JPH11228526A - Peptide derivative and pharmaceutically acceptable salt thereof, production method thereof and use thereof - Google Patents

Abstract

[PROBLEMS] To provide a novel compound useful as a prophylactic or therapeutic agent for periodontal disease by selectively inhibiting the proteolytic activity of arginine gingipain (RGP). SOLUTION: The general formula: (Wherein X represents -CHOH- or -CO-, A ¹ and A ² are the same or different and each represents a lysyl group or a histidyl group. R ¹ represents a hydrogen atom or a nitro group, and R ² represents a substituted group. Represents an oxycarbonyl group, and R ³ is —OR ⁴ or —NR ⁵
R ⁶ is shown. R ⁴ , R ⁵ or R ⁶ are the same or different and each represents a lower alkyl group. ), A pharmaceutically acceptable salt thereof, a production method thereof and a use thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide derivative, a pharmaceutically acceptable salt thereof, a method for producing the same, and a use thereof.

[0002]

2. Description of the Related Art Many periodontal diseases are considered to be a kind of infectious diseases caused by resident microorganisms in the periodontal region. In particular, the gram-negative anaerobic bacterium Porphyromonas gingivalis (P. gingivalis) is the most important etiological agent in adult periodontitis and rapidly progressive periodontitis. (J. Clin. Periodontol., 15 , 85-93,
1988, 316-323, 1988, J. Dent. Res., 63 , 441-451,
1984). In recent years, the P. It is known that proteases produced by gingivalis degrade its function, namely, collagen and other periodontal tissue components, and serum proteins involved in the body's defense system. (Greiner D., Mayrand D .: Biology of th
e Species Porphyromonas gingivalis, Edited by Shah
HN, Mayrand D. and Genco RJ, pp227-243, CRC
Press, Boca Raton, Ann Arbor, London, Tokyo, 199
3). This P. Arg-gingipain (RGP), a proteolytic enzyme with trypsin-like protease activity produced by gingivalis, also degrades neutrophil damage activity and collagen, a major component of periodontal tissue. It is thought to be involved in colonization of bacteria, development of periodontitis and destruction of periodontal tissue (Yamamoto, Nihon Pharmaceutical Journal, 105 , 345-355 (199)
5)).

[0003] Conventionally, for the prevention and treatment of periodontal disease, agents that inhibit the growth of bacteria, for example, antibiotics such as cyclohexazine, tranexamic acid, tetracycline, minocycline, natural products such as camillet tincture, ratania tincture, etc. Is used. However, these drugs have various problems such as a problem in safety and an unpleasant odor. JP-A-5-97708 discloses a therapeutic agent for periodontal disease containing an ATPase inhibitor, a cysteine protease inhibitor or the like as an active ingredient, but the anti-periodontal effect of the therapeutic agent is satisfactory. It wasn't good. On the other hand, substances that inhibit RGP are disclosed in
No. 81380 only discloses high molecular weight polyphenols.

[0004]

An object of the present invention is to determine the onset and progress of periodontal disease. That gingivalis is closely related; Focusing on the fact that RGP, which is a proteolytic enzyme, contributes to gingivalis-related components of periodontal disease, and selectively inhibits the proteolytic activity of RGP, thereby preventing and treating periodontal disease. An object of the present invention is to provide a novel compound useful as an agent or the like.

Another object of the present invention is to provide a method for producing the above compound and its use.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to obtain an effective agent for preventing and treating periodontal diseases, and as a result, a novel peptide derivative which inhibits the activity of RGP which is a proteolytic enzyme. The present invention was completed based on this.

That is, the present invention provides a compound represented by the general formula

[0008]

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(Wherein X represents —CHOH— or —CO—, A ¹ and A ² are the same or different and each represents a lysyl group or a histidyl group. R ¹ represents a hydrogen atom or a nitro group, ² represents a substituted oxycarbonyl group; R ³ represents —OR
⁴ or an -NR ⁵ R ^6. R ⁴ , R ⁵ or R ⁶ are the same or different and each represents a lower alkyl group. ) And a pharmaceutically acceptable salt thereof.

Further, the present invention provides a compound represented by the general formula

[0011]

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(Wherein X represents —CHOH— or —CO—, A ¹ and A ² are the same or different and each represents a lysyl group or a histidyl group. R ¹ represents a hydrogen atom or a nitro group; ² represents a substituted oxycarbonyl group; R ³ represents —OR
⁴ or an -NR ⁵ R ^6. R ⁴ , R ⁵ or R ⁶ are the same or different and each represents a lower alkyl group. A ³ and A ⁴
Is the same or different and represents a lysyl derivative in which N ^ε is protected by a protecting group or a histidyl derivative in which N ^im is protected by a protecting group, and R ⁷ is a hydrogen atom, a nitro group or a protecting group. ) And a pharmaceutically acceptable salt thereof, which undergoes a deprotection reaction.

[0013]

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Wherein X, A ¹ , A ² , R ¹ , R ² and R ³
Is the same as above. )) And a method for producing the pharmaceutically acceptable salt thereof.

Further, the present invention provides an arginine dipain inhibitor comprising a peptide derivative represented by the general formula (1) and a pharmaceutically acceptable salt thereof as an active ingredient, a peptide represented by the general formula (1) A drug for periodontal disease containing a derivative and a pharmaceutically acceptable salt thereof as an active ingredient, and a peptide derivative represented by the general formula (1) and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof The present invention also relates to a composition containing a carrier.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The substituted oxycarbonyl group represented by R ² in the general formula (1) includes, for example, an aralkyloxycarbonyl group such as benzyloxycarbonyl, diphenylmethoxycarbonyl, p-methoxybenzyloxycarbonyl, and ethoxycarbonyl. And tert-butoxycarbonyl and the like, preferably an aralkyloxycarbonyl group, more preferably a benzyloxycarbonyl group.

In the general formula (1), the lower alkyl group represented by R ⁴ , R ⁵ or R ⁶ includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl and the like. Examples thereof include 1 to 6 linear or branched alkyl groups, and a methyl or ethyl group is preferable.

Among the compounds of the present invention represented by the general formula (1), preferred are the peptide derivatives wherein A ¹ and A ² are lysyl groups and R ³ is --NR ⁵ R ⁶ in the general formula (1). It is a pharmaceutically acceptable salt. Further, a more preferred compound is that, in the general formula (1), A ¹ and A ² are a lysyl group, R ¹ is a hydrogen atom, R ² is an aralkyloxycarbonyl group, and R ³ is —NR ⁵ R ⁶ . Peptide derivatives and pharmaceutically acceptable salts thereof.

The pharmaceutically acceptable salt of the compound of the present invention is not particularly limited, and examples thereof include an acid addition salt obtained by acting a pharmaceutically acceptable acid. Examples of the acid addition salts include inorganic acid salts such as hydrochloride and sulfate, formate, trifluoroacetate, acetate, tartrate, maleate, fumarate, and the like.
Salts such as organic acid salts such as succinate and methanesulfonate are exemplified. Further, the compound of the present invention or a pharmaceutically acceptable salt thereof may be in the form of a solvate represented by a hydrate.

