiesmuller, G., Jung, and
W, G, BesslerImonunobiol
ogy, 135. 1900 (1985), W
G, Be5slet, M, Cox, A, Le
x, B, 5uhr, K, HWiesmullet
and G, Jung, 1.1mmunolog
y, 135°1900 (1985)].

In order to protect the cysteine moiety from cariesization in the condensation reaction, the inventors proposed
By using a cysteinyl intermediate, a novel S-(
2,3-bis(palmitoyloxy)-(2R and 2S
)-propyl)N-valmitoyl-(R)-cysteinyl-(S)-seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine [the following general formulas (1) and (
3) and its derivative N-(2-trichloroethoxy-carbonyl) [the following general formulas (2) and (4
) was synthesized.

CI(2, OR CH20R CH2 Troc-Cys-3er-3rr-Asn-Ala-
0) 1 [In the formula, Troc is -COOCH2CCA'3R is the same as above. ] R-Cys-3er-3er-Asn-Ala-0)1
0-C-H C)+2 OR [wherein R is the same as above. Troc-Cys-3er-3er-Asn-Ala-
Indicates OFI.

R-Cys-3er-8er-^5n-Ala-OFI
[In the formula, R represents 100 (CH2)+ a CH3.

] ]0-C- CH2OR [wherein R and Troc are the same as above. The above general formula (
Polypeptides 1) to (4) are produced according to the method shown below.

(%))10-C-Fl Troc 0-C-H Troc CH2OR CH2-Cys-OH CH20R RO-, C-H Troc CH2-Cys-3er (Bu') Ser(Bu
)-Asn-Ala-OBuH20R (13)→RO-C-H C)12-Cys-3er(Bu')-3et(B
u') 85n-Ala-OBu CI (2OR -111÷RO-C-)1 CH2-Cys-8er(Bu')-3er(Bu
)-Asn-Ala-OBu' [wherein R and Tr
oc is the same as above. ] For each of the above-mentioned reactions, the reaction conditions used when synthesizing ordinary peptides can be appropriately applied. A specific example is as follows.

In the above reaction scheme, the compound of formula (5) used as a starting material is K, H, Wiesmuller
[The literature is mentioned above]. N-protection of the compound of formula (5) with trichloroethoxychloroformate (3 volumes) in pyridine followed by reduction with dithioerythritol (4 volumes) in chloroform in the presence of triethylamine (3 volumes). A compound of formula (7) is produced.

A compound of formula (9) was prepared by formula (9) in dimethylformamide in the presence of N,N-diisopropylethylamine (4 volumes).
It is produced by reacting the compound of formula (7) with the compound of formula (8).

Compounds of formula (10) 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. It is produced by esterifying the compound of 9).

The compound of formula (11) can be prepared by formula (11) using trifluoroacetic acid.
It is produced by deprotecting the tert-butyl group of the compound 10).

The compound of formula (13) is K, H, Wiesmulle
The compound of formula (11) and formula (12) were combined in dimethylformamide using dicyclohexylcarbodiimide (1 volume) and 1-hydroxybenzotriazole (2 volumes) as coupling reagents according to the method of R et al. ) is produced by coupling with the compound of

The compound of formula (2) is produced by treating all the protecting groups of the tcrt-butyl groups of the compound of formula (13) obtained above with trifluoroacetic acid.

Moreover, the compound of formula (14) is produced by treating the trichloroethoxycarbonyl group of the compound of formula (13) with zinc in acetic acid to remove it.

The compound of formula (15) is prepared by acylating the compound of formula (14) 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 tcrt-butyl groups of the compound of formula (15) obtained above with trifluoroacetic acid.

The compound of formula (3) and the compound of formula (4) can be produced by using formula I21 0-C-H CF+208 in place of the compound in the above reaction scheme and carrying out each reaction shown in the above reaction scheme. be done.

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, gunpowder, powders, liquids, suspensions, emulsions, granules, capsules, halves, 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,
The tablets can be enteric-coated, film-coated, double-layered, or multilayered. When forming the powder into a gunpowder form, 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 stasis, and when formulated into these forms, diluents commonly used in the art 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, intracranially, 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 Synthesis of (R)-(+)-1-0-acetyl-2-0-befuji-3-0-tosylglycerol (S)-(+)-
1-0-acetyl-2-〇-benzylglycerol 13
．． Pyridine 60z/ was added to 44 g, and 19.45 g of p-4 luenesulfonyl chloride was added under ice cooling, and the mixture was stirred overnight. The reaction solution was poured into ice water and extracted with dichloromethane. The organic layer was washed with IN hydrochloric acid (150 zl x 3 times) and saturated brine, dried over magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was chromatographed on silica gel. (developing solvent; isopropyl ether: chloroform = 1:1)
0) to obtain 21.75 g of the target compound (yield: 98
%) was obtained.

