USOO648.9291B1 (12) United States Patent (10) Patent No.: US 6,489,291 B1 Suzuki et al. (45) Date of Patent: Dec. 3, 2002

(54) TOPICAL COMPOSITION FOR SKIN Patent Abstracts of Japan, vol. 1997, No. 04, Apr. 30, 1997 & JP 08 333230 A (KAO CORP), Dec. 17, 1996, Abstract. (75) Inventors: Yasuto Suzuki, Tochigi (JP); Mikako Database WPI, Section Ch, Week 200031, Derwent Publi Watanabe, Tochigi (JP); Naoko Tsuji, cations Ltd., London, GB; Class B05, AN 2000–359593 Tochigi (JP); Shigeru Moriwaki, XP002901566 & JP 2000 11914.4A (KAO CORP), Apr. 25, Tochigi (JP); Shinya Amano, Tochigi 2000, Abstract. (JP) * cited by examiner (73) Assignee: Kao Corporation, Tokyo (JP) Primary Examiner Dameron L. Jones ASSistant Examiner-Lauren O. Wells (*) Notice: Subject to any disclaimer, the term of this (74) Attorney, Agent, or Firm-Oblon, Spivak, McClelland, patent is extended or adjusted under 35 Maier & Neustadt, P.C. U.S.C. 154(b) by 0 days. (57) ABSTRACT (21) Appl. No.: 09/678,615 A topical skin composition containing a dipeptide com (22) Filed: Oct. 4, 2000 pound represented by, formula (1) or a Salt of the dipeptide: (30) Foreign Application Priority Data (1) Oct. 5, 1999 (JP) ...... 11-283794 R1 ti (51) Int. Cl." ...... A01N 37/18: A01N 37/00; N- CH-CON-CH-(CH2) X AO1N 37/30; A61K 38/00; A61K 31/185; R2 k A61K 31/205; A61K 31/16; A61K 6/00; A61K 7/00; A61K 31/74 wherein R' represents a, hydrogen atom, an alkyl group, an (52) U.S. Cl...... 514/2; 424/401; 424/78.03; alkanoyl group, or -CH(R)COOR7 (wherein R represents 514/554; 514/627 a hydrogen atom or a lower alkyl group, and R7 represents (58) Field of Search ...... 514/562, 533, a hydrogen atom, a lower alkyl group, a lower alkenyl 514/547, 569, 570, 575, 524, 443, 2,553, group, or an aralkyl group); 554, 627; 424/401, 78.03 R represents a hydrogen atom or an alkyl group which may have a Substituent; (56) References Cited R represents a lower alkyl group or a phenyl group; U.S. PATENT DOCUMENTS R" represents a hydrogen atom or a lower alkyl group, and 4.513,009 A 4/1985 Roques et al. may form a heterocyclic ring together with R and an 6,075,052 A 6/2000 Suzuki et al. adjacent nitrogen atom; 6,171,595 B1 1/2001 Suzuki et al. R represents a hydrogen atom, an alkyl group which may have a Substituent, or an aralkyl group which may have FOREIGN PATENT DOCUMENTS a Substituent, and may form the heterocyclic ring EP O O54862 6/1982 together with R"; JP 2000-1191.44 4/2000 X represents -COOR (wherein R represents a hydro gen atom, a lower alkyl group, a lower alkenyl group, OTHER PUBLICATIONS or an aralkyl group) or -SO3H, and JP abstract 08333230. Dec. 17, 1996.* n is an integer of 0-4; and a pharmaceutically acceptable JPAbstract 40634.5797. Dec. 20, 1994.* carrier therefor. Chemical Abstracts, vol. 98, No. 3, Jan. 17, 1983, Abstract No. 11055W. 20 Claims, No Drawings US 6,489,291 B1 1 2 TOPCAL COMPOSITION FOR SKN R represents a hydrogen atom or an alkyl group which may have a Substituent; R represents a lower alkyl group or a phenyl group; BACKGROUND OF THE INVENTION R" represents a hydrogen atom or a lower alkyl group, and 1. Field of the Invention may form a heterocyclic ring together with R and an The present invention relates to a topical composition for adjacent nitrogen atom; the skin (hereinafter may be simply referred to as a topical R represents a hydrogen atom, an alkyl group which may skin composition) exhibiting effects of retarding aging of the have a Substituent, or an aralkyl group which may have skin or rejuvenating the Skin and of Suppressing hair growth. a Substituent, and may form the heterocyclic ring 1O together with R"; 2. Background Art X represents -COOR (wherein R represents a hydro Studies have revealed that aging of the skin is mainly gen atom, a lower alkyl group, a lower alkenyl group, caused by advancing age, drying, oxidation, or Sunlight (i.e., or an aralkyl group) or -SOH, and UV rays). Aging of the skin is recognized by a decrease in n is an integer of 0-4; and a pharmaceutically acceptable collagen or elastin in the dermis of the skin; a decrease in 15 carrier therefor. mucopolysaccharides, including hyaluronic acid; or the The present invention also provides, as a novel compound presence of cells which are damaged by UV rayS. which has not yet been described in any literature, For the prevention of wrinkle formation, however, Suffi N-(carboxymethyl)phenylalanyl-f-alanine, among dipep cient effects have not yet been attained, for example, by a tide compounds represented by formula (1), or