JP2934905B2 - New lipopeptide and antitumor agent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel lipopeptide and an antitumor agent.

2. Description of the Related Art Bacterial cell wall peptidoglycan and lipopolysaccharide localized on the cell wall outer membrane have various immunity-enhancing (adjuvant) activities against mammals, pyrogenicity, anti-ulcer activity, non-specific infection protection activity, and the like. It is known to express biological activity. Such a cell surface substance has various functions and biological activities such as regulating the immune function of the living body in a very small amount, recognizing a host or a cell, a receptor function, adhesion or inducing cell differentiation. . In recent years, there has been much interest in basic research for elucidating the molecular structure and function, or the essence of activity expression at the molecular level, and for its effective use.

Bacteria are broadly divided into Gram-negative and Gram-positive bacteria, and the main difference between the two lies in the cell surface structure. The outer membrane of Escherichia coli, a representative strain of Gram-negative bacteria, contains a large amount of several proteins, and the proteins OmpA, OmpF, and 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 outer membrane assembly on the peptidoglycan layer. OmpF and OmpC are important proteins in outer membrane function and structure. In addition, lipopeptides present between the cell membrane and cytoplasm of Gram-negative bacteria have recently been attracting attention as substances that exhibit B-cell mitogenic activity by stimulating B cells. Furthermore, it has been reported that five substances have strong activity in the peptide part of lipopeptide. Although a method for synthesizing this substance has been reported, it is limited to a racemic synthesis method. This synthesis method uses a racemic glycerol derivative as a starting material,
After acylating the N-terminal of cysteine, a peptide synthesis reaction is performed. However, when a peptide is synthesized using this method, racemization of the cysteine moiety may occur, and this is not a preferable method.

Under such circumstances, a simple and reliable method for synthesizing optically active lipopeptides has been desired.

Means for Solving the Problems Accordingly, the present inventors have conducted intensive studies to solve the above problems, and as a result, used an optically active glycerol compound obtained by an enzymatic method as a starting material, and used N-terminal of cysteine as trichloroethoxy. By protecting with a carbonyl group, racemization of the cysteine moiety was prevented, new natural and non-natural lipopeptides were successfully synthesized, and a derivative with further enhanced activity was successfully synthesized. Completed the invention.

Lipopeptides present in Gram-negative bacteria are potent activators of B lymphocytes [V. Braun. Biochem.
s. Acta., 415 , 335 (1975)]. In order to analyze the molecular structure that expresses the biological activity of B lymphocytes, an analog synthesis of the N-terminal part of this lipopeptide was attempted. As a result, S- (2,3-bis (palmitoyloxy)-(2RS)
-Propyl) -N-palmitoyl- (R) -cysteinyl- (S) -seryl- (S) -seryl- (S) -asparginyl- (S) -alanine activates B lymphocytes in vivo and in vitro. Were found [WGBess
ler, RBJohnson, KHWiesmuller and G.Jung, Hoppe.Se
yer 2, Physiol. Chem., 363, 767 (1982), RB Johnson,
S.Kohl, KHWiesmuller, G.Jung, and WGBessler, Imonu
nobiology, 135 , 1900 (1985), WGBessler, M. Cox, A. Le
x, B.Suhr, KHWiesmuller and G.Jung, J.Immunology, 13
5,1900 (1985)].

The present inventors have developed a novel S by using N- (trichloroethoxy-carbonyl) cystinyl intermediate to protect the racemization of the cysteine moiety in the condensation reaction.
-(2,3-bis (palmitoyloxy)-(2R and 2S)
-Propyl) -N-palmitoyl- (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 the formula, R ₁ represents —CO (CH ₂ ) ₁₄ CH ₃ . ] [Wherein R ₂ ′ represents —COOCH ₂ CCl ₃ . R ₁ is the same as above. ] [Wherein R ₁ is the same as above. ] Wherein R ₁ and R ₂ ′ are the same as above. The polypeptides of the above general formulas (1) to (4) are produced according to the following method.

Reaction process formula Wherein R ₁ and R ₂ ′ are the same as above. In each of the above reactions, reaction conditions for synthesizing ordinary peptides can be appropriately applied. Specific examples are as follows.

In the above reaction scheme, the compound of formula (5) used as a starting material is prepared by the method of KH Wiesmuller et al. N-protection of the compound of formula (5) is performed with trichloroethoxychloroformate (3 volumes) in pyridine, followed by triethylamine (3
In the presence of (v), reduction with dithioerythritol (4 v) in chloroform produces the compound of formula (7).

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

The compound of formula (10) is prepared by adding palmitoyl chloride (2 volumes) and N, N-diisopropylethylamine (4 volumes) in methylene chloride in the presence of a catalytic amount of 4-dimethylaminopyridine.
) And esterifying the compound of formula (9).

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

The compound of the formula (13) can be prepared by the method of KH Wiesmuller et al. [Literature is mentioned above] using dicyclohexylcarbodiimide (1 volume) and 1-hydroxybenzotriazole (2 volumes) as coupling reagents in dimethylformamide. And a compound of the formula (12).