Colorless oil reference example 2 21.68 g of (R)-(+)-1-0-acetyl-2-0-benzyl-3-〇-tosylglycerol obtained in the above reference example 1 was mixed with 25% ammonia water 10 1 / A mixed solvent of 150 y/ml of methanol (this was added and stirred overnight at room temperature. 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
-(+) -2-0-benzyl-1-0-) 18.43 g of silglycerol was added, 2 g of 5% Pd-C (50% water content) was added, and catalytic reduction was performed 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 zl 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 [αco D=6. 0' (C=1.15,
Chloroform) IR (KBr, νmax): 3334 (OH)c
m-' Reference Example 5 (S)-(+)-1-0-acetyl-2-〇=benzylglycerol 15g and diisopropylethylamine 1
3 g of methoxymethyl chloride was dissolved in dichloromethane, and 6.5 g of methoxymethyl chloride was dissolved in dichloromethane and added dropwise while stirring under water cooling. 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). Target compound 3
3 g (yield: 97%) was obtained.

Reference Example 6 (S)-1,-0-acetyl- obtained in Reference Example 5 above
18 g of 2-0-benzyl-3-0-methoxymethylglycerol was dissolved in an ethanol solution of 2.7 g of sodium hydroxide and stirred for 1 hour under water cooling. After neutralizing with 6N hydrochloric acid, ethanol was distilled off and the residue was extracted with dichloromethane. After drying the dichloromethane layer with magnesium sulfate,
Concentrate under reduced pressure to obtain 15 g of the target compound (yield: 100%)
I got it.

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 6N hydrochloric acid 1511, and 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 ( Yield: 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.

Pd-C (50% water content) 1.0g and ethanol 501
1 was treated in the same manner as in Reference Example 3 to obtain 6.54 g of the target compound.
(yield: 99%).

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

Yellow crystal mp 2 35-37°C [α]D=+6.0° (C=1.35, chloroform) IR (KBr, νmax): 3334 (OH)c
m-' Reference Example 11 45 g of 60% perchloric acid was added to 15 g of cystine to dissolve it, 350 z/m of tert-butyl acetate was added, and the mixture was stirred for 60 hours. 2M NaOH and IMNaHCOa 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 diferf-butyl ester U formula (7)
0.53 g of cystine di-tert-butyl ester obtained in Reference Example 11 above, dichloromethane and 0.50 g of pyridine were added, and while stirring on water cooling, 1.02 g of trichloroethyl chloroformate dissolved in dichloromethane was added dropwise. Stir for 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-2,2,1-trichloroethoxycarbonylcystine di-1 crt-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, the target compound 1, O] was obtained without purification.
I got g.

Reference Example 14 After dissolving 2.4 g of N-benzocarboxy-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% Na HCOs.
The organic layer was washed with saturated brine, dried over magnesium sulfate, and then concentrated. The residue was subjected to silica gel chromatography (developing solvent: n-hexane:ethyl acetate-10:1)
2.02 g (yield near 2%) of the target compound was obtained.

Reference Example 15 Synthesis of L-alanine tert-butyl ester hydrochloride 400 mg of ethanol and Pd-C (50% water content) were added to 3.76 g of benzocarboxy-L-alanine-terl-butyl ester obtained in Reference Example 14 above. In addition, 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 (
A yield of 87% was obtained.

White crystal reference example 16 L-alanine obtained in reference example 15 (e) 0.72 g of 1-butyl ester hydrochloride, 0.41 g of N-methylmorpholine and dimethylformamide were added, and while stirring,
1.06 g of benzocarboxy-asharaquine, dicyclohexylcarfosiimide (hereinafter abbreviated as "DCC")
0.91 g and 0.91 g of 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, and 4%
The organic layer was washed with NaHC03 and saturated brine, dried over magnesium sulfate, filtered, and the filtrate was concentrated. Add the minimum amount of chloroform to the residue to dissolve it, add n-hexane to this until no precipitate occurs, filter the precipitate with suction through a glass filter, and dry it (re-precipitation method).
, 1.07 g (yield: 68%) of the target compound was obtained.