a Salt thereof. collagen-containing cosmetic composition. In addition, a Various other objects, features and many of the attendant number of research projects have focused on Skin aging advantages of the present invention will be readily appre caused by exposure to UV rayS. However, cosmetic com ciated as the same becomes better understood by reference positions SuperSeding UV absorbing agents or UV protect to the following detailed description of the preferred ing agents have not yet been developed. embodiments. There is a trend towards a preference for hairleSS bodies, 25 DETAILED DESCRIPTION OF PREFERRED particularly, hairleSS arms or legs, for reasons of aesthetic EMBODIMENTS appearance. Various methods have been tried in the pursuit In formula (1), an alkyl group represented by R' is of body hair removal, for example, a mechanical hair preferably a C1-C6 alkyl group, may be a linear or branched removal method making use of a shaver or hair-tweezers, a alkyl group, and is more preferably a methyl group, an ethyl method for removing body hair from the hair root by use of group, an n-propyl group, an isopropyl group, an n-butyl a hair removing agent, and a method for removing body hair group, an isobutyl group, or a t-butyl group. through chemical action of a hair removing agent. An alkanoyl group represented by R' is preferably a However, the aforementioned hair removing methods C1-C6 alkanoyl group, maybe a linear or branched alkanoyl physically or chemically Stimulate the Skin, and are limited group, and is more preferably an acetyl group, a propionyl by the unsatisfactory duration of the hairless State. 35 group, or a butyryl group. Therefore, there is a need for the development of a method A lower alkyl group represented by R. R", R, R", or R which facilitates removal of body hair. is preferably a C1-C6 alkyl group, may be a linear or branched alkyl group, and is more preferably a methyl SUMMARY OF THE INVENTION group, an ethyl group, an n-propyl group, an isopropyl In View of the foregoing, an object of the present inven 40 group, an n-butyl group, an isobutyl group, or a t-butyl tion is to provide a topical composition for the skin exhib grOup. iting effects with respect to retarding aging of the skin, Such A lower alkenyl group represented by R7 or R is pref as wrinkle formation, or rejuvenating the Skin, and of erably a C2-C6 alkenyl group, more preferably a vinyl Suppressing hair growth. group or a propenyl group. The present inventors have found that a dipeptide com 45 An aralkyl group represented by R or R is a C7-C18 pound having a Specific structure or a Salt thereof exhibits aralkyl group. Specific examples include a phenyl C1-C6 excellent effects with respect to Suppressing wrinkle alkyl group, a biphenyl C1-C6 alkyl group, and a naphthyl formation, preventing a reduction in Skin elasticity, and C1-C6 alkyl group. Of these, a phenyl C1-C6 alkyl group Suppressing hair growth, and that the compound or the Salt 50 is preferable, and a benzyl group is more preferable. is effectively employed in a topical Skin composition for An alkyl group which may have a Substituent, represented retarding aging of the Skin or Suppressing hair growth. by R, may be a C1-C6 alkyl group, a carboxy C1-C6 alkyl Accordingly, the present invention provides a topical skin group, or a C1-C6 alkoxycarbonyl C1-C6 alkyl group. Of composition comprising a dipeptide compound represented these, a C1-C6 alkyl group is preferable. R is more by formula (1) or a salt of the dipeptide: preferably a methyl group, an ethyl group, an n-propyl 55 group, an isopropyl group, an isobutyl group, or a t-butyl (1) grOup. R1 An alkyl group which may have a Substituent, represented ti-R by R, may be a C1-C6 alkyl group, a carboxy C1-C6 alkyl N-CH-CON-ti- (CH2) X 60 group, or a C1-C6 alkoxycarbonyl C1-C6 alkyl group. Of R2 k these, a C1-C6 alkyl group and a carboxy C1-C6 alkyl group are preferable. R is more preferably a methyl group, wherein R' represents a hydrogen atom, an alkyl group, an an isopropyl group, an isobutyl group, a t-butyl group, a alkanoyl group, or -CH(R)COOR7 (wherein R represents carboxymethyl group, or a carboxyethyl group. a hydrogen atom or a lower alkyl group, and R7 represents 65 A heterocyclic ring which is formed by Rand R may be a hydrogen atom, a lower alkyl group, a lower alkenyl pyrrolidine or piperidine. group, or an aralkyl group); An integer n is preferably 0, 1, or 2. US 6,489,291 B1 3 4 In formula (1), R is more preferably a hydrogen atom, a -continued

