US 20100152288A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0152288 A1 OGATA et al. (43) Pub. Date: Jun. 17, 2010
(54) NOVEL A-LIPOIC ACID DERIVATIVES AND Publication Classification APPLICATIONS THEREOF (51) Int. Cl. (75) Inventors: Kazumi OGATA, Osaka (JP); gt. 5. CR Takahiro Sakaue, Hyogo (JP); ( .01) Kazuhiko Ito, Hyogo (JP) A61O 90/00 (2009.01) Correspondence Address: (52) U.S. Cl...... 514/494; 556/120,556/130 MERCHANT & GOULD PC P.O. BOX 2903 MINNEAPOLIS, MN 55402-0903 (US) (57) ABSTRACT Provided are novel C.-lipoic acid derivatives represented by (73) Assignee: Senju Pharmaceutical Co. Ltd., the following formula (I), which have a tyrosinase inhibiting Osaka (JP) activity, a melanin production Suppressing activity and an (21) Appl. No.: 12/715.131 elastase inhibiting activity. y x- - - 9 (22) Filed: Mar. 1, 2010 (I) O Related U.S. Application Data (63) Continuation of application No. 10/472.726, filed on A. Sep. 17, 2003, now Pat. No. 7,700,080, filed as appli- S S cation No. PCT/JP2002/002577 on Mar. 18, 2002. NM1 (30) Foreign Application Priority Data (In the formula, M denotes a metal, and A denotes an amino Mar. 19, 2001 (JP) ...... 2001-078571 acid which is bound via N.) US 2010/0152288 A1 Jun. 17, 2010
NOVEL A-LPOIC ACID DERVATIVES AND a pharmacologically acceptable salt thereof, wherein the APPLICATIONS THEREOF N-(6,8-dimercaptooctanoyl)amino acid metal chelate com pound is selected from the group consisting of N-(6,8-dimer CROSS-REFERENCE TO RELATED captooctanoyl)-C.-amino acid metal chelates, N-(6,8-dimer APPLICATIONS captooctanoyl)-3-amino acid metal chelates, N-(6.8- 0001. This application is a Continuation Application of dimercaptooctanoyl)-y-amino acid metal chelates, N-(6.8- Ser. No. 10/475,726 filed in the U.S.A. on Sep. 17, 2003, dimercaptooctanoyl)-8-aminoacid metal chelates, and N-(6. which is a National Stage Application of PCT/JP2002/02577, 8-dimercaptooctanoyl)-e-amino acid metal chelates, filed Mar. 18, 2002, which claims benefit of Serial No. 2001 0009 (3) the N-(6,8-dimercaptooctanoyl)amino acid 078571, filed Mar. 19, 2001 in Japan and which applications metal chelate compound of the above-identified formula (I) or are incorporated herein by reference. A claim of priority to all, a pharmacologically acceptable salt thereof, wherein the to the extent appropriate is made. N-(6,8-dimercaptooctanoyl)amino acid metal chelate com pound is selected from the group consisting of N-(6,8-dimer TECHNICAL FIELD captooctanoyl)-aminoethanesulfonic acid metal chelates, N-(6,8-dimercaptooctanoyl)glycine metal chelates, N-(6.8- 0002 The present invention relates to novel C-lipoic acid dimercaptooctanoyl)aspartic acid metal chelates, N-(6.8- derivatives, pharmacologically acceptable salts thereof and dimercaptooctanoyl)-6-aminohexanoic acid metal chelate, applications thereof. N-(6,8-dimercapto-octanoyl)-y-amino-n-butyric acid metal chelates, N-(6,8-dimercaptooctanoyl)-phenylalanine metal BACKGROUND ART chelates, N-(6,8-dimercaptooctanoyl)anthranilic acid metal 0003 Alfa-lipoic acid (also known as thioctic acid or 6.8- chelates, N-(6,8-dimercaptooctanoyl)methionine metal che dithiooctanoic acid), a coenzyme occurring in mitochondria, lates, and N-(6,8-dimercaptooctanoyl)cysteine metal che has anti-oxidative activity and draws attention as a therapeu lates, tic agent for a variety of pathologic conditions induced by 0010 (4) the N-(6,8-dimercaptooctanoyl)amino acid oxidative stress, such as arteriosclerosis and cataract. Its metal chelate compound of the above-identified formula (I) reduced State compound, dimercaptooctanoic acid, acts to wherein the metal is Zinc, or a pharmacologically acceptable restore reduced forms of glutathione or vitamin C back from salt thereof, their oxidized forms. 