The amino acids constituting the compound of the present invention may be either L-form or D-form. Preferably,
L-body.

The compound of the present invention represented by the general formula (1)
For example, it can be produced according to the following reaction scheme 1 based on the production method of the present invention.

<Reaction process formula 1>

[0023]

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(Where X, A ¹ , A ² , R ¹ , R ² ,
R ³ , A ³ , A ⁴ and R ⁷ are the same as described above. ).

In the above general formula (2), N ^ε represented by A ³ and A ⁴ is a lysyl derivative or N
^The protecting group of the histidyl derivative in which ^im is protected with a protecting group is not particularly limited as long as it is removed under a reaction condition such that R ² is stable. For example, t-butoxycarbonyl group, p-methoxy Examples include a carbobenzoxy group and a trityl group.

The protecting group represented by R ⁷ is not particularly limited as long as it can be removed under the reaction conditions under which R ² is stable. For example, t-butoxycarbonyl group, 1-adamantyloxycarbonyl group And a 2,2,5,7,8-pentamethylchroman-6-sulfonyl group, and preferably 2,2,5,7,8-pentamethylchroman-6.
It is a sulfonyl group.

[Step (i)] The compound represented by the general formula (2) is treated with a dilute acid in a suitable solvent or without a solvent to form a protective group for A ³ , A ⁴ and R ^7. By selective removal, the compound of the present invention represented by the general formula (1) is obtained.
The reaction conditions are not particularly limited as long as R ² is stable.

The solvent used in the reaction is not particularly limited as long as it does not participate in the reaction, and examples thereof include chloroform, dichloromethane, dioxane, and tetrahydrofuran. Examples of the dilute acid include mineral acids such as hydrochloric acid and sulfuric acid, and organic acids such as trifluoroacetic acid and p-toluenesulfonic acid. The amount of the dilute acid is about 1 to 1000 times, preferably 1 to 100 times the molar amount of the compound represented by the general formula (2). In order to promote the reaction, anisole, thioanisole and the like may be added as a reaction accelerator. The reaction time is 0.1
About 100 hours, preferably 0.5 to 5 hours. The reaction temperature is about 0 to 100 ° C, preferably 0 to 40 ° C.

The compound represented by the general formula (2) used as a raw material in the reaction scheme 1 can be prepared by a method generally used in the field of peptide synthetic chemistry, for example, "Biochemical Experiment Course, edited by The Biochemical Society of Japan (Corporation)". 1. Protein chemistry IV, 207-400
Page, 1977, Tokyo Chemical Doujin Co., Ltd. ". Specifically, it can be produced according to the following reaction schemes 2 and 3.

<Reaction process formula 2>

[0031]

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(In each formula, A ³ , A ⁴ , R ² and R ³ are the same as described above. R ^7a represents a hydrogen atom or a protecting group, R ⁸ represents an aralkyloxycarbonyl group, and R ⁹ represents a lower group. Represents an alkyl group).

In the above reaction scheme, the protecting group for R ^7a includes, for example, t-butoxycarbonyl group, 1-adamantyloxycarbonyl group, 2,2,5,7,8-pentamethylchroman-6-sulfonyl group And the like, and preferably a 2,2,5,7,8-pentamethylchroman-6-sulfonyl group.

The aralkyloxycarbonyl group for R ⁸ includes, for example, benzyloxycarbonyl, diphenylmethoxycarbonyl and the like, and preferably benzyloxycarbonyl.

The lower alkyl group for R ⁹ includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl and the like.
And 6 linear or branched alkyl groups, preferably a methyl or ethyl group.

[Step (ii)] The compound represented by the general formula (3) is condensed with a known compound represented by the general formula (4) in a suitable solvent or without a solvent,
A compound represented by the general formula (5) is obtained.

The solvent used in the reaction is not particularly limited as long as it does not participate in the reaction. For example, N, N-
Dimethylamide, tetrahydrofuran, methylene chloride,
Examples thereof include dioxane, ethyl acetate, 1-methyl-2-pyrrolidone and the like, and these may be used alone or in combination of two or more. When condensing, for example, a method using a condensing agent such as N, N-dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, a mixed acid anhydride method using isobutyl chloroformate, etc. An azide method, an active ester method and the like can be used, and a method using a condensing agent is preferable. The amount of the condensing agent is about 0.5 to 10 times the molar amount of the compound represented by the general formula (3), preferably 1 to 10 times.
~ 2 times the molar amount. In order to promote the reaction, an additive such as 1-hydroxybenzotriazole or N-hydroxy-5-norbornene-2,3-dicarboximide may be added to the condensing agent as a reaction accelerator. Good. The reaction time is about 0.1 to 100 hours, preferably 0.5 to 20 hours. The reaction temperature is
About -50 to 100C, preferably -20 to 40.
° C.

The compound represented by the general formula (5) obtained by this step is isolated or isolated without isolation.
i) can be used.

[Step (iii)] The compound of the general formula (5) obtained in the step (ii) is reacted with the compound of the general formula (6) in a suitable solvent or without solvent. By doing so, a compound represented by the general formula (2a) is obtained.

The solvent used in the reaction is not particularly limited as long as it does not participate in the reaction. For example, N, N-
Dimethylamide, tetrahydrofuran, methylene chloride,
Examples thereof include dioxane, ethyl acetate, 1-methyl-2-pyrrolidone and the like, and these may be used alone or in combination of two or more. The amount of the compound represented by the general formula (6) is 0.5 to 0.5 to the compound represented by the general formula (5).
The molar amount is about 10 times, preferably 0.9 to 2 times. The reaction time is about 0.1 to 100 hours, preferably 0.5 to 20 hours. The reaction temperature is -50 to
The temperature is about 100 ° C, preferably -20 to 40 ° C.

The compound represented by the general formula (2a) obtained in this step is isolated or isolated without isolation.
It can be used for (i).

The compound represented by the general formula (3) is, for example, 6-nitroguanidino-3- (S)-(1,1-dimethylethoxy) methanamide which is disclosed in JP-T-8-502493. Starting from 2-hydroxyhexanoic acid ester, N ^α −
It is obtained by converting a t-butoxycarbonyl group into an R ⁸ group and further converting a nitro group into an R ^7a group.

<Reaction process formula 3>

[0044]

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(In each formula, A ³ , A ⁴ , R ² , R ³ and R ⁹ are the same as described above.)

[Step (iv)] In the same manner as in the step (iii) of the reaction scheme 2, the compound represented by the general formula (5) is disclosed in JP-A-8-502493. Using the compound represented by the general formula (7), the compound represented by the general formula (8) is obtained.

The compound represented by the general formula (8) obtained in this step can be isolated or isolated without the following step.
(v) can be used.

[Step (v)] In the same manner as in the step (ii) of the reaction scheme 2, the compound represented by the general formula (IV) obtained in the step (iv) is used instead of the compound represented by the general formula (3). Using the compound represented by 8), a compound represented by the general formula (2b) is obtained.