C1-C6 alkyl group, a C1-C6 alkanoyl group, or -CH(R) (compound 5) COOR7 (wherein R represents a hydrogen atom or a C1-C6 alkyl group, and R7 represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, or a phenyl C1-C6 alkyl group). R is more preferably a hydrogen atom or a C1-C6 alkyl group. R is more preferably a C1-C6 alkyl group or a phenyl group. R" is more preferably a hydrogen atom or a C1-C6 alkyl group. R is more preferably a hydrogen atom, a C1-C6 alkyl group, a phenyl C1-C6 alkyl group, or a carboxy C1-C6 alkyl group. X is more prefer ably -COOR (wherein R represents a hydrogen atom, a (compound 6) C1-C6 alkyl group, a C2-C6 alkenyl group, or a phenyl C1-C6 alkyl group) or -SOH. An integer n is more 15 preferably 0, 1, or 2. Examples of salts of a dipeptide compound (1) include ul NH OH: alkali metal Salts, alkaline earth metal Salts, amine Salts, Me NH n amino acid Salts, and acid addition Salts. Of these, alkali O O metal Salts and amino acid Salts are preferable. A dipeptide (compound 7) compound (1) may have optical activity, and its steric configuration may be any of R, S, and a racemic mixture. The compound may also be in the form of a hydrate. 25

Among dipeptide compounds of formula (1), the more M e OH preferred compounds are as follows: n N N s Me O O (compound 1) (compound 8)

H HO N OH: 35 N H H N1a N N OH: O O O H (compound 2) O O (compound 9) 40

O H HO N-- 45 r NH OH: O O (compound 3) (compound 10)