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

The compound of the formula (14) is produced by removing the trichloroethoxycarbonyl group of the compound of the formula (13) by treatment with zinc in acetic acid.

The compound of formula (15) is prepared by acylating the compound of formula (14) with palmitoyl chloride and N, N-diisopropylethylamine in methylene chloride.

The compound of the formula (1) is produced by treating all the protecting groups for the tert-butyl group in the compound of the formula (15) obtained above with trifluoroacetic acid.

The compound of the formula (3) and the compound of the formula (4) are replaced by a compound of the formula To perform each reaction shown in the above reaction scheme.

The target compound obtained by the method shown in each of the above reaction schemes can be separated from the reaction system by ordinary separation means and can be further purified. As the separation and purification means, for example, a distillation method, a recrystallization method, a column chromatography, an ion exchange chromatography, a gel chromatography, an affinity chromatography, a preparative thin layer chromatography, a solvent extraction method and the like can be adopted. .

The thus obtained active ingredient compound is effective as an antitumor agent, and the agent is used in the form of a general pharmaceutical preparation. The preparation is prepared using a commonly used diluent or excipient such as a filler, a bulking agent, a binder, a humectant, a disintegrant, a surfactant and a lubricant. Various forms can be selected as the pharmaceutical preparation according to the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspensions, emulsions,
Granules, capsules, suppositories, injections (solutions, suspensions, etc.)
And the like. In molding into tablets, various carriers well-known in the art can be widely used as carriers. Examples thereof include lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, excipients such as silicic acid, water, ethanol, propanol, simple syrup, glucose solution, starch solution, Gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, binders such as polyvinylpyrrolidone, dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters,
Disintegrating agents such as sodium lauryl sulfate, stearic acid monoglyceride, starch, and lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oil; quaternary ammonium bases; and absorption promoters such as sodium lauryl sulfate;
Humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid;
Lubricants such as purified talc, stearates, boric acid powder, and polyethylene glycol can be used. Further, the tablet can be made into a tablet coated with a usual coating, if necessary, such as a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet or a double tablet or a multilayer tablet. In molding into the form of pills, a wide variety of carriers conventionally known in this field can be used. Examples thereof include excipients such as glucose, lactose, starch, cocoa butter, hardened vegetable oils, kaolin, talc, binders such as gum arabic, tragacanth, gelatin, ethanol, disintegrants such as laminaran, agar, etc. Can be used. In the case of molding into a suppository form, conventionally known carriers can be widely used. Examples thereof include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. Capsules are prepared according to a conventional method, usually by mixing the active ingredient compound with the various carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules and the like. When prepared as an injection, liquid preparations, emulsions and suspensions are preferably sterilized and isotonic with blood, and are commonly used as diluents in the art when molded into these forms. Can be used, for example, water, ethyl alcohol, macrogol, propylene glycol,
Ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. In this case, a sufficient amount of salt, glucose or glycerin to prepare an isotonic solution may be included in the pharmaceutical preparation, and a usual solubilizer, buffer, soothing agent, etc. may be added. You may. Further, a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent and the like and other pharmaceuticals can be contained in the pharmaceutical preparation as required.

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

The administration method of the antitumor agent of the present invention is not particularly limited, and the antitumor agent is administered by a method according to various preparation forms, patient age, gender and other conditions, degree of disease, and the like. For example, tablets, pills, solutions,
In the case of suspensions, emulsions, granules and capsules, they are administered orally. In the case of an injection, it is administered intravenously, alone or in combination with a normal replenisher such as glucose or amino acid, and if necessary, intramuscularly, intradermally, subcutaneously or intraperitoneally. In the case of suppositories, they are administered rectally.

The dose of the antitumor agent of the present invention is appropriately selected depending on the usage, the age of the patient, gender and other conditions, the degree of the disease, and the like.
Usually, the amount of the active ingredient compound is about 1 kg per body weight per day.
It is good to be about 0.6-50mg. It is desirable that the active ingredient compound is contained in the dosage unit form in the range of about 10 to 1000 mg.

EXAMPLES Hereinafter, the present invention will be further clarified with reference to Reference Examples and Examples.

Reference Example 1 Synthesis of (R)-(+)-1-O-acetyl-2-O-benzyl-3-O-tosylglycerol (S)-(+)-1-O-acetyl-2-O-benzyl Add 13.44 g of glycerol to 60 ml of pyridine, and add
-Toluenesulfonyl chloride (19.45 g) was added and the mixture was stirred overnight. The reaction solution was poured into ice water, extracted with dichloromethane, and the organic layer was washed with 1N hydrochloric acid (150 ml × 3 times) and saturated saline, dried over magnesium sulfate, filtered, and the solution was concentrated under reduced pressure. Purification by chromatography (developing solvent; isopropyl ether: chloroform = 1: 10) gave 21.75 g (yield: 98%) of the target compound.