0011 (5) a medicament comprising the compound or a 0004. A class of C-lipoic acid derivatives, C-lipoylamino pharmacologically acceptable salts thereof defined in one of acids, in which C-lipoic acid is bound to glycine, methionine, (1)–(4) above, glutamic acid, Valine or the like, respectively are known 0012 (6) a tyrosinase inhibiting agent comprising the (Japanese Patent Publication No. S42-1286, and its corre compound or a pharmacologically acceptable Salt thereof sponding U.S. Pat. No. 3,238,224). Japanese Patent Applica defined in one of (1)–(4) above, tion Publication No. 2000-169371 discloses a salt of C-lipoy 0013 (7) a melanin production Suppressing agent com laminoethylsulfonic acid with imidazole and its use as an prising the compound or a pharmacologically acceptable salt enhancer of glutathione reductase activity. thereof defined in one of (1)–(4) above, 0005. Upon the above background, the present inventors 0014 (8) a prophylactic or therapeutic agent for blotches Succeeded in efficient synthesis of metal chelate compounds and freckles or a Suntan of the skin comprising the compound and their pharmacologically acceptable salts of reduced or a pharmacologically acceptable salt thereof defined in one forms (dihydro-forms) of novel C-lipoylamino acids, found of (1)–(4) above, that these compounds possess a tyrosinase inhibiting activity, 0015 (9) a whitening agent comprising the compound ora a melanin production Suppressing activity and an elastase pharmacologically acceptable salt thereof defined in one of inhibiting activity, and completed the present invention (1)–(4) above, through further studies. 0016 (10) a skin-beautifying agent comprising the com DISCLOSURE OF INVENTION pound or a pharmacologically acceptable salt thereof defined in one of (1)–(4) above, 0006 Thus, the present invention relates to: 0017 (11) an elastase inhibiting agent comprising the 0007 (1) an N-(6,8-dimercaptooctanoyl)amino acid metal compound or a pharmacologically acceptable Salt thereof chelate compound represented by the following formula (I), defined in one of (1)–(4) above, 0018 (12) an anti-wrinkle agent comprising the com pound or a pharmacologically acceptable salt thereof defined (I) in one of (1)–(4) above, 0019 (13) a cosmetic preparation comprising the com pound or a pharmacologically acceptable salt thereof defined in one of (1)–(4) above, 0020 (14) a method for inhibition of tyrosinase compris ing administering to a human an effective amount of the compound or a pharmacologically acceptable Salt thereof wherein M denotes a metal, and A denotes an amino acid defined in one of (1)–(4) above, which is bound via N, or a pharmacologically acceptable salt 0021 (15) a method for suppression of melanin produc thereof (hereinafter referred to as the present compound), tion comprising administering to a human an effective 0008 (2) the N-(6,8-dimercaptooctanoyl)amino acid amount of the compound or a pharmacologically acceptable metal chelate compound of the above-identified formula (I) or salt thereof defined in one of (1)–(4) above, US 2010/0152288 A1 Jun. 17, 2010
0022 (16) a method for prophylaxis or therapeutic treat include Y-amino-n-butyric acid (GABA) and carnitine. ment of blotches and freckles or a Suntan of the skin compris Examples of 6-amino acids include 5-aminolevulinic acid, ing administering to a human an effective amount of the and 5-aminovaleric acid. Examples of e-amino acids include compound or a pharmacologically acceptable salt thereof 6-aminohexanoic acid. Among those amino acids, anthranilic defined in one of (1)–(4) above, acid, aminoethanesulfonic acid, methionine, phenylalanine, 0023 (17) a method for whitening the skin comprising y-amino-n-butyric acid and 6-aminohexanoic acid are pre administering to a human an effective amount of the com ferred. pound or a pharmacologically acceptable salt thereof defined 0037. As a metal in the metal chelate compounds of the in one of (1)–(4) above, present invention, preferably employed are Zinc and cobalt, 0024 (18) a method for beautifying the skin comprising which are divalent metals, iron, which is a divalentortrivalent administering to a human an effective amount of the com metal, and tetravalent germanium. Among them, Zinc, which pound or a pharmacologically acceptable salt thereof defined is a divalent metal, is particularly preferred. The present com in one of (1)–(4) above, pound is an easily purified, stable compound. 