50

"r 55 O O O O

(compound 4) Phe-Gly (compound 11); Phe-B-Ala (compound 12); Phe Phe (compound 13); Phe-Leu (compound 14); Phe-Ala 60 (compound 15); and Phe-Asp (compound 16): Phe-Tau (compound 17). Of these, compound 1 and compound 12 are more pref erable. A compound of formula (1) in which R" is a hydrogen 65 atom may be Synthesized through a process described in "K. Ienalga, K. Higashihara, and H. Kimura, Chem. Pharm. Bull., 35, 1249–1254 (1987).” A compound of formula (1) in US 6,489,291 B1 S 6 which R" is not a hydrogen atom but is any of the afore The topical skin composition of the present invention may mentioned groups may be Synthesized through, for example, be used as a skin composition for pharmaceutical use or a the following process: cosmetic composition. Examples of pharmaceutical compo Sitions include a-variety of ointments containing a pharma CH-R3 R' ceutically active ingredient. Such an ointment may comprise an oily base, or an oil-in-water or water-in-oil emulsion R1-Y R-Si-in-co-ch-ch X -- base. No particular limitation is imposed on the Species of (2) k oily base, and, for example, vegetable oil, animal oil, Synthetic oil, fatty acid, or natural or Synthetic glyceride may (3) be employed. No particular limitation is imposed on the R1 CH-R3 R' Species of pharmaceutically active ingredient, and, for example, analgesic and anti-inflammatory agents, antipru N-CH-CON-H- (CH2) X ritic drugs, astringent agents, or hormones may be appro R2 l priately employed in accordance with needs. (1) 15 Examples of cosmetic compositions include lotions, emulsions, creams, ointments, Sticks, Solutions in organic (wherein Y represents a halogen atom, a p-toluenesulfonyl Solvents or purified water, packs, gels-and aerosols. Namely, group, or a methanesulfonyloxy group; R represents any of the cosmetic composition may be employed as a lotion, an the aforementioned groups other than a hydrogen atom; and oil essence, an O/W-type or, W/O-type cream, a pack, a R° through R, n, and X are the same as described above). foundation, a skin-cleansing agent, a tonic, a bathing agent, Namely, a compound (2) is reacted with a compound (3) or an aeroSol. in the presence or absence of a base, and if necessary, hydrolysis by use of a base Such as Sodium hydroxide, or EXAMPLES catalytic hydrogenation by use of a catalyst Such as palla dium carbon may be: carried out, to thereby yield a com 25 Referential Example 1 pound (1) of the present invention. Phenylalanyl-B-alanine benzyl ester hydrochloride (10.0 Examples of bases which may be employed in the reac g, 0.028 mol) and benzyl bromoacetate (14.6 g., 0.062 mol) tion include Sodium hydroxide, potassium hydroxide, potas were dissolved in tetrahydrofuran (200 mL). To the resultant Sium carbonate, Sodium carbonate, Sodium Solution, triethylamine Suspended in tetrahydrofuran was hydrogencarbonate, and triethylamine. Of these, potassium added, and the resultant mixture was stirred for 42 hours. carbonate and triethylamine are preferable, and triethy After completion of the reaction, the resultant mixture was lamine is more preferable. subjected to extraction with ethyl acetate (100 mL), and the Examples of Solvents which may be employed in the organic layer was washed with a Saturated aqueous Solution reaction include water, dimethylform amide, of Sodium hydrogencarbonate and Saturated brine and then tetrahydrofuran, benzene, toluene, Xylene, and hexane. Of 35 dried over anhydrous Sodium Sulfate. Subsequently, the these, toluene and benzene are preferable, and toluene is Solvent was removed, and the resultant residue was Sub more preferable. The reaction temperature is -30 to 200 C., jected to column chromatography with a Solvent mixture of preferably 10 to 90° C., more preferably 40 to 70° C. ethyl acetate and n-hexane Serving as an eluent. Thereafter, Adipeptide compound (1) or a Salt thereof is incorporated the solvent was removed, to thereby obtain a dibenzyl ester into the topical skin composition of the present invention in 40 (8.8 g., yield: 66%). The ester was suspended in methanol, an amount of 0.0001–40 wt.% (hereinafter “wt.%” will be and then Subjected to catalytic hydrogenation by use of simply referred to as “%”), preferably 0.01-20%. palladium carbon (10%). The resultant crystals were sub The topical Skin composition of the present invention is jected to filtration, and then recrystallized from water, to more preferably used for preventing and ameliorating unfa thereby obtain compound 1 (1.34g, yield: 35%). vorable skin conditions caused by aging of the Skin, Such as 45 Wrinkles, flabbiness, and reduction of elasticity; or for NMR(DMSO-d) 8: 2.30(t, 2H, J-7 Hz), 2.75(dd, 1H, Suppressing hair growth. J=8, 14 Hz), 2.87(dd, 1H, J=6, 14 Hz), 3.10(dd, 2H, J=17, 25 The topical skin composition of the present invention may Hz), 3.22(dt, 2H, J=7.7 Hz), 3.33(t, 1H, J-7 Hz), 7.10-7.36 further contain a keratinization-ameliorating agent, to (m, 5H), 7.99(t, 1H, J=6 Hz). thereby enhance the effect of retarding aging of the skin, 50 Such as wrinkle formation, or the effect of Suppressing hair Referential Example 2 growth. Examples of Such keratinization-ameliorating Phenylalanyl-y-aminobutyric acid methyl ester hydro agents include Sphingosine derivatives. chloride (1.4g, 0.0047 mol) and ethyl bromoacetate (0.8 g. The topical skin composition of the present invention may 0.0056 mol) were dissolved in tetrahydrofuran (80 mL). To appropriately contain, in addition to the aforementioned 55 the resultant Solution, triethylamine Suspended in tetrahy ingredients, a variety,of ingredients or carriers which are drofuran was added, and the resultant mixture was Stirred for usually employed in cosmetics, quasidrugs, and drugs. 