White powder reference example 17 Ethanol and 5% Pd-C (containing 50%) were added to 0.20 g of benzocarboxy-asparaginyl-L-alanine tert-butyl ester obtained in Reference Example 16 above, and the mixture was heated in a hydrogen stream for 3 hours. Catalytic reduction was carried out in the presence of The reaction solution was filtered, the reaction solution was concentrated, and the target compound was reduced to 0.13
g (yield: 97%) was obtained.

White powder reference example 18 Benzocarboxy-Q -jcrt-butyl-seri Asparaginyl-L-alanine 1ert-butyl ester obtained in the above reference example 17 0.20 g1 benzocarboxy-0-1crt-butyl-L Serine 0.23
A mixture of G, 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, filter with suction through a glass filter, concentrate the furnace liquid under reduced pressure, add ethyl acetate to the residue, filter the suction again, concentrate the furnace liquid, and dissolve the residue in dichloromethane. and 4% NaHC
After washing with O3 and saturated saline, the organic layer was dried and filtered.
The furnace liquid was concentrated under reduced pressure, and the residue was purified by the same reprecipitation method as in Reference Example 16, yielding 0.31 g of the target compound (yield near 6%).
) was obtained.

Reference Example 19 0-jcrt-butyl-ceryl-L-asparagine formation benzocarboxy-0-te obtained in Reference Example 18 above
rt-butyl-ceryl-asparaginyl-
A mixture of 80 g of alanine 1erl-butyl ester o, ethanol, and 0.1 g of 5% Pd-C (50% water content) was subjected to catalytic reduction for 3 hours in the presence of a hydrogen stream. The reaction solution was filtered, and the reaction solution was concentrated under reduced pressure to obtain 0.5 of the target compound.
8 g (yield: 97%) was obtained.

Synthesis of Reference Example 20 O-terj-butyl-cyclo-ceryl-cyclo-asparaginyl-cycloalanine tert obtained in Reference Example 19 above
-butyl ester 1.32g, benzocarboxy-0-
1erf-butyl-thi-serine 0.97g, DCCo,
A mixture of 74 g, HOBtO, 50 g, and dimethylformamide was stirred for 15 hours. The reaction solution was treated and purified in the same manner as in Reference Example 18, and 1.05 g of the target compound (
A yield of 47% was obtained.

Reference Example 21 Benzocarboxy-0-te obtained in Reference Example 19 above
rt-butyl-ceryl-0-tert-butyl-ceryl-asparaginyl-L-alanine-tert
0.78 g t-butyl ester, ethanol and 5% P
d-C (50% water content) 0.1g (7) mixture for 3 hours,
Catalytic reduction was carried out in the presence of a hydrogen stream. The reaction solution was filtered, and the filtrate solution was concentrated under reduced pressure to obtain 0.55 g of the target compound (yield:
88%).

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

A mixture of 0.68 g of N-2,2,2-1-lichloroethoxycarbonylcysteine di-tert-butyl ester obtained in Reference Example 13, 1.0 g of N,N-diisopropylethylamine, and dimethylformamide was heated at room temperature for 15 hours. Stirred. Add dichloromethane to the reaction solution,
After washing with an aqueous solution containing an equivalent amount of 0.8 zl of hydrochloric acid and saturated brine, the organic layer was dried over magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was subjected to silica gel chromatography (developing solvent: chloroform: Methanol =
15:1) to obtain 0.49 g of the target compound (yield: 5
9%).

Yellowish brown oil [α]D=-4,5°IR (KBr, νmax): 3405 (OH, NH
)1731 (COO) cm-' S-(2,3-dihydroxy-28-propyl)-N-2,2,2-trichloroethoxycarbonyl-(R)-cysteine tert-
Butyl ester was obtained.

[α]o=+9.16°IR (KBr, νmax): 3414 (OH, N
H) 1723 (COO) cm-' Reference Example 23 S-(2,3-dihydroxy-2R-propyl)-N-2,2,2 trichloroethoxycarbonyl-(R)-cysteine obtained in Reference Example 22 above A dichloromethane solution of 0.88 g of valmitoyl chloride was added dropwise to a mixture of 0.64 g of terf-butyl ester, dichloromethane, 0.046 g of 4-dimethylaminopyridine, and 0.78 g of diisopropylethylamine while cooling with water and stirring.
Stirred for 5 hours. Add dichloromethane to the reaction solution to make 5%
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. Add chloroform:methanol=1:3 to the residue and recrystallize to obtain 1.0 g of the target compound.
(yield near 3%) was obtained.