Colorless oil Reference Example 2 Synthesis of (R)-(+)-2-O-benzyl-1-O-tosylglycerol (R)-(+)-1-O-acetyl-2 obtained in Reference Example 1 above. 21.68 g of -O-benzyl-3-O-tosylglycerol was added to a mixed solvent of 10 ml of 25% aqueous ammonia and 150 ml of methanol, followed by stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure, and dichloromethane was added to the residue. The extract was washed with purified water and saturated brine, dried over magnesium sulfate, concentrated, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developing solvent; chloroform: ethanol = 20: 1) to obtain 18.43 g of the desired compound ( Yield: 96%).

Colorless oil Reference Example 3 Synthesis of (R)-(-)-3-tosyloxy-1,2-propanediol (R)-obtained in Reference Example 2 above in 100 ml of ethanol.
18.43 g of (+)-2-O-benzyl-1-O-tosylglycerol was added, and 2 g of 5% Pd-C (containing 50% water) was added.
The catalytic reduction was carried out in the presence of a hydrogen stream for a period of time. The reaction solution was passed, and the solution was concentrated under reduced pressure.
%).

Colorless oil Reference Example 4 Synthesis of (R)-(−)-3-iodo-1,2-propanediol [compound of formula (8)] (R)-(−)-3 obtained in Reference Example 3 above. A mixture of 1.9 g of tosyloxy-1,2-propanediol, 5 ml of acetone and 3.63 g of sodium iodide in a sealed tube at 90 ° C.
Stirred for hours. Pass the reaction solution through a glass filter,
The solution was concentrated under reduced pressure, ether was added to the residue, and the mixture was stirred, extracted, filtered again, and 0.1N sodium thiosulfate was added to the solution until the color disappeared.The organic layer was dried over magnesium sulfate, filtered, and filtered. Was concentrated, n-hexane was added to the residue, and the mixture was stirred. The crystal part was passed through a glass filter and dried to obtain 1.05 g of the desired compound (yield: 65%).

Yellow crystal mp: 35-37 ° C. [α] _D = −6.0 ° (C = 1.15, chloroform) IR (KBr, νmax): 3334 (OH) cm ⁻¹ Reference Example 5 (S) -1-O-acetyl- 2-O-benzyl-3-O
Synthesis of -methoxymethylglycerol (S)-(+)-1-O-acetyl-2-O-benzylglycerol 15 g and diisopropylethylamine 13 g
Was dissolved in dichloromethane, and 6.5 g of methoxymethyl chloride was dissolved in dichloromethane and added dropwise while stirring under ice cooling. The mixture was stirred overnight at room temperature, washed with water and saturated saline, 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 of the desired compound (yield: 97
%).

Reference Example 6 Synthesis of (R) -2-O-benzyl-3-O-methoxymethylglycerol (S) -1-O-acetyl- obtained in Reference Example 5 above.
18 g of 2-O-benzyl-3-O-methoxymethylglycerol was dissolved in a solution of 2.7 g of sodium hydroxide in ethanol and stirred for 1 hour under ice cooling. After neutralization 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 of the desired compound (yield: almost 100%).

Reference Example 7 (S) -1-O-tosyl-2-O-benzyl-3-O-
Synthesis of methoxymethylglycerol (R) -2-O-benzyl- obtained in Reference Example 6 above
15 g of 3-O-methoxymethylglycerol and 10 g of triethylamine were dissolved in dichloromethane. Under ice cooling and stirring, p-toluenesulfonyl chloride was dissolved in dichloromethane and added dropwise. After stirring overnight, the mixture was washed with water and saturated saline. The organic layer is dried over magnesium sulfate,
After that, the solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent; hexane: ethyl acetate = 5: 1) to obtain 25 g of the desired compound (yield: 98%).

Reference Example 8 Synthesis of (S) -1-O-tosyl-2-O-benzyl-glycerol (S) -1-O-tosyl-2 obtained in Reference Example 7 above.
-O-benzyl-3-O-methoxymethylglycerol
Add 25 g to a methanol solution containing 15 ml of 6N hydrochloric acid, add 60 g
Stirred at C for 8 hours. After neutralization with 2N sodium hydroxide solution, methanol is distilled off, the residue is extracted with dichloromethane, the organic layer is washed with water and saturated saline, dried over magnesium sulfate, and concentrated under reduced pressure to give 21 g of the desired compound ( Yield: 98
%).

White amorphous Reference Example 9 Synthesis of (S) -1-O-tosylglycerol (S) -1-O-tosyl-2 obtained in Reference Example 8 above.
9.05 g of -O-benzyl-glycerol, 1.0 g of Pd-C (containing 50% water) and 50 ml of ethanol were 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 Synthesis of (S)-(+)-3-iodo-1,2-propanediol 1.9 g of (S) -1-O-tosyl-glycerol obtained in Reference Example 9 above, 5 ml of acetone and 3.63 g sodium iodide
Was treated in the same manner as in Reference Example 4 to give 1.05 g of the desired compound (yield:
65%).