The compound represented by the general formula (2b) obtained by this step can be isolated or isolated without the above step.
It can be used for (i).

The compound represented by the general formula (2a) or (2b) obtained by the above reaction scheme 2 or 3 is subjected to an oxidation reaction in a suitable solvent or without a solvent to give the compound of the general formula (2) X in the represented compound can be converted from -CHOH- to -CO-. This oxidation reaction is carried out, for example, by a method such as Dess-Martin oxidation using a Dess-Martin reagent, or modified Moffat oxidation using dimethyl sulfoxide-1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride-dichloroacetic acid as a reagent. Is used.

The solvent used in the reaction is not particularly limited as long as it does not participate in the reaction. For example, N, N-
Dimethylamide, tetrahydrofuran, methylene chloride,
Examples thereof include dioxane, ethyl acetate, 1-methyl-2-pyrrolidone and the like, and these may be used alone or in combination of two or more. The amount of the reagent is about 0.3 to 100 mol, preferably 0.5 to 10 mol, per mol of the compound represented by the general formula (2a) or (2b). The reaction time is about 0.1 to 100 hours, preferably 0.5 to 50 hours. The reaction temperature is between -20 and 100
° C, preferably 0 to 40 ° C.

The compound of the present invention and each compound obtained by the above method can be purified by a general separation means in synthetic chemistry such as recrystallization, distillation, various column chromatography and the like.

The peptide derivatives of the general formula (1) and pharmaceutically acceptable salts thereof of the present invention are closely related to the onset and progress of periodontal disease. It selectively and strongly inhibits RGP, a proteolytic enzyme produced by gingivalis. In addition, the peptide derivative of the present invention is considered to be extremely safe, including metabolites in the body, because its structural component is composed of naturally occurring amino acids or its derivatives that are highly safe.

Accordingly, the peptide derivative represented by the general formula (1) and a pharmaceutically acceptable salt thereof are useful as an argin-zindipain inhibitor and a drug for periodontal disease containing the peptide derivative as an active ingredient. . Here, the agents for periodontal disease include prophylactic and therapeutic agents for periodontal disease.

The peptide derivative represented by the general formula (1) and a pharmaceutically acceptable salt thereof are also useful as a composition containing the peptide derivative and a pharmaceutically acceptable carrier. For example, the peptide derivative of the present invention or a pharmaceutically acceptable salt thereof is mixed with a pharmaceutically acceptable carrier, and a gel preparation for oral cavity, a mucoadhesive ointment for oral cavity, a periodontal pocket insert, a gingival adhesive preparation, etc. It can be administered as an oral preparation or an oral hygiene such as toothpaste, mouthwash, troche and the like.

As the pharmaceutically acceptable carrier, appropriate components usually used according to the dosage form can be used. For example, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose , Liquid paraffin, white petrolatum, platinum base, Eudragit L, sodium alginate, propylene glycol alginate, pullulan, tragacanth, xanthan gum, chitosan, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, ethyl methacrylate, dimethyl Amino acetate, cellulose acetate, collagen, atelocollagen, gelatin, glycerin, triacetin, macrogol 400, Resorbate 60, polyoxyl stearate 40, butyl paraoxybenzoate, cetanol, glyceryl monostearate, calcium carbonate, magnesium carbonate, dibasic calcium phosphate, carrageenan, sodium dioctylsulfosuccinate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, hinokitiol, allantoin , Glycyrrhizin,
Gum arabic, starch, corn starch, saccharin sodium, stevioside, glucose, lactose, magnesium stearate, monopotassium phosphate, dipotassium phosphate, menthol, eucalyptus oil, peppermint, spearmint, pigment, sodium fluoride, sodium monofluorophosphate An anti-inflammatory agent such as fluoride, lysodium chloride and azulene, and commonly used components such as sodium chloride can be appropriately compounded.

The method of administering the composition containing the peptide derivative of the present invention or a pharmaceutically acceptable salt thereof includes the following.
For example, in the case of a gel preparation or an ointment preparation, it may be applied to the gingiva, in the case of a tape preparation, it may be attached to the gingiva, and in the case of a pocket insert, it may be inserted into a periodontal pocket.

The dosage varies depending on the degree of spread of periodontal disease, but an appropriate amount of a preparation containing 0.001% by weight or more as a peptide derivative of the present invention or a pharmaceutically acceptable salt thereof is usually 1 day. It may be administered, applied or washed more than once.

[0059]

EXAMPLES The present invention will be described below in detail with reference to Reference Examples, Examples and Test Examples, but the present invention is not limited to these Examples. In each of the chemical structural formulas shown in Reference Examples and Examples, the symbols “*” and “*” attached to the asymmetric carbon atom to which the OH group is bonded indicate that the absolute configuration between the paired compounds is different from each other. The opposite is shown.

Reference Example 1 Synthesis of the following compound (1)

[0061]

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6-nitroguanidino-3- (S) -produced according to the method described in JP-T-8-502493.
1.9 g (5.2 mm) of (1,1-dimethylethoxy) methnamimido-2-hydroxyhexanoic acid methyl ester
ol), 0.2 ml of anisole and 19 trifluoroacetic acid
The mixture was stirred for 15 minutes under ice cooling and at room temperature for 45 minutes. After the reaction, the reaction solution was concentrated under reduced pressure, and n-hexane 20 was added to the residue.
ml was added. After removing the n-hexane layer by decantation, the obtained residue was washed with 20 ml of tetrahydrofuran.
And neutralized with triethylamine under ice-cooling. This tetrahydrofuran solution was treated with N ^α -carbobenzoxy-N
2.57 g of ^ε- t-butoxycarbonyl-L-lysine
(6.8 mmol), 1.1 ml of triethylamine (7.
8 mmol) and 1.0 ml of isobutyl chloroformate
(7.8 mmol) was added to 30 ml of a mixed acid anhydride solution in tetrahydrofuran, and the mixture was stirred under ice cooling for 0.5 hour. After 2 ml of purified water was added to stop the reaction, a saturated saline solution was added to the reaction solution, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with 5% aqueous citric acid, saturated saline, 5% aqueous sodium hydrogen carbonate and saturated saline, and dried over sodium sulfate. The solvent is distilled off, and the residue is subjected to silica gel column chromatography (developing solvent; chloroform: ethanol = 1).
(Eluted with 0: 1 (v / v)). Powdered from petroleum ether, 2.85 g of the above compound (87.7 yield)
%). The physical properties are shown below.

Mass: FAB (+); m / e 664 (M + K) ⁺ .

¹ H-NMR (CDCl ₃ ) δ: 8.46 (1H,
brs), 7.38-7.23 (6H, m), 6.92 (1H, m), 5.96 and 5.7
8 (1H, brd), 5.06 (2H, m), 4.84 and 4.77 (1H, brs),
4.39 (1H, m), 4.27 and 4.203 (1H, brs), 4.08 (1H,
m), 3.79 and 3.77 (3H, s), 3.45-3.08 (4H, m), 1.82-
1.27 (19H, m).

IR (KBr) cm ^-1 : 3334, 1708, 1704,
1693, 1680, 1666, 1660, 1536, 1531, 1273, 1256.