White crystals mp+43~45°C Same as in Reference Example 23 above.
-1lichloroethoxycarbonyl(R)-cysteine
Tert-butyl ester was obtained.

mp: 45-46°C Reference Example 24 S-(2,3-bis(palmitoyloxy)-2R-propyl)-N-2,2,2- obtained in Reference Example 23 above
1 Lichloroethoxycarbonyl (R)-cysteine 1
2 xi of trifluoroacetic acid was added to 0.41 g of erj-butyl ester and stirred for 17 hours. Dichloromethane was added to the reaction mixture, washed with purified water, dried over magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 0.37 g of the target compound.

White powder mp: 31 to 33°C S-(2,3-bis(palmitoyloxy)-28-propyl)N-2,2,2-
Trichloroethoxycarbonyl-(R)-cysteine was obtained.

mp: 31 to 32°C Reference Example 25 S-(2,3-bis(palmitoyloxy)-2R-propyl)-N2.2.24 obtained in Reference Example 24 above
Lycroethoxycarbonyl-(R)-cysteine 0.
32 g, 0-1erl-butyl-L-seryl-0-tert-butyl-L-seryl- obtained in Reference Example 21 above
-Asparaginyl L-alanine-1e “1-butyl ester 0.21 g, DCCo, 09 g, HOBtO, 0
6 g 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]D=→-3,8° (Chloroform) TR (KB
r, νmax): 3288 (NH).

1737 (Coo), 1641°1541 (CONH) cm-1 5-(2,3-bis(palmitoyloxy)-28-propyl)N-2,2,2- in the same manner as in Reference Example 25 above
1-lichloroethoxycarbonyl(R,)-cysteinyl-〇-fert-butyl(S)-seryl-0-te
rt-butyl-(S)--1=lilu(S)-asparaginyl-(S)-alanine-ter-dobutyl ester was obtained.

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

1739 (Coo), 1639°1, 541 (CONH) cm” Reference Example 26 Synthesis of compound (14) S-(2,3-bis(palmitoyloxy)-2R-propyl obtained in Reference Example 25 above) )-N2.2.2-)
Dichloroethoxycarbonyl (R)-cysteinyl-〇-jert-butyl-(S)-seryl-0-tert-
Butyl-(S)-seryl(S)-asparaginyl-(S
)-alanine tert-butyl ester 0.1'5g,
A mixture of 0.75 g of zinc dust 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: 96%
) was obtained.

mp: 118 to 121°C S-(2,3-bis(palmitoyloxy)-28-propyl)-(R)-cysteinyl-tert-butyl-(S)-seryl- 0-tert-butyl(S)-seryl-(S)-asparaginyl(S)
-Alanine-terl-butyl ester was obtained.

Reference Example 27 5-(2,3-bis(palmitoyloxy)-2R-propyl)(R)-cysteinyl-〇-terf-butyl-(S)-seryl-0- obtained in Reference Example 26 above
tert-butyl-(S)-seryl-(S)-asparaginyl-(S)-alanine tert-butyl ester 0
．． A mixture of 06g, dichloromethane, 6.1mg of DMAP and 26mg of diisopropylethylamine was cooled with water.
While stirring, 14 mg of valmitoyl chloride was dissolved in dichloromethane and added dropwise, followed by stirring for 5 hours. The reaction solution was treated in the same manner as in Reference Example 23, and the residue was recrystallized from chloroform/methanol=1:3 to obtain 54 mg of the target compound (yield: 6%).

White crystals mp: 187-189°C S-(2,3-bis(palmitoyloxy)-28-propyl)N-valmitoyl-(R)-cysteinyl-01erl-butyl-(S )
-Ceryl-0-1erl-butyl-(S)-seryl-(
S)-Asparaginyl (S)-alanine-16N-butyl ester was obtained.

mp: 194-195°C Example 1 Synthesis of the compound (1) (hereinafter referred to as rKAB-IJ)] S-(2,3-bis(palmitoyloxy)-2R-propyl obtained in Reference Example 27 above) )-N-valmitoyl-(R)-cysteinyl-0fert-butyl-(S
)-Ceryl-0-tert-butyl-(S)-Ceryl-
(S)-asparaginyl-(S)-alanine-tert
- 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°C [α]D=+56. 5° (C=1.02, chloroform) IR (KBr, νmax): 3284 (OH, N
H) 1732 (COO), 1627°1550 (CONH) cm-'FABMASS
: m/z (M+H)'' 1270 Example 2 5-(2,3-bis(palmitoyloxy)-2R-propyl)-N-2,2,2- obtained in Reference Example 25 above
Trichloroethoxycarbonyl-(R)-cysteinyl-〇-tert-butyl-(S)seryl-0-ter
t-Butyl-(S)-seryl-(S)-asparaginyl-(S)-alanine tert-butyl ester 90+1
Trifluoroacetic acid was added to 1 g and stirred for 1 hour. The reaction solution was treated in the same manner as in Reference Example 24, and the residue was dissolved in chloroform:
Recrystallized with methanol 1:1 to obtain the target compound 32m
g (yield: 45%) was obtained.