24 hours. After completion of the reaction, the resultant Examples of Such ingredients or carriers include mixture was Subjected to extraction with ethyl acetate (20 humectants, powders, gelation agents, thickeners, mL), and the organic layer was washed with a Saturated Surfactants, emulsifiers, anti-inflammatory agents, 60 aqueous Solution of Sodium hydrogencarbonate and Satu antioxidants, pH regulating agents, chelating agents, rated brine, and then dried over anhydrous Sodium Sulfate. preservatives, dyes, perfumes, UV absorbing-agents, UV Subsequently, the Solvent was removed, and the resultant protecting agents, existing skin-aging preventive or retard residue was Subjected to column chromatography with a ing agents Such as collagen, and existing hair-growth Sup Solvent mixture of ethyl acetate and n-hexane Serving as an pressing agents. The composition may be produced through 65 eluent. Thereafter, the solvent was removed, to thereby a conventional method in accordance with the application obtain an ethyl ester (0.5 g, yield: 31%). The ester was form. dissolved in methanol, and an aqueous Solution of Sodium US 6,489,291 B1 7 8 hydroxide (2.2 equivalent) was added to the methanol by phenylalanyl-aspartic acid dimethyl ester (7.4 g., 0.024 Solution, and the resultant mixture was stirred for 15 hours. mol) and that the amount of ethyl bromoacetate was changed Thereafter, the solvent was removed, and the resultant into 6.0 g (0.036 mol), to thereby obtain compound 5 (1.0 g, residue was neutralized with hydrochloric acid. Then the yield: 12%). Solvent was removed, to thereby obtain compound 2 (0.18 g, NMR(DMSO-d) 8: 2.54–2.70(m, 2H), 2.77(dd, 1H, J-7, yield: 41%). 14 Hz), 2.93(dd, 1H, J=6, 14 Hz), 3.10(d. 1H, J=17 Hz), NMR(DMSO-d) 8: 1.52(tt, 2H, J–7, 7 Hz), 2.09(t, 2H, 3.28(d. 1H, J=17 Hz), 3.44(dd, 1H, J=6, 7 Hz), 4.45–4.55(m, J=8 Hz), 2.86(dd, 2H, J=7, 13 Hz), 3.01(dd, 2H, J=8, 14 Hz), 1H), 7.19–7.29(m, 5H), 8.28(d. 1H, J=8 Hz). 3.10(dt, 2H, J=3, 16 Hz), 3.44(t, 1H, J=7 Hz), 7.04-7.49(m, 5H), 8, 15(t, 1H, J=16 Hz). Referential Example 7 Phenylalanyl-B-alanine (17.4 g., 0.074 mol) was sus Referential Example 3 pended in pyridine (120 g), and the Suspension was cooled The procedure of Referential Example 2 was repeated, to 15° C. Acetic anhydride (30 g) was added dropwise to the except that phenylalanyl-y-aminobutyric acid methyl ester resultant Suspension over one hour. After completion of hydrochloride was replaced by phenylalanyl-leucine methyl 15 addition, the temperature of the resultant mixture was ester hydrochloride (1.0g, 0.00304 mol) and that the amount returned to room temperature, and the mixture was Stirred of ethyl bromoacetate was changed to 0.6 g (0.00365 mol), for 13 hours, and then the reaction was terminated. The to thereby obtain compound 3 (0.34g, yield: 33%). resultant reaction mixture was cooled to 5 C., and ethanol NMR(DMSO-d) 8: 0.81(d,3H, J=6 Hz), 0.86(d,3H, J=6 (50 mL) was added to the reaction mixture, and the mixture Hz), 1.40-1.68(m, 3H), 2.76(dd, 1H, J=8, 14 Hz), 2.93(dd, was stirred for 30 minutes, and then the solvent was 1H, J=6, 14 Hz), 3.08(d. 1H, J=17 Hz), 3.23(d. 1H, J=17 removed. The resultant residue was Subjected to extraction Hz), 3.42(dd, 1H, J=6, 8 Hz), 4.16-4.28(m, 1H), 7.17-7.29 with ethyl acetate (500 mL), and washed with 5% hydro (m, 5H), 8.12(d. 1H, J=8 Hz). chloric acid, distilled water, and Saturated brine, Successively, and then dried over anhydrous Sodium Sulfate. Referential Example 4 25 Subsequently, the Solvent was removed, and the resultant Phenylalanyl-B-alanine ethyl ester (10g, 0.038 mol) and residue was Subjected to column chromatography with a triethylamine (10.3 g, 0.1 mol) were Suspended in toluene Solvent mixture of chloroform and methanol Serving as an (45 g), and the Suspension was heated to 60° C. To the eluent. Thereafter, the solvent was removed, to thereby resultant suspension, ethyl bromoacetate (9.3 g, 0.057 mol) obtain compound 6 (11.5 g, yield: 56%). Suspended in toluene (5 g) was added dropwise over 0.5 NMR(DMSO-d) 8: 1.74(s, 3H), 2.30(dd, 2H, J–7, 7 Hz), hours, and then the resultant mixture was Stirred for three 2.70(dd, 1H, J=10, 14 Hz), 2.91 (dd, 1H, J=5, 14 Hz), hours. After completion of reaction, the resultant reaction 3.12-3.36(m, 2H), 4.35-446((m, 1H), 7.13–7.28(m, 5H), mixture was Subjected to extraction with ethyl acetate (20g). 8.02(t, 1H, J=6 Hz), 8.67(d. 1H, J=8 Hz), 12.22(br. S, 1H). The organic layer was washed with a 2.5% aqueous Solution 35 of citric acid. Subsequently, the Solvent was removed, to Referential Example 8 thereby obtain N-(carboethoxymethyl)phenylalanyl-B- Phenylalanyl-B-alanine (1 g, 0.0042 mol) was suspended alanine ethyl ester (10.7 g, yield: 80.3%). The compound in water (2 g). To the resultant Suspension, palladium carbon was dissolved in methanol, and a 48% acqueous Solution of (10%) (1 g) and a 36% aqueous solution of formaldehyde sodium hydroxide (6.6 g., 0.08 mol) was added to the 40 (7.1 g, 0.042 mol) were added, and the resultant mixture was methanol Solution, and the resultant mixture was stirred for stirred in an H atmosphere at 10 atm. for 24 hours. After three hours. After completion of reaction, the Solvent was completion of reaction, the Solvent was removed. The result removed, and the resultant residue was dissolved in water ant residue was purified through column chromatography, to (20 mL), and then washed with ethyl acetate (10 g). thereby obtain compound 7 (12.8 mg, yield: 8.1%). Subsequently, hydrochloric acid was added to the aqueous 45 NMR(DMSO-d) 8: 1.92–2.32(m, 2H), 2.81(s, 6H), layer, So as to attain a pH of 3.5. After being cooled, the 2.89-3.32(m, 2H), 3.11(dd, 2H, J=6, 13 Hz), 3.94(dd, 1H, resultant Solution was Subjected to filtration, to thereby J=4, 10 Hz), 7.00-7.41(m, 5H), 8.44(t, 1H, J=6 Hz), obtain crude crystals. The crystals were recrystallized from 11.82–12.52(br. s, 1H). a 20% aqueous Solution of isopropyl alcohol, to thereby Referential Example 9 obtain compound 1 (6.0 g, yield: 67%). 50 Phenylalanyl-B-alanine (0.5 g., 0.0021 mol) was sus Referential Example 5 pended in water (5 g). To the resultant Suspension, palladium The procedure of Referential Example 4 was repeated, carbon (10%) (0.5 g) and propionaldehyde (0.15g, 0.027 except that phenylalanyl-l-alanine ethyl ester was replaced mol) in methanol (5 g) were added, and the resultant mixture by phenylalanyl-alanine ethyl ester (3.7 g., 0.014 mol) and 55 was stirred in a H2 atmosphere for 24 hours. After comple that the amount of ethyl bromoacetate was changed into 3.5 tion of reaction, the Solvent was removed. The resultant g (0.021 mol), to thereby obtain compound 4 (1.4g, yield: residue was recrystallized from methanol, to thereby obtain 33%). compound 8 (0.13 g, yield: 22.2%). NMR(DMSO-d) 8: 1.24(d, 3H, J-7 Hz), 2.78(dd, 1H, NMR(DMSO-d) 8: 0.75(t, 3H, J-7 Hz), 1.30(ddt, 2H, J=8, 14 Hz), 2.96(dd, 1H, J=5, 14 Hz), 3.08(d. 1H, J=17 Hz), 60 J=7, 7, 14 Hz), 2.12–2.44(m, 2H), 2.28(dd, 2H, J=7, 7 Hz), 3.24(d. 1H, J=17 Hz), 2.46(dd, 1H, J=7, 7 Hz), 4.29(dq, 1H, 2.65(dd, 1H, J=8, 14 Hz), 2.81 (dd, 1H, J=6, 13 Hz), 3.16(dd, J–7, 7 Hz), 7.13–7.35(m, 5H), 8.22(d. 1H, J-7 Hz), 5.5–8.5 1H, J=6,8Hz), 3.13–3.33(m, 2H), 7.05–7.35(m,5H), 7.89(t, (br. S, 2H). 1H, J=6 Hz).