Yellow crystal mp: 35-37 ° C [α] _D = + 6.0 ° C (C = 1.35, chloroform) IR (KBr, νmax): 3334 (OH) cm ^-1 Reference Example 11 Cystine di-tert-butyl ester [formula (5) Synthesis of compound] To 15 g of cystine, add 45 g of 60% perchloric acid and dissolve
350 ml of tert-butyl was added and stirred for 60 hours. 2 in the reaction solution
After adding MNaOH and 1M NaHCO ₃ to adjust the pH to 7.8, the mixture was extracted with diethyl ether, washed with saturated saline, dried over magnesium sulfate, and filtered.
14.8 g (yield: 67%) was obtained.

Dilute yellow oil Reference Example 12 N-2,2,2-trichloroethoxycarbonylcystine
Synthesis of di-tert-butyl ester [compound of formula (6)] 0.53 g of cystine di-tert-butyl ester obtained in Reference Example 11, 0.50 g of dichloromethane and pyridine were added, and trichloroethyl was stirred under ice cooling. Chloroformate
1.02 g was dissolved in dichloromethane and added dropwise, followed by stirring for 3 hours. Dichloromethane was added to the reaction solution, and the mixture was washed with 1N hydrochloric acid and saturated saline, and the organic layer was dried over magnesium sulfate, filtered, and 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 Synthesis of N-2,2,2-trichloroethoxycarbonylcysteine di-tert-butyl ester [compound of formula (7)] N-2,2,2-trichloro compound obtained in Reference Example 12 above Ethoxycarbonylcystine di-tert-butyl ester cysteine (0.85 g), dithioerythritol (1.0 g), triethylamine (0.48 g) and chloroform were added, and the mixture was stirred for 2 hours while replacing with argon gas. Chloroform was added to the reaction solution,
After washing with 5% citric acid and purified water under argon gas replacement,
The organic layer was dried over magnesium sulfate, filtered, and the solution was concentrated under reduced pressure. Because of the susceptibility to air oxidation, 1.01 g of the target compound was obtained without purification.

Reference Example 14 Synthesis of benzocarboxy-L-alanine-tert-butyl ester After dissolving 2.4 g of N-benzocarboxy-L-alanine in dichloromethane, a catalytic amount of sulfuric acid was added, and isobutylene gas was blown in under ice cooling for 4 to 5 minutes. Stir for 3 days. The reaction solution was neutralized by adding triethylamine, and concentrated under reduced pressure. The residue was dissolved by adding ethyl acetate, washed with 4% NaHCO ₃ and brine, dried over magnesium sulfate, and concentrated. The residue is subjected to silica gel chromatography (developing solvent; n
-Hexane: ethyl acetate = 10: 1) to give the desired compound
2.02 g (yield: 72%) was obtained.

Reference Example 15 Synthesis of L-alanine tert-butyl ester / hydrochloride To 3.76 g of the benzocarboxy-L-alanine-tert-butyl ester obtained in Reference Example 14 above, ethanol and Pd were added.
400 mg of -C (containing 50% water) was added, and catalytic reduction was performed in the presence of a hydrogen stream for 3 hours. The reaction mixture was filtered, a hydrochloric acid-ethanol solution containing an equivalent of 0.47 g of hydrochloric acid was added to the reaction mixture, and the mixture was concentrated under reduced pressure to obtain 2.12 g of the desired compound (yield: 87%).

White crystal Reference Example 16 Benzocarboxy-L-asparaginyl-L-alanine
Synthesis of tert-butyl ester 0.72 g of L-alanine tert-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 benzocarboxy-L-asparagine was added. 0.91 g of dicyclohexylcarbodiimide (hereinafter abbreviated as “DCC”) and 0.61 g of 1-hydroxybenzotriazole were added, and the mixture was stirred for 15 hours. A small amount of ethyl acetate was added to the reaction solution, and the mixture was suctioned.
The solution was concentrated, and dichloromethane was added to the residue.
Washed with citric acid, purified water, 4% NaHCO ₃ and saturated saline,
The organic layer was dried over magnesium sulfate, filtered, and concentrated. A minimum amount of chloroform that can be dissolved in the residue is added and dissolved, n-hexane is added to the residue until no precipitate is generated, the precipitate is suctioned through a glass filter, and dried (reprecipitation method). Yield: 68%).

White powder Reference Example 17 Synthesis of L-asparaginyl-L-alanine tert-butyl ester Benzocarboxy-L-asparaginyl-L-alanine tert-butyl ester 0.20 obtained in Reference Example 16 above.
g and ethanol and 5% Pd-C (containing 50%).
The catalytic reduction was carried out in the presence of a hydrogen stream for a period of time. The reaction solution was passed, and the solution was concentrated to obtain 0.13 g (yield: 97%) of the target compound.