White powder mp: 205-207°C [α]D = +9,20° (Cm1.00, chloroform) νmax): 3300 (O) (, NH) (Coo
), 1662° (CONH) c+n-': m/z (M+H) ” 1205tR (KBr
.

FABMASS Example 3 Iodo-1,2-prono(1 instead of diol) (S)-(+)-3-iodo-1,2- obtained in Reference Example 10
The target compound was obtained in the same manner as in Example 1 except that propanediol was used.

White powder mp: 21 [α]D = 0-212°C 28,3° (Cm0.86, chloroform) νmax): 3296 (○H, NH) (COO)
, 1639° (CONH) Cm": m/z (M+H)" 1270IR (KBr
.

Synthesis of FABMASS Example 4 (R)-(-)-3(S) obtained in Reference Example 4 above
- Synthesis of alanine [compound of formula (4), hereinafter referred to as [KAB-4J]] Reference example instead of (R) -(-) -3iodo-1,2-propanediol obtained in Reference Example 4 above The target compound was obtained in the same manner as in Example 2 except that (S)-(+)-3-iodo-1,2-propanediol obtained in Example 10 was used.

White powder mp: 204-207°C [α]o=+16.6° (C21.00, chloroform) IR (KBr, νmax): 3302 (OH, NH
)1737 (Coo), 1629°1538 (CONH) cm' FABMASS : m/z (M+H)” 1205Next, pharmacological test examples are given.

■ Measurement of mitogen activity The spleens were removed from LPS-reactive C3H/He 7 mice and LPS-unresponsive C3H/HeJ mice, spleen cells were isolated, and red blood cells were cultured with 0.83% NH4CA' solution. was hemolyzed and 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 solution containing % live fetal serum were added to 5 x 10 cells per well, and a test sample (compound of the present invention) and a positive control standard were added to each well. Synthetic lipid A preparation (506, manufactured by Daiichi Kagaku Co., Ltd.) or S, LT derived from typhimutium
-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 filter paper using a cell harvester, and after drying, liquid scintillation was performed. The amount of radioactivity (3) J-thymidine uptake, average count/min) of the cells was measured using a 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 (81, stimulae on
1ndex).

The radioactivity levels obtained in the control test in Table 1 are shown below in Table 1 (1) (when using C3H/He cells) and Table 1 (2) (in the case of using C3H/He cells).
(in case of J cell use) are shown respectively.

Table 1 From Table 1 above, LPS or 506 was observed to have the ability to rejuvenate C3H/He cells, but no reaction was observed in C3H/HeJ cells from LPS-unresponsive mice.

On the other hand, since all of the compounds of the present invention have been found to have the ability to transform into tile cells of mice of both of the above-mentioned strains, it has become clear that their effects are different from those of LPS. Furthermore, among the compounds of the present invention, the compound obtained in Example 2 (KAB-2) having two valmitoyl groups had particularly strong activity.

■, Lethal toxicity test using galactosamine-loaded mice Galactosamine hydrochloride (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 640 ■/kg was intraperitoneally administered to C57B L/6 mice, and the compounds of the present invention (KAB-1 to KAB-4) were immediately suspended. The suspension 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 examined.

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, which indicates the toxicity. It is clear that the

■, Cell proliferation inhibitory activity test C3H/He mice with 3% thioglycollate solution 2.0
xll 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 culturing,
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 (4x10a
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
Absorbance was measured at 550 mm using an LrSA reader, 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.

■Anti-tumor effect (tumor necrosis factor (TNF) effect) test B ALB/C7 mice with MethA tumor cells 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, the tumor was excised, the average tumor weight (g+SD) was determined, and the tumor cell growth inhibition rate was determined according to the following formula.

Suppression rate (%) = (1-A/B)xlo.

A: 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 was revealed that among the compounds of the present invention, KAB-2 has a significant tumor cell proliferation suppressive effect.

(that's all)

Claims (5)

[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) An antitumor agent containing the lipopeptide according to claims 1 to 4 as an active ingredient.