Reference Example 2 The following compounds (2-1) and (2)
Synthesis of -2)

[0067]

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2.30 g of compound (1) obtained in Reference Example 1
To a 23 ml methanol solution of (3.7 mmol) was added 22 ml of an aqueous solution of 221 mg (5.5 mmol) of sodium hydroxide under ice-cooling, and the mixture was stirred for 3 hours. 1N reaction solution
The pH was adjusted to 4 with hydrochloric acid, the solvent was distilled off, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed with saturated saline,
Dried over sodium sulfate. The solvent was distilled off, and diethyl ether was added to the residue to form a powder, thereby obtaining 1.94 g (yield 85.8 g) of the carboxylic acid derivative of the compound (1). To 1.60 g (2.6 mmol) of the obtained carboxylic acid derivative,
N ^alpha - carbobenzoxy -N ^{epsilon-t-butoxycarbonyl--L-} lysine dimethylamide 847 mg (3.1m
mol), 1-hydroxybenzotriazole hydrate 4
24 mg (3.1 mmol), 1-ethyl-3- (3-
Dimethylaminopropyl) carbodiimide hydrochloride 594
mg (3.1 mmol), N-methylmorpholine 263
mg (2.6 mmol) in 16 ml of DMF at room temperature 6
Stirred for hours. After the reaction, a saturated saline solution was added to the reaction solution, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed sequentially with 5% aqueous citric acid, saturated aqueous sodium chloride, 5% aqueous sodium bicarbonate, and saturated aqueous sodium chloride, and then dried over sodium sulfate. The solvent is distilled off, and the residue is subjected to silica gel column chromatography (developing solvent;
Purification by elution with chloroform: methanol = 10: 1 (v / v)) and an Rf value of 0.39 on thin layer chromatography (developing solvent; chloroform: methanol = 1).
0: 1 (v / v)), and powdered from petroleum ether to obtain 579 mg of compound (2-1) (yield: 25.
7%). Further, a fraction having an Rf value of 0.35 (developing solvent; chloroform: methanol = 10: 1 (v / v)) was collected, powdered from diethyl ether, and the compound (2-
918 mg (yield 40.7%) of 2) was obtained. Further, 349 mg (yield 15.5%) of a mixture of both compounds was obtained. The physical properties are shown below.

(I) Compound (2-1): Melting point: 95-99 ° C.

Mass: FAB (+); m / e 905 (M + K) ⁺ .

¹ H-NMR (CDCl ₃ ) δ: 8.10 (1H,
brs), 7.81-7.69 (1H, brd), 7.32 (5H, m), 6.86 (1H,
m), 5.98 (1H, brs), 5.11-5.02 (2H, m), 4.89-4.60
(3H, m), 4.36-4.12 (3H, m), 3.45 (1H, m), 3.11 (8H,
m), 2.93 (3H, s), 1.80-1.27 (34H, m).

IR (KBr) cm ^-1 : 3334, 1721, 1710,
1704, 1692, 1679, 1666, 1659, 1650, 1641, 1632, 16
15, 1601, 1547, 1536, 1530, 1519, 1514, 1503, 145
3, 1444, 1413, 1408, 1403, 1392, 1367, 1268, 1254,
1170.

Specific rotation: [α] ²⁵ _D = −22.53 °
(C = 1.003, chloroform).

(Ii) Compound (2-2): Melting point: 91-95 ° C.

Mass: FAB (+); m / e 905 (M + K) ⁺ .

¹ H-NMR (CDCl ₃ ) δ: 8.30 (1H,
brs), 7.70-7.52 (2H, m), 7.33-7.23 (5H, m), 6.13
(1H, brs), 5.17-4.74 (5H, m), 4.30 (1H, m), 4.18
(1H, m), 4.12 (1H, m), 3.33-2.91 (12H, m), 1.72-1.3
5 (34H, m).

IR (KBr) cm ^-1 : 3334, 1722, 1710,
1704, 1692, 1679, 1666, 1659, 1650, 1642, 1632, 16
14, 1548, 1536, 1530, 1519, 1514, 1503, 1453, 144
4, 1413, 1409, 1403, 1392, 1367, 1268, 1254, 117
0.

Specific rotation: [α] ²⁵ _D = -13.01 °
(C = 0.507, chloroform).

Reference Example 3 Synthesis of the following compound (3)

[0080]

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Compound (2-1) 50 obtained in Reference Example 2
0 mg (0.58 mmol) of a 10% acetic acid-methanol solution (20 ml) is mixed with 10% palladium carbon (250 mg).
Was added thereto and subjected to catalytic hydrogen reduction under normal pressure for 20 hours. After the reaction,
The insoluble material was removed by filtration, and the solvent was distilled off. 165 μl of carbobenzoxyl chloride was added to a mixed solution of the obtained residue in 2 ml of dioxane and 2 ml of saturated aqueous sodium hydrogen carbonate under ice-cooling.
(1.154 mmol) was added and reacted for 10 hours. After the reaction, the reaction solution was concentrated by half, purified water was added, and the mixture was washed with diethyl ether. After concentrating the aqueous layer, the resulting residue was
It was dissolved in 0% aqueous methanol and purified by column chromatography (developing solvent; eluted with 60% aqueous methanol) using MCI gel (Mitsubishi Chemical Corporation, CHP-20 (75 to 150 μ)) as a carrier. The obtained fraction was concentrated under reduced pressure, and powdered from diethyl ale.
g (78.9% yield). The physical properties are shown below.

Melting point: 100-104 ° C.

Mass: FAB (+); m / e 822 (M + H) ⁺ .

¹ H-NMR (CD ₃ OD) δ: 7.35-7.29
(5H, m), 5.12-5.04 (2H, m), 4.84 (1H, m), 4.28-4.27
(1H, m), 4.07-4.04 (2H, m), 3.21-3.01 (9H, m), 2.9
5 (3H, s), 1.81-1.25 (34H, m).

IR (KBr) cm ^-1 : 3345, 1721, 1709,
1704, 1691, 1689, 1679, 1674, 1666, 1660, 1650, 16
42, 1633, 1615, 1548, 1536, 1530, 1519, 1514, 150
3, 1407, 1403, 1367, 1275, 1252, 1172.

Specific rotation: [α] ²⁵ _D = −30.20 °
(C = 1.013, methanol).

Reference Example 4 Synthesis of the following compound (4)

[0088]

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Compound (2-2) 85 obtained in Reference Example 2
400 mg of 10% palladium carbon was added to 30 ml of 4 mg (0.99 mmol) of a 10% acetic acid-methanol solution.
Was added thereto and subjected to catalytic hydrogen reduction under normal pressure for 20 hours. After the reaction,
The insoluble material was removed by filtration, and the solvent was distilled off. To a mixed solution of 3 ml of dioxane and 3 ml of saturated aqueous sodium hydrogen carbonate solution of the obtained residue was added, under ice-cooling, 211 μl of carbobenzoxycyclolide.
(1.48 mmol) was added and reacted for 10 hours. After the reaction, the reaction solution was concentrated by half, purified water was added, and the mixture was washed with diethyl ether. After concentrating the aqueous layer, the resulting residue was
MCI gel (Mitsubishi Chemical Corporation,
Purification was performed by column chromatography using CHP-20 (75 to 150 μ)) as a carrier (developing solvent; eluted with 55% aqueous methanol). The obtained fraction is concentrated under reduced pressure,
After freeze-drying, 544 mg of the above compound (67.2% yield).
I got The physical properties are shown below.