White powder Reference Example 18 Benzocarboxy-O-tert-butyl-L-seryl-L
Synthesis of -Asparaginyl-L-alanine tert-butyl ester 0.20 g of L-asparaginyl-L-alanine tert-butyl ester obtained in the above Reference Example 17, 0.23 g of benzocarboxy-O-tert-butyl-L-serine, dimethyl Formamide, 1-hydroxybenzotriazole 0.12 g
And 0.16 g of DCC were stirred at room temperature for 15 hours. A small amount of ethyl acetate was added to the reaction solution, and the mixture was filtered with suction through a glass filter. The liquid was concentrated under reduced pressure, ethyl acetate was added to the residue, the mixture was suctioned again, the liquid was concentrated, and the residue was dissolved in dichloromethane. % NaHCO ₃ , saturated saline, and then the organic layer was dried. The mixture 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 desired compound.
(Yield: 76%) was obtained.

Reference Example 19 O-tert-butyl-L-seryl-L-asparaginyl-
Synthesis of L-alanine tert-butyl ester Benzocarboxy-O-tert obtained in Reference Example 18 above
-Butyl-L-seryl-L-asparaginyl-L-alanine tert-butyl ester 0.80 g, ethanol and 5
A mixture of 0.1 g of% Pd-C (containing 50% water) was subjected to catalytic reduction for 3 hours in the presence of a hydrogen stream. The reaction solution was passed, and the solution was concentrated under reduced pressure to obtain 0.58 g of the desired compound (yield: 97%).

Reference Example 20 Benzocarboxy-O-tert-butyl-L-seryl-O
-Tert-butyl-L-seryl-L-asparaginyl-L
Synthesis of -alanine-tert-butyl ester 1.32 g of O-tert-butyl-L-seryl-L-asparaginyl-L-alanine tert-butyl ester obtained in Reference Example 19 above, benzocarboxy-O-tert-butyl- A mixture of 0.97 g of L-serine, 0.74 g of DCC, 0.50 g of HOBt and dimethylformamide was stirred for 15 hours. Treatment and purification of the reaction solution was the same as in Reference Example 18, and 1.05 g of the target compound was obtained.
(Yield: 47%) was obtained.

Reference Example 21 O-tert-butyl-L-seryl-O-tert-butyl-L
-Seryl-L-asparaginyl-L-alanine-tert-
Synthesis of butyl ester Benzocarboxy-O-tert obtained in Reference Example 19 above
-Butyl-L-seryl-O-tert-butyl-L-seryl-L-asparaginyl-L-alanine-tert-butyl ester 0.78 g, ethanol and 5% Pd-C (50% aqueous)
0.1 g of the mixture was subjected to catalytic reduction in the presence of a hydrogen stream for 3 hours. The reaction solution was passed, and the solution was concentrated under reduced pressure to obtain 0.55 g of the desired compound (yield: 88%).

Reference Example 22 S- {2,3-dihydroxy-2R-propyl} -N-2,2,2
-Trichloroethoxycarbonyl- (R) -cysteine
Synthesis of tert-butyl ester [compound of formula (9)] 0.43 g of (R)-(−)-3-iodo-1,2-propanediol obtained in Reference Example 4 above and obtained in Reference Example 13 above. A mixture of 0.62 g of N-2,2,2-trichloroethoxycarbonylcysteine di-tert-butyl ester, 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 the mixture was washed with an aqueous solution containing an equivalent amount of hydrochloric acid (0.8 ml) and saturated saline, and the organic layer was dried over magnesium sulfate.
The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent; chloroform: methanol = 15: 1) to obtain 0.49 g of the desired compound (yield: 59%).

Yellow-brown oil [α] _D = -4.5 ° IR (KBr, νmax): 3405 (OH, NH) 1731 (COO) cm ^-1 S- ｛2,3-dihydroxy-2S- Propyl {-N-2,2,2-trichloroethoxycarbonyl- (R) -cysteine tert-butyl ester was obtained.

[Α] _D = + 9.16 ° IR (KBr, νmax): 3414 (OH, NH) 1723 (COO) cm ^-1 Reference Example 23 S- {2,3-bis (palmitoyloxy) -2R-propyl}- N-2,2,2-trichloroethoxycarbonyl-
(R) -cysteine tert-butyl ester [formula (10)
Of S- {2,3-dihydroxy-2 obtained in Reference Example 22 above.
R-propyl} -N-2,2,2-trichloroethoxycarbonyl- (R) -cysteine tert-butyl ester 0.64
g, dichloromethane, 4-dimethylaminopyridine 0.046
g and a mixture of 0.78 g of diisopropylethylamine were stirred under ice cooling while a dichloromethane solution of 0.88 g of palmitoyl chloride was added dropwise, and the mixture was stirred for 5 hours. Dichloromethane was added to the reaction solution, and the mixture was washed with 5% citric acid, purified water, 5% NaHCO ₃ and saturated saline, and the organic layer was dried over magnesium sulfate. After that, the solution was concentrated under reduced pressure. Chloroform: methanol = 1: 3 was added to the residue and recrystallization was performed to obtain 1.0 g of the target compound (yield: 73%).