0025 (19) a method for inhibiting elastase comprising 0038 Examples of pharmacologically acceptable salts of administering to a human an effective amount of the com the present compound include alkali metal salts such as pound or a pharmacologically acceptable salt thereof defined Sodium salt and potassium salt and alkali earth metal salts in one of (1)–(4) above, Such as calcium salt and magnesium salt. Any other salts may 0026 (20) a method for prevention or therapeutic treat be employed as desired for the purpose of the present inven ment of wrinkles comprising administering to a human an tion insofar as they are pharmacologically acceptable. effective amount of the compound or a pharmacologically 0039. Further, monohydrates, dihydrates, /2 hydrates, /3 acceptable salt thereof defined in one of (1)–(4) above, hydrates, 4 hydrates, 2/3 hydrates, 3/2 hydrates and% hydrates 0027 (21) use of the compound or a pharmacologically are also included in the present invention. acceptable salt thereof defined in one of (1)–(4) above for the 0040. In general, for the synthesis of C-lipoylamino acids, manufacture of a medicament, intermediate compounds, an amino acid is esterified on its 0028 (22) use of the compound or a pharmacologically carboxyl group to get the group protected, then converted to acceptable salt thereof defined in one of (1)–(4) above for the an acid amide with C-lipoic acid using a dehydration conden manufacture of a tyrosinase inhibiting agent, sation agent, and finally Subjected to saponification. How 0029 (23) use of the compound or a pharmacologically ever, synthesis is difficult by this method where aminoethane acceptable salt thereof defined in one of (1)–(4) above for the Sulfonic acid is employed. manufacture of a melanin production Suppressing agent, 0041. The present compound may be synthesized as 0030 (24) use of the compound or a pharmacologically desired by, or according to, e.g., the following scheme of acceptable salt thereof defined in one of (1)–(4) above for the synthesis. manufacture of a prophylactic or therapeutic agent for blotches and freckles or a Suntan of the skin, 0031 (25) use of the compound or a pharmacologically O acceptable salt thereof defined in one of (1)–(4) above for the OH amino acid manufacture of a whitening agent, -- mixed acid 0032 (26) use of the compound or a pharmacologically anhydride method acceptable salt thereof defined in one of (1)–(4) above for the S-S manufacture of a skin-beautifying agent, O 0033 (27) use of the compound or a pharmacologically H acceptable salt thereof defined in one of (1)–(4) above for the M manufacture of a elastase inhibiting agent, 0034 (28) use of the compound or a pharmacologically acceptable salt thereof defined in one of (1)–(4) above for the manufacture of an anti-wrinkle agent, and 0035 (29) use of the compound or a pharmacologically acceptable salt thereof defined in one of (1)–(4) above for the SH--~~s. SH manufacture of a cosmetic preparation. 0036. The present compound is of a structure consisting of O C-lipoic acid that is amide-bonded with an amino acid and a chelated metal therewith, and is a novel compound that has not been found in a literature. In the present invention, amino S ~~~S N 1 acid means C.-amino acid, 3-amino acid, Y-amino acid, M Ö-amino acid and e-amino acid, which have in their molecule a carboxyl group together with an amino group, and aminom (I) ethylcyclohexanoic acid and anthranilic acid, as well as ami noethanesulfonic acid (taurine), which has in the molecule a (In the formulas, A and Mare as defined hereinbefore.) Sulfonic acid group together with an amino group. Examples 0042. As a result of a study for an efficient production of of C.-amino acids include glycine, alanine, Valine, leucine, C-lipoylamino acid, an intermediate for the present com isoleucine, serine, threonine, tyrosine, cysteine, methionine, pound, it was found that the aimed C-lipoylamino acid is aspartic acid, asparagine, glutamic acid, glutamine, arginine, obtained in high yield when synthesized by the mixed acid lysine, histidine, phenylalanine, tryptophan, etc. Examples of anhydride method (MA). Briefly, C.