Melting point: 94-98 ° C.

Mass: FAB (+); m / e 822 (M + H) ⁺ .

¹ H-NMR (DMSO-d ₆ ) δ: 7.46
(1H, brs), 7.36-7.31 (5H, m), 6.76 (1H, brs), 5.04
-4.95 (2H, m), 4.66-4.63 (1H, m), 4.09 (1H, m), 3.9
6 (1H, m), 3.92-3.88 (1H, m), 3.05-2.82 (12H, m),
1.64-1.09 (34H, m).

IR (KBr) cm ^-1 : 3354, 1684, 1672,
1642, 1527, 1456, 1405, 1367, 1274, 1252, 1172.

Specific rotation: [α] ²⁵ _D = −13.51 °
(C = 1.006, methanol) Reference Example 5 Synthesis of the following compound (5)

[0095]

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30 ml of a dichloromethane solution of 349 mg (0.40 mmol) of a mixture of the compounds (2-1) and (2-2) obtained in Reference Example 2 and 680 mg (1.61 mmol) of Dess-Martin reagent was stirred at room temperature for 13 hours. rear,
2 ml of purified water was added to the reaction solution, and the mixture was stirred for 0.2 hours. The insoluble material was removed by filtration, and the dichloromethane layer was washed successively with 5% aqueous sodium hydrogen carbonate and saturated saline, and dried over sodium sulfate. The solvent was distilled off, and the residue was subjected to silica gel column chromatography (developing solvent; chloroform: ethanol =
12: 1 (v / v)). Powdered from petroleum ether, 191 mg of the above compound (yield 54.
8%). The physical properties are shown below.

Melting point: 81-84 ° C. Mass: FAB (−); m / e 863 (M−H) ⁻ .

¹ H-NMR (CDCl ₃ ) δ: 7.86 (1H,
brs), 7.52 (1H, brs), 7.33 (5H, m), 5.88 (1H, brs),
5.39 (1H, brs), 5.07 (2H, s), 4.87-4.83 (1H, m),
4.75 (1H, m), 4.17 (1H, m), 3.49-3.29 (2H, m), 3.1
0-2.91 (10H, m), 1.98 (1H, m), 1.75-1.33 (33H,
m).

IR (KBr) cm ^-1 : 3332, 1722, 1710,
1704, 1692, 1680, 1666, 1659, 1650, 1642, 1632, 16
21, 1614, 1548, 1536, 1530, 1519, 1514, 1503, 145
3, 1367, 1254, 1170.

Reference Example 6 Synthesis of the following compound (6)

[0101]

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6-Nitroguanidino-3- (S)-produced according to the method described in JP-T-8-502493.
14.2 g of (1,1-dimethylethoxy) methnamimido-2-hydroxyhexanoic acid methyl ester (39.
1 mmol) of 10% acetic acid-methanol solution 20 ml
Then, 4.0 g of 10% palladium carbon was added thereto, and subjected to catalytic hydrogen reduction under normal pressure for 32 hours. After the reaction, insolubles were removed by filtration, and the solvent was distilled off. A mixture of the obtained residue and 150 ml of trifluoroacetic acid was stirred for 1.3 hours. The reaction solution was concentrated under reduced pressure, and 50 ml of n-hexane was added to the residue. n
After removing the hexane layer by decantation, 7.3 ml (51.0 mmol) of carbobenzoxychloride was added to a mixed solution of the obtained residue in 180 ml of dioxane and 200 ml of saturated aqueous sodium hydrogen carbonate under ice-cooling.
Stirred for hours. The reaction solution was concentrated, 2N hydrochloric acid was added, and the mixture was washed with diethyl ether. The aqueous layer was purified by column chromatography (developing solvent; eluted with 30% aqueous methanol) using MCI gel (Mitsubishi Chemical Corporation, CHP-20 (75 to 150 μ)) as a carrier. The obtained fraction was concentrated and dried under reduced pressure to obtain 11.5 g (83.5%) of amorphous crystals. 10.5 g (29.8 mmol) of the obtained crystals,
A mixture of 2.0 g (50 mmol) of sodium hydroxide and 200 ml of 50% aqueous methanol was stirred for 55 minutes. The reaction solution was adjusted to pH 7 with 1N hydrochloric acid and concentrated under reduced pressure. The obtained residue was dissolved in 1N hydrochloric acid, and column chromatography using MCI gel as a carrier (developing solvent: 30 to 40%
(Eluted with methanol water). The obtained fraction was concentrated and dried under reduced pressure, and 7.32 g of amorphous crystals (72.
6%). 500 mg of the obtained crystals (1.44 mm
ol) 2N sodium hydroxide 3ml, acetone 3ml
To a mixture of 2,2,5,7,8-pentamethylchroman-6-sulfonyl chloride under ice-cooling.
2 mmol of an acetone solution of
Stirred for hours. After the reaction, the reaction solution was adjusted to pH 7 with 5% citric acid.
And concentrated under reduced pressure. The residue was acidified with 5% citric acid and extracted with ethyl acetate. The ethyl acetate layer was washed with saturated saline and dried over sodium sulfate. The solvent was distilled off, and the residue was converted to a cyclohexylamine salt in methanol,
Powdered from diethyl ether. 1N
After desalting with hydrochloric acid and extraction with ethyl acetate, the ethyl acetate layer was dried over sodium sulfate and the solvent was distilled off. The obtained residue is subjected to silica gel column chromatography (developing solvent;
Chloroform: methanol: purified water = 10: 3: 1 (v
/ V) eluted with the lower solution). Powdered from diethyl ether, 154 mg of the above compound (yield 1
7.7%). The physical properties are shown below.

Mass: FAB (+); m / e 627
(M + Na) ⁺ .

¹ H-NMR (DMSO-d ₆ ) δ: 7.35
7.29 (5H, m), 7.09 and 6.84 (1H, d, J = 8Hz), 6.74 (1
H, brs), 6.41 (1H, brs), 5.03-4.99 (2H, m), 3.93-
3.89 (1H, m), 3.77-3.72 (1H, m), 3.03-2.97 (2H, m),
2.60-2.47 (8H, m), 2.03 (3H, s), 1.79-1.74 (2H,
m), 1.48-1.26 (10H, m).

IR (KBr) cm ^-1 : 3435, 3354, 1722,
1710, 1626, 1621, 1580, 1550, 1453, 1299, 1238, 11
66, 1110.