Referential Example 6 65 Referential Example 10 The procedure of Referential Example 4 was repeated, The procedure of Referential Example 4 was repeated, except that phenylalanyl-B-alanine ethyl ester was replaced except that phenylalanyl-B-alanine ethyl ester was replaced US 6,489,291 B1 10 by leucyl-B-alanine ethyl ester (2.0 g, 0.009 mol) and that the amount of ethyl bromoacetate was changed into 2.2 g TABLE 1-continued (0.013 mol), to thereby obtain compound 9 (1.6 g., yield: 45%). Test Compound Concentration Wrinkle Index NMR(DMSO-d) 8: 0.85(d, 6H, J-7 Hz), 1.37(dd, 2H, Compound 4 O.1% 1.75 + 0.12 Compound 5 O.1% 2.21 - 0.14 J=7, 6 Hz), 1.48–1.68(m, 1H), 2.37(t, 2H, J=7 Hz), 3.02(d, Compound 6 O.1% 2.57 O.2O 1H, J=17 Hz), 3.15(d. 1H, J=17 Hz), 3.27–3.21(m, 3H), Compound 7 O.1% 2.34 - 0.10 8.26(t, 1H, J=6 Hz). Compound 8 O.1% 2.03 - 0.09 Compound 9 O.1% 2.11 - 0.11 Referential Example 11 1O Compound 10 O.1% 1.95 + 0.12 Compound 11 1% 2.82 - 0.18 Phenylalanyl taurine (2.5 g., 9.18 mmol) and sodium Compound 12 1% 1.98 O2O Compound 13 1% 2.64 - 0.16 hydroxide (0.58 g., 0.0144 mol) were dissolved in distilled Compound 14 1% 2.82 - 0.22 water (25 g), and the solution was heated to 50° C. To the Compound 15 1% 2.72 - 0.16 resultant Solution, bromoacetic acid (1.5 g., 0.011 mol) in 15 Compound 16 1% 2.50 - 0.15 distilled water (3 g) and an aqueous Solution of Sodium Compound 17 1% 1.99 O.12 hydroxide were added dropwise simultaneously over 0.5 hours, so as to attain a pH of 10.5-11.5, and the resultant mixture was stirred for three hours. After completion of The results shown in Table 1 reveal that dipeptide com reaction, concentrated hydrochloric acid was added to the pounds of formula (1) exhibit excellent effects with respect resultant reaction mixture, So as to attain a pH of 3. to Suppressing wrinkle formation and retarding aging of the Thereafter, the solvent was removed, to thereby obtain an skin or rejuvenating the skin. N-(carboethoxymethyl)phenylalanyl taurine-inorganic Salt mixture (7.15 g). The resultant mixture (1 g) was Subjected Test Example 2 to ODS-gel column chromatography, to thereby obtain N-(carboethoxymethyl)phenylalanyl taurine (0.15 g). The 25 compound was further treated through fractionation HPLC, Maintenance of Skin Elasticity in Rat to thereby yield compound 10 (48 mg). Three-week-old SD male rats were classified into three NMR (DMSO-d) 8: 2.35(dd, 2H, J=7.0, 6.5 Hz), groups (five rats per group): a group of rats to which an 80% 2.97-326(m, 4H), 3.74(d, 2H, J=3.3 Hz), 4.01(t, 1H, J=6.7 ethanol Solution containing a test compound was applied to Hz), 7.19-7.32(m, 5H), 8.29(t, 1H, J=5.5 Hz), 9.41(bs, 1H). pads of hind limbs; a group of rats to which a solvent (80% Test Example 1 ethanol) was applied to the pads; and a group of non-treated rats. Each pad was exposed to UV-B rays (less than 1 MED), Suppression of Wrinkle Formation in Hairless 35 and then the test compound-containing Solution or the Mouse Solvent was applied to the pad in an amount of 10 ul. The above procedure was carried out three times a week (every The back of each hairless mouse (HR/ICR, 6 weeks old at the start of the test, 5 mice per group) was Subjected to a other day of the week days) for six weeks. single exposure of UV-B rays at a dose of less than 1 MED Skin elasticity was determined using a cutometer (model: using a health light lamp (model: SE20, product of Toshiba 40 SES575, product of Courage Kazaka) as follows. The skin Corporation). Immediately after the exposure, an 80% etha of the pad was subjected to Suction at 500 hPa for three nol Solution (100 uD) containing a 0.1% test compound was Seconds, and then released for three Seconds. The displace applied to the back. The above. procedure was carried out ment over Six Seconds was measured, to thereby obtain Ue daily for 20 weeks. The radiation energy was measured and Uf values. The measurement was repeated five times for using a UV-radiometer (model: UVR-305/365D, product of 45 each pad. Tokyo Optical). In the control group, only 80% ethanol was applied to the back of each mouse during the test. After the The linearity of elastic fibers was analyzed according to test, wrinkle formation was visually observed, and the the method of Imokawa et al. (J. Invest. Dermatol., 105, degree of wrinkle formation was evaluated against the 254-258(1995)) through image analysis of an SEM micro following ratings (wrinkle index). The results are shown in 50 graph. Specifically, Samples for Scanning electron microS Table 1. copy (SEM) were prepared by fixing the pad of a rat with (Wrinkle index) MERCOX (product of Dainippon Ink and Chemicals, Inc.) 0: No wrinkle formation. under reflux, followed by digestion with formic acid. From 1: A few shallow wrinkles were formed. SEM micrographs (x1000) of each of the samples, ten 55 typical micrographs were Selected and enlarged copies were 2: A Slight amount of wrinkles was formed. made. Each of the enlarged micrographs was divided into 16 3: Some wrinkles were formed. uniform regions. From each of the regions, any one of the 4: Deep wrinkles were formed. elastic fibers was Selected and traced on a transparent film 60 with a line having a fixed width (width:8 pixels). When the TABLE 1. area Surrounded by the line tracing the elastic fiber is called Test Compound Concentration Wrinkle Index A, and the longitudinal length and lateral length of the minimum rectangle Surrounding the trace are called B and C, Control 3.06 - 0.13 Compound 1 O.1% 1.25 + 0.11 respectively, the linearity of the elastic fiber is represented Compound 2 O.1% 2.01 - 0.14 65 by A/(BxC). For example, when the trace of the elastic fiber Compound 3 O.1% 182 0.09 is linear, the linearity becomes 1. The results are shown in Table 2. US 6,489,291 B1 11 12