White crystals mp: 43-45 ° C. S- {2,3-bis (palmitoyloxy) -2S-propyl} -N-2,2,2-trichloroethoxycarbonyl- (R)- Cysteine tert-
The butyl ester was obtained.

mp: 45-46 ° C. Reference Example 24 S- {2,3-bis (palmitoyloxy) -2R-propyl} -N-2,2,2-trichloroethoxycarbonyl-
Synthesis of (R) -cysteine [compound of formula (11)] S- {2,3-bis (palmitoyloxy) -2R-propyl} -N-2,2,2-trichloro obtained in Reference Example 23 above 2 ml of trifluoroacetic acid was added to 0.41 g of ethoxycarbonyl- (R) -cysteine tert-butyl ester, and the mixture was stirred for 1 hour. Dichloromethane was added to the reaction solution, washed with purified water, dried over magnesium sulfate, and thereafter, the solution was concentrated under reduced pressure to obtain 0.37 g of the desired compound.

White powder mp: 31-33 ° C S- {2,3-bis (palmitoyloxy) -2S-propyl} -N-2,2,2-trichloroethoxycarbonyl- (R)-as in Reference Example 24 above. Cysteine was obtained.

mp: 31-32 ° C. Reference Example 25 S- {2,3-bis (palmitoyloxy) -2R-propyl} -N-2,2,2-trichloroethoxycarbonyl-
(R) -cysteinyl-O-tert-butyl- (S) -seryl-O-tert-butyl- (S) -seryl- (S) -asparaginyl- (S) -alanine-tert-butyl ester [formula (13) The compound of S- {2,3-bis (palmitoyloxy) -2R-propyl} -N-2,2,2-trichloroethoxycarbonyl- (R) -cysteine obtained in Reference Example 24 above was prepared. g, O-tert-butyl-L-seryl- obtained in Reference Example 21 above.
O-tert-butyl-L-seryl-L-asparaginyl-
L-alanine-tert-butyl ester 0.21 g, DCC 0.09
g, HOBt 0.06 g and dimethylformamide were added, and the mixture was stirred for 15 hours. The reaction solution was treated in the same manner as in Reference Example 18, and the obtained residue was recrystallized from chloroform: methanol = 1: 3 to obtain 0.25 g of the desired compound (yield: 50%).

White crystal mp: 132-134 ° C [α] _D = + 3.8 ° (chloroform) IR (KBr, νmax): 3288 (NH), 1737 (COO), 1641, 1541 (CONH) cm ^-1 Reference example 25 above S- {2,3-bis (palmitoyloxy) -2S-propyl} -N-2,2,2-trichloroethoxycarbonyl- (R) -cysteinyl-O-
tert-butyl- (S) -seryl-O-tert-butyl-
(S) -Seryl- (S) -asparaginyl- (S) -alanine-tert-butyl ester was obtained.

mp: 146 to 148 ° C [α] _D = -6.61 ° (chloroform) IR (KBr, νmax): 3288 (NH), 1739 (COO), 1639, 1541 (CONH) cm ^-1 Reference Example 26 S- ｛2 , 3-Bis (palmitoyloxy) -2R-propyl}-(R) -cysteinyl-O-tert-butyl-
(S) -Seryl-O-tert-butyl- (S) -seryl-
Synthesis of (S) -asparaginyl- (S) -alanine-tert-butyl ester [compound of formula (14)] S- {2,3-bis (palmitoyloxy) -2R-propyl obtained in Reference Example 25 above ｝ -N-2,2,2-trichloroethoxycarbonyl- (R) -cysteinyl-O-te
rt-butyl- (S) -seryl-O-tert-butyl-
A mixture of (S) -seryl- (S) -asparaginyl- (S) -alanine-tert-butyl ester 0.15 g, zinc dust 0.75 g and acetic acid was stirred at room temperature for 15 hours. Had a reaction solution, dichloromethane was added to the solution, after neutralization with saturated NaHCO _3, the organic layer was washed purified water, with saturated brine, dried over magnesium sulfate, filtered, the liquid was concentrated, the target compound 0 .
13 g (yield: 96%) was obtained.

mp: 118-121 ° C In the same manner as in Reference Example 26 above, S- {2,3-bis (palmitoyloxy) -2S-propyl}-(R) -cysteinyl-O-tert-butyl- (S) -seryl- O-tert-butyl- (S) -seryl- (S) -asparaginyl-
(S) -alanine-tert-butyl ester was obtained.

Reference Example 27 S- {2,3-bis (palmitoyloxy) -2R-propyl} -N-palmitoyl- (R) -cysteinyl-O-
tert-butyl- (S) -seryl-O-tert-butyl-
Synthesis of (S) -seryl- (S) -asparaginyl- (S) -alanine-tert-butyl ester [compound of formula (15)] S- {2,3-bis (palmitoyl) obtained in Reference Example 26 above Oxy) -2R-propyl}-(R) -cysteinyl-O-tert-butyl- (S) -seryl-O-tert-butyl- (S) -seryl- (S) -asparaginyl- (S)
-Alanine-tert-butyl ester 0.06 g, dichloromethane, DMAP 6.1 mg and diisopropylethylamine 26 mg
While stirring the mixture under ice cooling, palmitoyl chloride 14 mg
Was dissolved in dichloromethane, added dropwise, and stirred 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 the title compound 54m
g (yield: 76%) was obtained.