Reference Example 7 Synthesis of the following compound (7)

[0107]

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Compound (6) obtained in Reference Example 6 (700 m)
g (1.16 mmol) was added to 381 mg of N ^ε -t-butoxycarbonyl-L-lysine dimethylamide (1.3 mg).
9 mmol), 203 mg (1.5 mmol) of 1-hydroxybenzotriazole hydrate, 1-ethyl-3-
A solution of (3-dimethylaminopropyl) carbodiimide hydrochloride (290 mg, 1.5 mmol) and N-methylmorpholine (117 mg, 1.28 mmol) in DMF (25 ml) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction, a saturated saline solution was added to the reaction solution, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed sequentially with 5% aqueous citric acid, saturated aqueous sodium chloride, 5% aqueous sodium bicarbonate, and saturated aqueous sodium chloride, and then dried over sodium sulfate. The solvent is distilled off, and the residue is subjected to silica gel column chromatography (developing solvent; chloroform: ethanol = 15: 1).
(Eluted with (v / v)).
g (63.3% yield). The physical properties are shown below.

Mass: FAB (+); m / e 860 (M + H) ⁺ .

¹ H-NMR (CDCl ₃ ) δ: 7.58-7.57
(1H, m), 7.32-7.29 (5H, m), 6.28-6.02 (2H, m), 5.7
5 and 5.55 (1H, m), 5.11-5.04 (2H, m), 4.81 (2H, m
m), 4.46-4.05 (2H, m), 3.34-3.04 (7H, m), 2.91 (3
H, s), 2.63-2.56 (8H, m), 2.10 (3H, s), 1.81 (2H,
m), 1.61-1.24 (25H, m).

IR (KBr) cm ^-1 : 3340, 1705, 1701,
1636, 1550, 1455, 1403, 1394, 1368, 1298, 1251, 11
67, 1111, 610.7.

Reference Example 8 Synthesis of the following compound (8)

[0113]

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Compound (7) 621m obtained in Reference Example 7
A mixture of 210 mg of 10% palladium carbon, 3 drops of acetic acid and 100 ml of methanol was added to g (0.72 mmol), and the mixture was subjected to catalytic hydrogen reduction at 3.0 kg / cm ² for 5 hours. After the reaction, insolubles were removed by filtration, and the solvent was distilled off. The obtained residue and 73 mg of N-methylmorpholine (0.72 mm
ol) in 2 ml of DMF was added to N ^α -carbobenzoxy-
N ^{epsilon-t-butoxycarbonyl} -L- lysine 330mg
(0.87 mmol), 117 mg (0.87 mmol) of 1-hydroxybenzotriazole hydrate, 166 mg (0.87 mmol) of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride in DMF 5m
The solution was added dropwise under ice-cooling and stirred at room temperature for 12 hours. After the reaction, a saturated saline solution was added to the reaction solution, and the mixture was extracted with ethyl acetate.
The ethyl acetate layer was washed successively with 5% aqueous citric acid, saturated saline, 5% aqueous sodium hydrogen carbonate and saturated saline, and dried over sodium sulfate. After evaporating the solvent, the residue was purified by silica gel column chromatography (developing solvent; eluted with chloroform: ethanol = 25: 2 (v / v)) to obtain 632 mg (yield: 80.5%) of the above compound. The physical properties are shown below.

Mass: FAB (+); m / e 108
8 (M + H) ⁺ .

¹ H-NMR (CDCl ₃ ) δ: 7.71-7.69
(1H, m), 7.30 (5H, m), 6.97 (1H, m), 6.31 (2H, br
s), 6.13-6.03 (1H, m), 5.10-4.80 (5H, m), 4.30-4.1
5 (3H, m), 3.20-2.91 (12H, m), 2.61 (2H, m), 2.57
(3H, s), 2.55 (3H, s), 2.09 (3H, s), 1.79 (2H, m),
1.71-1.24 (40H, m).

IR (KBr) cm ^-1 : 3344, 2934, 1691,
1656, 1639, 1548, 1479, 1454, 1402, 1393, 1367, 12
96, 1251, 1168, 1111, 610.6.

Reference Example 9 Synthesis of the following compound (9)

[0119]

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Compound (8) obtained in Reference Example 8 510m
g (0.47 mmol) and 794 mg of Dess-Martin reagent
After stirring 20 ml of a dichloromethane solution of (1.87 mmol) at room temperature for 5 hours, 2 ml of purified water was added to the reaction solution,
Stirred for 0.5 hour. The insoluble material was removed by filtration, and the dichloromethane layer was washed successively with 5% aqueous sodium hydrogen carbonate and saturated saline, and dried over sodium sulfate. The solvent was distilled off, and the residue was purified by silica gel column chromatography (developing solvent; eluted with chloroform: ethanol = 16: 1 (v / v)). Trituration from petroleum ether gave 215 mg of the above compound (42.3% yield). The physical properties are shown below.

Melting point: 86-90 ° C.

Mass: FAB (−); m / e 108
4 (M−H) ⁻ .

¹ H-NMR (DMSO-d ₆ ) δ: 8.53
(1H, brd, J = 7.6Hz), 8.28 (1H, brd, J = 6.6Hz), 7.35-
7.26 (6H, m), 6.77-6.75 (2H, m), 6.40 (1H, brs),
5.04-4.97 (2H, m), 4.92 (1H, m), 4.67-4.62 (1H,
m), 4.02-3.97 (1H, m), 3.03-2.83 (12H, m), 2.59-2.
47 (8H, m), 2.03 (3H, s), 1.78-1.75 (2H, m), 1.62-
1.14 (40H, m).

IR (KBr) cm ^-1 : 3348, 1722, 1711,
1704, 1692, 1679, 1666, 1659, 1650, 1642, 1632, 16
21, 1613, 1581, 1573, 1564, 1548, 1536, 1531, 151
9, 1513, 1503, 1494, 1453, 1444, 1392, 1367, 1296,
1251, 1168, 1111.

Example 1 Synthesis of the following compound (10)

[0126]

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Compound (3) obtained in Reference Example 3 105m
g (0.128 mmol) in 0.1 ml of anisole,
A mixture of 10 ml of trifluoroacetic acid was added, and the mixture was
And stirred at room temperature for 1.5 hours. After the reaction, the reaction mixture was concentrated under reduced pressure. Diethyl ether was added to the residue, and the precipitated crystals were collected by filtration. The obtained crystals were dissolved in water-containing methanol, 1 ml of 4N hydrochloric acid was added, and the reaction solution was evaporated to dryness twice. Then, the reaction solution was dissolved in purified water, and MCI gel (CHP-20, manufactured by Mitsubishi Chemical Corporation) was used. Purification was performed by column chromatography (developing solvent; eluted with 5% aqueous methanol) using 75-150 μ) as a carrier. After the obtained fraction was concentrated under reduced pressure, 0.5 ml of 1N hydrochloric acid was further added and freeze-dried to obtain 73.3 mg of the hydrochloride of the above compound. The physical properties are shown below.

Melting point: 124-128 ° C.

Mass: FAB (+); m / e 622 (M + H) ⁺ .