TABLE 2 Test Compound Concentration Ue value Uf value Linearity No treatment Solvent only Compound 1 O.1% Compound 3 O.1% Compound 4 O.1% Compound 5 O.1% Compound 6 O.1% Compound 7 O.1% Compound 8 O.1% Compound 9 O.1% Compound 10 O.1% Compound 12 1% Compound 17 1% *p < 0.05 (vs. solvent only) "p < 0.05 (vs. no treatment)

The results shown in Table 2 reveal that dipeptide com pounds of formula (1) exhibit excellent effects with respect TABLE 3-continued to preventing the lowering of skin elasticity due to UV-B rays and also preventing degradation of the three Hair-growth dimensional Structure of the elastic fibers which cause the Test compound Concentration suppression ratio lowering, and thus the compounds can maintain Skin elas 25 Compound 16 1% 75.2% ticity. Compound 17 1% 79.5% Test Example 3 The results shown in Table 3 reveal that dipeptide com Hair Growth Suppression in Mouse pounds of formula (1) exhibit excellent effects with respect to Suppressing hair growth. The hair on a portion of the backs (size: 2 cmx4 cm) of groups of six-week-did C3H mice, each group consisting of five mice, was carefully shaved So as not to injure the skin Formulation Example 1 using an electric clipper and electric Shaver. To the thus 35 Shaved portion, a Sample was applied in an amount of 100 till twice a day for four weeks. Test compounds were In accordance with the formulation described below, a dissolved in a solvent (80% ethanol) to attain the concen cream for retarding aging of the skin was produced through trations shown in Table 3, to thereby prepare Samples. In the a conventional method. The cream exhibited an excellent control group, only the Solvent was applied to the shaved 40 effect with respect to retarding aging of the skin. portion. After three weeks, in order to observe hair regrowth, the Shaved portion was photographed at a certain magnification, and the ratio of the area of hair regrowth (i.e., the ratio of the area of hair regrowth to that of the shaved (%) area) was compared with that of the control group using an 45 Compound 1 or 12 O.2 image analyzer. The hair growth Suppression ratio (%) was Stearic acid 2.O Cetanol 4.0 represented by a relative value when the ratio of the area of Squalene 8.0 the hair regrowth in the control group was taken as 100. The Vaseline 5.0 results are shown in Table 3. Hydrogenated palm oil 4.0 Polyoxyethylene sorbitan monostearate (20 E.O.) 1.4 50 Oleophilic glycerin monostearate 2.4 TABLE 3 Butyl paraben O1 Glycerin 3.0 Hair-growth 10.0% Potassium hydroxide O.2 Test compound Concentration suppression ratio Perfume O1 Compound 1 O.1% 82.4% 55 Purified water Balance Compound 2 O.1% 73.3% Compound 3 O.1% 72.3% Total 1OO.O Compound 4 O.1% 80.5% Compound 5 O.1% 71.0% Compound 6 O.1% 58.3% Compound 7 O.1% 69.6% 60 Compound 8 O.1% 77.8% Formulation Example 2 Compound 9 O.1% 77.1% Compound 10 O.1% 79.2% Compound 11 1% 32.9% Compound 12 1% 90.4% In accordance with the formulation described below, a Compound 13 1% 78.5% pack for preventing and retarding aging of the skin was Compound 14 1% 70.9% 65 produced through a conventional method. The pack exhib Compound 15 1% 68.4% ited excellent effects with respect to preventing and retard ing aging of the skin. US 6,489,291 B1 14 thereof, a topical skin composition comprising a dipeptide compound represented by formula (1) or a Salt of the dipeptide: (%) Compound 3 3.0 (1) Polyvinyl alcohol 2O.O Glycerin 5.0 R1 tle Ethanol 16.O N- CH-CON-CH-(CH2) X Perfume O1 Dye O1 R2 k Purified water Balance

Total 1OO.O wherein R' represents a hydrogen atom, an alkyl group, an Formulation Example 3 15 alkanoyl group, or -CH(R)COOR", wherein R represents In accordance with the formulation described below, a a hydrogen atom or a C- alkyl group, and R7 represents a hair growth Suppression lotion was produced. Specifically, a hydrogen atom, a C- alkyl group, a C2-alkenyl group, or Solution containing ingredients of A and a Solution contain an aralkyl group; ing ingredients of B were prepared Separately. The Solution B was added to the Solution A, and the resultant mixture was Stirred for homogeneity, to thereby produce a hair growth R represents a hydrogen atom or an alkyl group which suppression lotion. The lotion exhibited an excellent effect may have a Substituent; with respect to Suppressing hair growth. R represents a C- alkyl group or a phenyl group; 25 R" represents a hydrogen atom or a C- alkyl group, and may form a heterocyclic ring together with R and an (%) adjacent nitrogen atom; A. Polyoxyethylene hydrogenated castor oil O.8 Ethanol 3O.O R represents a hydrogen atom, an alkyl group which may B. Compound 1 or 12 1.O have a Substituent, or an aralkyl group which may have Sodium dodecyl sulfate O.12 a Substituent, and may form the heterocyclic ring Dodecylmethylamine oxide O.18 Isopropyl alcohol 15.O together with R"; Benzyl alcohol 12.O Glycerin 2.O X represents -SOH or -COOR, wherein R repre Purified water Balance 35 Sents a hydrogen atom, a C alkyl group, a C alkenyl group, or an aralkyl group; and Total 1OO.O n is an integer of 0-4; and a pharmaceutically acceptable carrier therefor. 2. The method according to claim 1, wherein, in formula Formulation Example 4 40 (1), R is a hydrogen atom, a C1-C6 alkyl group, a C1-C6 In accordance with the formulation described below, a alkanoyl group, or -CH(R)COOR", wherein R represents hair growth Suppression aerosol was produced. Specifically, a hydrogen atom or a C1-C6 alkyl group, and R7 represents ingredients of A were uniformly mixed and charged in a a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl container. The container was then filled with Solution B group, or a phenyl C1-C6 alkyl group; through a conventional method, to thereby produce a hair 45 growth Suppression aerosol. The aerosol exhibited an excel R is a hydrogen atom or a C1-C6 alkyl group; lent effect with respect to Suppressing hair growth. R is a C1-C6 alkyl group or a phenyl group; R" is a hydrogen atom or a C1-C6 alkyl group; 50 R is a hydrogen atom, a C1-C6 alkyl group, a phenyl (%) C1-C6 alkyl group, or a carboxy C1-C6 alkyl group; A. Compound 1 1.O Cetanol 1.2 X is -SOH or -COOR, wherein R represents a Propylene glycol 4.0 hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl Ethanol 8.O 55 group, or a phenyl C1-C6 alkyl group; and Purified water Balance B. Liquefied petroleum gas (propellant) 4.0 n is 0, 1, or2. 3. The method of claim 1 wherein the dipeptide com Total 1OO.O pound represented by formula (1) is N-(carboxymethyl) 60 phenylalanyl-B-alanine or phenylalanyl-B-alanine. The topical skin composition of the present invention 4. The method of claim 1, wherein said dipeptide com exhibits excellent effects with respect to retarding aging of the skin or rejuvenating the Skin and Suppressing hair pound is present in an amount of 0.0001 to 40 wt %. growth. 5. The method of claim 1, wherein said dipeptide com What is claimed is: 65 pound is present in an amount of 0.01 to 20 wt %. 1. A method for retarding aging of the Skin or rejuvenating 6. The method of claim 1, wherein said dipeptide is the Skin comprising applying to a Surface of skin in need Selected from the group consisting of: US 6,489,291 B1 15 16 -continued