White crystal mp: 187 to 189 ° C S- {2,3-bis (palmitoyloxy) -2S-propyl} -N-palmitoyl-
(R) -Cysteinyl-O-tert-butyl- (S) -seryl-O-tert-butyl- (S) -seryl- (S) -asparaginyl- (S) -alanine-tert-butyl ester was obtained.

Example 1 S- {2,3-bis (palmitoyloxy) -2R-propyl} -N-palmitoyl- (R) -cysteinyl-
(S) -seryl- (S) -seryl- (S) -asparaginyl- (S) -alanine [compound of formula (1);
B-1 ")]] S- {2,3-bis (palmitoyloxy) -2R-propyl} -N-palmitoyl- obtained in Reference Example 27 above.
(R) -Cysteinyl-O-tert-butyl- (S) -seryl-O-tert-butyl- (S) -seryl- (S) -asparaginyl- (S) -alanine-tert-butyl ester Acetic acid was added and stirred for 1 hour. The reaction solution was treated in the same manner as in Reference Example 24, and the residue was treated with chloroform:
Recrystallized from methanol = 1: 1, 33 mg of the desired compound
(Yield: 53%) was obtained.

White powder _{mp: 211~213 ℃ [α] D} = + 56.5 ° (C = 1.02, chloroform) IR (KBr, νmax): 3284 (OH, NH) 1732 (COO), 1627, 1550 (CONH) cm - ^{1 FABMASS: m / z (M} + H) + 1270 example 2 S- {2,3-bis (palmitoyloxy)-2R-propyl} -N-2,2,2-trichloroethoxycarbonyl - (R) - cysteinyl - ( S) -Seryl- (S) -seryl- (S)-
Synthesis of asparaginyl- (S) -alanine [compound of formula (2), hereinafter referred to as "KAB-2"] S- {2,3-bis (palmitoyloxy) -2R- obtained in Reference Example 25 above. Propyl} -N-2,2,2-trichloroethoxycarbonyl- (R) -cysteinyl-O-te
rt-butyl- (S) -seryl-O-tert-butyl-
Trifluoroacetic acid was added to 90 mg of (S) -seryl- (S) -asparaginyl- (S) -alanine-tert-butyl ester, and the mixture was 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 32 mg of the desired compound (yield: 45%).

White powder mp: 205-207 ° C [α] _D = + 9.20 ° (C = 1.00, chloroform) IR (KBr, νmax): 3300 (OH, NH) 1736 (COO), 1662, 1537 (CONH) cm ^{− 1} FABMASS: m / z (M + H) ⁺ 1205 Example 3 S- {2,3-bis (palmitoyloxy) -2S-propyl} -N-palmitoyl- (R) -cysteinyl-
(S) -seryl- (S) -seryl- (S) -asparaginyl- (S) -alanine [compound of formula (1);
B-3 ")]] (S)-obtained in Reference Example 10 in place of (R)-(-)-3-iodo-1,2-propanediol obtained in Reference Example 4 above. A target compound was obtained in the same manner as in Example 1 except that (+)-3-iodo-1,2-propanediol was used.

White powder mp: 210-212 ° C [α] _D = -28.3 ° (C = 0.86, chloroform) IR (KBr, νmax): 3296 (OH, NH) 1736 (COO), 1639, 1538 (CONH) cm ^-1 FABMASS: m / z (M + H) + 1270 example 4 S- {2,3-bis (palmitoyloxy)-2S-propyl} -N-2,2,2-trichloroethoxycarbonyl - (R) - cysteinyl - (S ) -Seryl- (S) -seryl- (S)-
Synthesis of asparaginyl- (S) -alanine [compound of formula (4), hereinafter referred to as “KAB-4”] The (R)-(−)-3-iodo-1,2 obtained in Reference Example 4 above. A target compound was obtained in the same manner as in Example 2 except that (S)-(+)-3-iodo-1,2-propanediol obtained in Reference Example 10 was used instead of -propanediol.

White powder _{mp: 204~207 ℃ [α] D} = + 16.6 ° (C = 1.00, chloroform) IR (KBr, νmax): 3302 (OH, NH) 1737 (COO), 1629, 1538 (CONH) cm - ¹ FABMASS: m / z (M + H) ⁺ 1205 Next, a pharmacological test example will be described.

I. Mitogen activity measurement LPS-reactive C3H / He mice and LPS-inactive C3H / HeJ
The spleen was excised from each mouse, splenocytes were isolated, and erythrocytes were hemolyzed and removed with a 0.83% NH ₄ Cl solution to obtain a lymphocyte fraction.