¹ H-NMR (D ₂ O) δ: 7.47-7.37 (5H,
m), 5.21-5.11 (2H, m), 4.79 (1H, m), 4.27-4.08 (3
H, m), 3.18 (3H, s), 3.10-2.97 (9H, m), 1.80-1.32
(16H, m).

IR (KBr) cm ^-1 : 3392, 3190, 3059,
2946, 1703, 1661, 1650, 1633, 1573, 1562, 1525, 14
69, 1455, 1255, 1128, 699, 634, 622, 607, 593, 57
9, 564, 550, 536.

Specific rotation: [α] ²⁵ _D = −26.12 °
(C = 0.513, methanol).

Example 2 Synthesis of the following compound (11)

[0134]

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84 mg of the compound (4) obtained in Reference Example 4
(0.102 mmol), a mixture of 0.1 ml of anisole and 10 ml of trifluoroacetic acid was added, and the mixture was cooled to 15
And stirred at room temperature for 1.5 hours. After the reaction, the reaction mixture was concentrated under reduced pressure. Diethyl ether was added to the residue, and the precipitated crystals were collected by filtration. The obtained crystals were dissolved in hydrated methanol, 1 ml of 4N hydrochloric acid was added, the reaction solution was evaporated to dryness twice, and then dissolved in purified water, and MCI gel (CHP-20, manufactured by Mitsubishi Chemical Corporation) was used. Purification was performed by column chromatography (developing solvent; eluted with purified water) using (75 to 150 μ)) as a carrier. After the obtained fraction was concentrated under reduced pressure, 0.5 ml of 1N hydrochloric acid was further added and freeze-dried to obtain 60.7 mg of the hydrochloride of the above compound. The physical properties are shown below.

Melting point: 123-126 ° C.

Mass: FAB (+); m / e 622 (M + H) ⁺ .

¹ H-NMR (CD ₃ OD) δ: 7.73 (1H,
d, J = 9Hz), 7.36-7.30 (5H, m), 5.12-5.04 (2H, m),
4.83 (1H, m), 4.28 (1H, m), 4.17-4.04 (2H, m), 3.2
3-3.12 (5H, m), 2.98-2.92 (7H, m), 1.84-1.35 (16H, m
m).

IR (KBr) cm ^-1 : 3388, 3253, 3051,
1698, 1662, 1656, 1644, 1633, 1526, 1469, 1457, 12
52, 599, 575.

Specific rotation: [α] ²⁵ _D = −19.86 °
(C = 0.574, methanol).

Example 3 Synthesis of the following compound (12)

[0142]

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Compound (5) obtained in Reference Example 5
g (0.174 mmol) in 0.1 ml of anisole,
A mixture of 10 ml of trifluoroacetic acid was added, and the mixture was added under ice cooling.
Stir for 5 minutes at room temperature for 1.5 hours. After the reaction, the reaction mixture was concentrated under reduced pressure. Diethyl ether was added to the residue, and the precipitated crystals were collected by filtration. The obtained crystals were dissolved in hydrated methanol, 1 ml of 4N hydrochloric acid was added, the reaction solution was evaporated to dryness twice, and then dissolved in purified water, and MCI gel (CHP-20, manufactured by Mitsubishi Chemical Corporation) was used. 75-150μ)) as a carrier (eluent: 10
% Methanol). After the obtained fraction was concentrated under reduced pressure, 0.5 ml of 1N hydrochloric acid was further added and freeze-dried to obtain 90.9 mg of a hydrochloride of the above compound. The physical properties are shown below.

Melting point: 131-134 ° C.

Mass: FAB (+); m / e 665 (M + H) ⁺ .

¹ H-NMR (CD ₃ OD) δ: 7.35-7.29
(5H, m), 5.09 (2H, s), 4.81 (1H, m), 4.29-4.02 (2H,
m), 3.22-2.91 (12H, m), 1.93-1.35 (16H, m).

IR (KBr) cm ^-1 : 3294, 3209, 2941,
1673, 1634, 1528, 1455, 1404, 1342, 1266, 1132, 10
61, 700.

Example 4 Synthesis of the following compound (13)

[0149]

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Compound (9) obtained in Reference Example 9 (440 m)
g (0.405 mmol) in 0.4 ml of anisole,
A mixture of 6 ml of trifluoroacetic acid was added, and the mixture was
For 1.5 minutes at room temperature. After the reaction, the reaction mixture was concentrated under reduced pressure. Diethyl ether was added to the residue, and the precipitated crystals were collected by filtration. The obtained crystals were dissolved in water-containing methanol, 1 ml of 4N hydrochloric acid was added, and the reaction solution was evaporated to dryness twice. Then, the reaction solution was dissolved in purified water, and MCI gel (CHP-20, manufactured by Mitsubishi Chemical Corporation) was used. Purification was performed by column chromatography (developing solvent; eluted with 10% methanol) using 75 to 150 μ)) as a carrier. After the obtained fraction was concentrated under reduced pressure, 1N hydrochloric acid (0.5 ml) was further added and freeze-dried to obtain 174 mg of the hydrochloride of the above compound. The physical properties are shown below.

Melting point: 102-106 ° C.

Mass: FAB (+); m / e 620 (M + H) ⁺ .

¹ H-NMR (CD ₃ OD) δ: 7.36 to 7.30
(5H, m), 5.12 (1H, d, J = 12Hz), 5.07 (1H, d, J = 12H
z), 4.80-4.77 (1H, m), 4.26-4.02 (2H, m), 3.22-3.1
0 (5H, m), 2.98-2.92 (7H, m), 1.98-1.36 (16H, m).

IR (KBr) cm ^-1 : 3371, 3180, 3042,
2949, 1660, 1649, 1520, 1469, 1456, 1405, 1256, 11
74, 620, 597, 584.

Test Example 1 Measurement of the inhibitory activity of the compound of the present invention on RGP RGP was performed according to the method of Kadowaki, Yamamoto et al. [T.
o, et al. The Journalof Biological Chemistry, 269 ,
21371-21378 (1994)] by Porphyromonasgingivali
s The one prepared from the supernatant of the culture filtrate of 381 was used.

The inhibitory activity on RGP was determined according to the method of Kadowaki and Yamamoto et al. [The Journal of Biological Chemis
try, 269 , 21371-21378 (1994)].
That is, 100 μl of 50 mM L-cysteine, 0.1 M
200 μl of a sodium phosphate buffer (pH 7.5)
0.05% bridge 35 (polyoxyethylene (2
3) 20 μl of 12.3 nM enzyme (RGP) solution containing lauryl ether (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), 80 μl of distilled water, dimethyl sulfoxide solution 1 of the compound of the present invention
00 μl of the mixture was incubated at 37 ° C. for 5 minutes. afterwards,
20μM of 7 - 0.1% dimethylsulfoxide containing (N ^alpha carbobenzoxy -L- phenylalanyl -L- arginyl) amino-4-methylcoumarin sulfoxide solution 500
μl was added and reacted at 40 ° C. After 10 minutes, 10 mM
0.1M sodium acetate buffer containing iodoacetic acid (p
H5.0) 1000 μl was added to the reaction mixture to stop the reaction, and the released 7-amino-4-methylcoumarin was excited at 380 nm using a fluorescence spectrophotometer at a wavelength of 460 nm.
The fluorescence intensity at the fluorescence wavelength of was measured. As a control, dimethyl sulfoxide was used in place of the dimethyl sulfoxide solution of the compound of the present invention, and the reaction was performed in the same manner, and the fluorescence intensity was measured. Table 1 shows the protease activity (50% inhibitory activity) of each compound calculated from the results.