5 | | HO N OH, Me N OH, n N N O H O O 1O Me O O

15 | | O N HO N-- N-1 n N-" 1. OH, h O O O H O 2O | HO N OH,

25 O H O O O HO sus

E 3O H O H O N HO 1. N Y-r-ol O H O O 35 Phe-Gly, Phe-B-Ala, Phe-Phe, Phe-Leu, Phe-Ala, Phe-Asp and mixtures thereof. 7. The method according to claim 1, wherein n=1-4. H O 8. The method according to claim 1, wherein n=1-2. 9. The method according to claim 1, wherein said com HO N sus 40 position is in the form of an ointment. r OH, 10. The method according to claim 1, wherein said O H O E composition is Selected from the group consisting of lotions, emulsions, creams, ointment, Sticks, Solutions in organic 45 Solvents, Solutions in purified water, packs, gels and aero Sols. 11. A method for Suppressing hair growth comprising applying to a Surface of Skin in need thereof, a topical skin composition comprising a dipeptide compound represented H O by formula (1) or a salt of the dipeptide: 50 HO N (1) R1 CH-R3 R' O O E OH N-CH-CON-H-CH-x " . R2 k O wherein R' represents a hydrogen atom, an alkyl group, an alkanoyl group, or -CH(R)COOR", wherein R represents a hydrogen atom or a C, alkyl group, and R7 represents a 60 hydrogen atom, a C- alkyl group, a C2-alkenyl group, or an aralkyl group; R represents a hydrogen atom or an alkyl group which may have a Substituent; ------ol R represents a C- alkyl group or a phenyl group; Me N n- 65 R" represents a hydrogen atom or a C- alkyl group, and may form a heterocyclic ring together with R and an adjacent nitrogen atom; US 6,489,291 B1 17 18 R represents a hydrogen atom, an alkyl group which may -continued have a Substituent, or an aralkyl group which may have a Substituent, and may form the heterocyclic ring together with R"; X represents -SOH or -COOR, wherein R represents H O a hydrogen atom, a C- alkyl group, a C- alkenyl group, or an aralkyl group; and HO Nulls n is an integer of 0-4; and a pharmaceutically acceptable 1. 1 OH, carrier therefor. O H O 12. The method according to claim 11, wherein, in for mula (1), R is a hydrogen atom, a C1-C6 alkyl group, a C1-C6 alkanoyl group, or -CH(R)COOR7, wherein R represents a hydrogen atom or a C1-C6 alkyl group, and R' represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 15 | O alkenyl group, or a phenyl C1-C6 alkyl group; HO Nulls R is a hydrogen atom or a C1-C6 alkyl group; OH, R is a C1-C6 alkyl group or a phenyl group; R" is a hydrogen atom or a C1-C6 alkyl group; O H O S. R is a hydrogen atom, a C1-C6 alkyl group, a phenyl O C1-C6 alkyl group, or a carboxy C1-C6 alkyl group; X is -SOH or -COOR wherein R represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl 25 group, or a phenyl C1-C6 alkyl group; and O | n is 0, 1, or 2. 13. The method of claim 11, wherein the dipeptide com us N OH, pound represented by formula (1) is N-(carboxymethyl) Me N phenylatanyl-B-alanine or phenylalanyl-B-alanine. H O O 14. The method of claim 11, wherein said dipeptide compound is present in an amount of 0.0001 to 40 wt %. 15. The method of claim 11, wherein said dipeptide compound is present in an amount of 0.01 to 20 wt %. 16. The method of claim 11, wherein said dipeptide is 35 | Selected from the group consisting of: Me N OH, n N N Me O O

40

| H HO N OH, 45 N-1N N OH, O H O O N N H O O

50 H | O HO N-N-N HO r N N N OH, 1. OH, O H O O O H O 55

H H O 60 HO N HO N 1. N-- r^No. O H O O O H O 65 Phe-Gly, Phe-B-Ala, Phe-Phe, Phe-Leu, Phe-Ala, Phe-Asp and mixtures thereof. 17. The method according to claim 11, wherein n=1-4. US 6,489,291 B1 19 20 18. The method according to claim 11, wherein n=1-2. emulsions, creams, ointment, Sticks, Solutions in organic 19. The method according to claim 11, wherein said Solvents, Solutions in purified water, packs, gels and aero composition is in the form of an ointment. Sols. 20. The method according to claim 11, wherein said composition is Selected from the group consisting of lotions, k . . . . UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 6,489,291 B1 Page 1 of 1 DATED : December 3, 2002 INVENTOR(S) : Yasuto Suzuki et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 14 Line 52, “R', should read -- R --. Column 17 Line 5, “R”, should read -- R --. Line 30, "phenylatanyl', should read -- phenylalanyl --.

Signed and Sealed this Twentieth Day of May, 2003

JAMES E ROGAN Director of the United States Patent and Trademark Office