10% for each well of a 96-well plate (Falcon)
The lymphocytes suspended in the RPMI-1640 culture medium containing fetal calf serum were added at 5 × 10 ⁵ cells / well, and a test sample (compound of the present invention) and a positive control standard were added to each well. Synthetic lipid A standard (506, manufactured by Daiichi Kagaku) or St
LT-2 LPS from yphimurium was added, and the lymphocytes in the plate were cultured in a carbon dioxide cell incubator at 37 ° C for 48 hours. Thereafter, 0.25 μCi of ³ H-thymidine was added per well to each well, and the culture was continued at 37 ° C. for 16 hours. After the culture was completed, the cultured cells were collected on paper using a cell harvester, dried, and then subjected to a liquid scintillation counter. radioactivity of cells in ⁽³ H- thymidine uptake, mean counts / min) was measured as an index of young rejuvenation determination of cells.

In the same manner as above, a control test was carried out without adding any test sample, and the radioactivity obtained by the control test (average count / min)
Is compared with the same radioactivity (average count / min) when each test sample is added, and the result is obtained according to the following formula, and this is calculated as a stimulation index (SI).
And

The obtained results are shown in Table 1 (1) (when using C3H / He cells) and Table 1 (2) (when using C3H / HeJ cells).
Are shown below.

From Table 1 above, LPS or 506 was found to have splenocyte blastogenesis on C3H / He cells, but C3H in LPS-insensitive mice.
No response was observed in / HeJ cells.

On the other hand, each of the compounds of the present invention was found to have blastogenic potential in mouse spleen cells of both strains, indicating that the action was different from that of LPS. Among the compounds of the present invention, the activity of the compound (KAB-2) obtained in Example 2 having two palmitoyl groups was particularly strong.

II. Lethal Toxicity Test Using Galactosamine-Loaded Mice Galactosamine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) at 640 mg / kg was intraperitoneally administered to C57BL / 6 mice, and the compound of the present invention (KA
The suspension of B-1 to KAB-4) was intravenously administered, and 24 hours later, the survival or death of the experimental mouse was determined, 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 performed on 506 as a comparative compound.

Incidentally, ND indicates that the experiment was not performed.

From the above table, it can be seen that 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 at a dose of 50 μg / mouse. It is clear that it is low.

III. Cell growth inhibitory activity test 2.0 ml of a 3% thioglycolate solution was intraperitoneally administered to C3H / He mice, and on the fourth day, peritoneal cells were collected.

1 × 10 ⁶ cells per well were added to each well of a 24-well microplate (manufactured by Falcon), and the cells were cultured at 37 ° C. for 2 hours in a carbon dioxide cell incubator. The cells were washed and removed, a predetermined amount of the compound of the present invention was added as a test sample to the obtained macrophages, and the cells were further cultured at 37 ° C for 24 hours. After completion of the culture, the culture supernatant (containing TNF) was collected by centrifugation.

Then, using a 96-well microplate (manufactured by Falcon), a two-step dilution series (RP
L929 cells (4 × 10 ⁴ cells / well) to which actinomycin was added were added to each well, and the cells were cultured at 37 ° C. for 24 hours. Cells were stained by washing with acid buffer and then adding crystal violet. After drying, the dye was released by adding ethanol, and the amount of the dye was determined by measuring the absorbance at 550 nm of an ELISA reader, and the cell growth inhibitory activity (TNF-producing ability) of the test compound was examined from this.

 The results obtained are shown in Table 3 below.

From Table 3 above, all the compounds of the present invention have a clear cell growth inhibitory activity as compared to LPS, and in particular, KAB-2
Had a strong activity.

IV. Antitumor activity (tumor necrosis factor (TNF) activity) test BALB / c mice were inoculated subcutaneously with 5 × 10 ⁵ MethA tumor cells, and on the 7th and 9th days, the compound of the present invention was added at 50 μM / mouse
g intravenously (test group, 5 animals per group). A control group (five animals per group) was provided with a group to which only physiological saline without the compound of the present invention was added.

Twenty 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 equation.

Inhibition rate (%) = (1-A / B) × 100 A: Average tumor weight of test group B: Average tumor weight of control group The obtained results are shown in Table 4 below.

From the above table, it was revealed that among the compounds of the present invention, KAB-2 exerts a significant tumor cell growth inhibitory effect.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-54-9224 (JP, A) Chem. Pept. Protein s. Proc. USSR-FRG Sy mp. , 4th (1984) p. 87-96 Pept. : Struct. Func t. Proc. Am. Pept. Sy mp. , 8th (1983) p. 179-182 Hoppe-Seylers Z. Physiol. Chem. , Vol. 7 (1982) p. 767-770 (58) Field surveyed (Int. Cl. ⁶ , DB name) C07K 7/06 A61K 38/08 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] (1) General formula [Wherein, R ₁ represents —CO (CH ₂ ) ₁₄ CH ₃ . R ₂ is -COOCH ₂ CCl ₃
Is shown. ] The lipopeptide represented by these. 2. An antitumor agent comprising the lipopeptide of claim 1 as an active ingredient.