Similarly, the measurement of the inhibitory activity of cathepsin B and cathepsin L was performed according to A. J. The method of Barrett et al. (Bi
ochemical Journal, 201 , 189-198 (1982)).

That is, the inhibitory activity against cathepsin B was determined by measuring 0.5 ml of 400 mM sodium phosphate buffer (pH 6.0) containing 8 mM dithiothreitol and 4 mM tetrasodium ethylenediaminetetraacetate, 5 μl of cathepsin B and 0 μl of cathepsin B. 0.8 ml of an enzyme solution containing 0.1% bridge 35 (polyoxyethylene (23) lauryl ether, trade name, manufactured by Wako Pure Chemical Industries, Ltd.) and 0.2 ml of the compound of the present invention were added, and the mixture was kept at 40 ° C. for 10 minutes. . Thereafter, a 0.1% aqueous solution of dimethyl sulfoxide containing 20 μM of 7- (N-carbobenzoxy-L-phenylalanyl-L-arginyl) amino-4-methylcoumarin was added.
5 ml was added and reacted at 40 ° C. for 10 minutes. After the reaction, 1
100 mM with 00 mM sodium monochloroacetate
The reaction was stopped by adding 2.0 ml of a sodium acetate buffer solution (pH 4.3), and the released 7-amino-4-methylcoumarin was excited with a fluorimeter at a fluorescence wavelength of 458 nm excited at a wavelength of 350 nm. It was measured. The proteolytic enzyme activity of each compound calculated from the results (50
% Inhibitory activity) are shown in Table 1.

The inhibitory activity on cathepsin L was determined according to the method for measuring cathepsin B described above.

That is, instead of cathepsin B in the above method, cathepsin L was added to a buffer solution of pH 5.5, and the released 7-amino-4-methylcoumarin was similarly measured for fluorescence intensity using a fluorometer. Table 1 shows the protease activity (50% inhibitory activity) of each compound calculated from the results.

[0161]

[Table 1]

Table 1 shows that the compound of the present invention has high activity and can selectively inhibit the enzymatic degradation activity of argin-gingipain (RGP).

Test Example 2 Measurement of Inhibitory Activity of RGP by Human Compound of the Invention by Degradation of Human Type I Collagen The inhibitory activity of RGP by the compound of the present invention on degradation of human type I collagen by RGP was measured as follows. A 1: 2 ratio (V / V) of 50 mM L-cysteine and 100 mM sodium phosphate buffer (pH 7.5)
V) 10 μl of the mixture, 0.36 mg / ml enzyme solution 10
μl and 10 μl of an aqueous solution of the compound of the present invention at 37 ° C.
For 10 minutes. Thereafter, 30 μl of 1000 μg / ml human type I collagen (manufactured by Seikagaku Corporation) was added,
The reaction was performed at 37 ° C. After 60 minutes, add 20 μl of the reaction solution to 10
0 μg / ml leupeptin and 10 mM EDTA-N
The reaction mixture 1μl containing a ₂ was stopped. Reaction solution 1
After heat treatment at 00 ° C for 5 minutes, 15 μl of the reaction solution is
The sample was subjected to S-polyacrylamide gel electrophoresis, and human type I collagen was measured under the following conditions. As a control, purified water was used in place of the aqueous solution of the compound of the present invention.

Gel: Multigel (4/20) 20,000
0-200,000 (Daiichi Pure Chemical Co., Ltd.), Loading buffer: 0.0625 M Tris-HCl buffer (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 5% 2-mercaptoethanol, 10% Bromophenol blue, electrode buffer: 0.025 M Tris-0.194 M glycine (pH 8.4), 0.1% sodium dodecyl sulfate, electrophoresis conditions: 20 mA constant current, electrophoresis time: about 2 hours, coloring: Coomassie Brilliant Blue R -250 (PAGE Blue 8
3).

As a result of the above measurement, the compound of the present invention (1
3) The human type by RGP at a low dose of 1 μg / ml
It was found that the degradation of I collagen was significantly inhibited.

[0166]

Industrial Applicability The compounds of the present invention specifically and highly activate the activity of proteolytic enzymes, particularly Arg-gingipain (RGP) produced by Porphyromonas gingivalis present in the oral cavity. It is useful, for example, as a prophylactic and therapeutic agent for periodontal disease.

Claims (7)

[Claims] 1. A compound of the general formula (Wherein X represents -CHOH- or -CO-, A ¹ and A ² are the same or different and each represents a lysyl group or a histidyl group. R ¹ represents a hydrogen atom or a nitro group, and R ² represents a substituted group. Represents an oxycarbonyl group, and R ³ is —OR ⁴ or —NR ⁵
R ⁶ is shown. R ⁴ , R ⁵ or R ⁶ are the same or different and each represents a lower alkyl group. And a pharmaceutically acceptable salt thereof. 2. A ¹ and A ² are lysyl groups, and R ³ is -N
The peptide derivative according to claim 1, which is R ⁵ R ⁶ , and a pharmaceutically acceptable salt thereof. 3. A ¹ and A ² are a lysyl group, R ¹ is a hydrogen atom, R ² is an aralkyloxycarbonyl group, and R ^{3 is}
Is -NR < ⁵ > R < ⁶ >, The peptide derivative of Claim 2 and its pharmaceutically acceptable salt. 4. A compound of the general formula (Wherein X represents -CHOH- or -CO-, A ¹ and A ² are the same or different and each represents a lysyl group or a histidyl group. R ¹ represents a hydrogen atom or a nitro group, and R ² represents a substituted group. Represents an oxycarbonyl group, and R ³ is —OR ⁴ or —NR ⁵
R ⁶ is shown. R ⁴ , R ⁵ or R ⁶ are the same or different and each represents a lower alkyl group. A ³ and A ⁴ are the same or different, and a lysyl derivative in which N ^ε is protected by a protecting group or N ^im
Represents a histidyl derivative protected with a protecting group, and R ⁷ represents a hydrogen atom, a nitro group or a protecting group. ) And a pharmaceutically acceptable salt thereof, which undergoes a deprotection reaction. (Wherein X, A ¹ , A ² , R ¹ , R ² and R ³ are the same as described above) and a method for producing a pharmaceutically acceptable salt thereof. 5. An arginine comprising a peptide derivative represented by the general formula (1) and a pharmaceutically acceptable salt thereof as an active ingredient.
Zingipain inhibitor. 6. A drug for periodontal disease comprising a peptide derivative represented by the general formula (1) and a pharmaceutically acceptable salt thereof as an active ingredient. 7. A composition comprising a peptide derivative represented by the general formula (1), a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.