-lipoic acid dissolved in B-amino acids include 3-alanine. Examples of Y-amino acids an organic solvent (e.g., chloroform, tetrahydrofuran, aceto US 2010/0152288 A1 Jun. 17, 2010 nitrile, etc.) is reacted with a mixed acid anhydride reagent Such as a halogenated carbonic acid ester (such as ethyl car bonyl chloride, butyl carbonyl chloride), isobutyloxycarbo O O O nyl chloride, diethylacetyl chloride, trimethylacetyl chloride A. e A. and the like to forman mixed acid anhydride of C-lipoic acid Sal-S - 7" s-s at -15 to -5°C. in the presence of tertiary amine (such as Zh H triethylamine, tributylamine, N-methylmorpholine (NMM)). The length of time of the reaction is from 1-2 minutes to (In the formulas, A is as defined hereinbefore.) several tens of minutes. Then, an amino acid dissolved in 0049. The present compound is useful for prophylaxis and alcohol, water or a mixture solution thereof in the presence of treatment of a variety of disorders caused by oxidative stress a base (such as Sodium hydroxide, potassium hydroxide, or a in mammals (e.g., bovine, horse, rabbit, mouse, rat, human), tertiary amine Such as triethylamine or tributylamine) is e.g., ischemic cardiovascular disease, cerebralischemia, arte added and reacted. Subsequent recrystallization from a Suit riosclerosis, diabetes mellitus and cataract. able solvent, e.g., water or alcohol, gives C.-lipoylamino acid 0050. When the present compound is used as a medical in high yield. drug, one of the species of the present compound, or two or more of them in combination, may be employed as desired 0043 Reduction of thus obtained C-lipoylamino acid with according to purpose and need. a metal and an acid gives, via a dihydro compound, a chelate 0051. The present compound is used as desired orally or compound of the present invention in highyield. Examples of parenterally as a drug for the treatment of the aforementioned acids employed in the reduction of a stable C-lipoylamino disorders. As for preparation forms, it may be provided in any acid includes inorganic acids Such as hydrochloric acid and form by a known method, e.g., Solid preparations such as Sulfuric acid and organic acids such as acetic acid and citric tablets, granules, powder, capsules, or liquid preparations acid. In the case of a Zinc chelate compound, the two —SH Such as injection or eye drops. These preparations may con groups (mercapto group) in its molecule are thought to bind tain conventional additives as desired, such as diluents, bind with one Zinc atom. ers, thickening agents, dispersing agents, reabsorption 0044) Melanin in the skin is produced by melanocytes. enhancers, buffers, Surfactants, solubilizing agents, preserva Tyrosinase has long been known as a sole rate-controlling tives, emulsifiers, isotonizers, stabilizers, pH adjusting enzyme which regulates melanin production. Tyrosinase is agents, etc. catalyzed by three enzymes, i.e., tyrosine hydroxylase, 3,4- 0.052 The doses of the present compound, when used as a dihydroxyphenylalanine (DOPA) oxidase and 5,6-dihy medical drug, will vary according to the employed species of droxyindole (DHI) oxidase, which play important roles in the present compound, the body weight and age of a given patient, the disorder to be treated and its condition and the early as well as late reactions in melanin production. As way of administration. When used for ischemic cardiovascu evident from the test examples mentioned below, the present lar diseases, cerebral ischemia, arteriosclerosis or diabetes compound Suppresses melanin production by inhibiting tyro mellitus, the doses for an adult are about 1 mg to about 30 mg sinase, the rate-controlling enzyme, via inhibition of tyrosine per day given at a time in the case of an injection and about 1 hydroxylase. mg to about 100 mg at a time, administered several times a 0045. On the other hand, elastase is an enzyme which day, in the case of an oral preparation. When used as an splits elastin, an elastic structure protein having a number of anti-cataract drug, eye drops containing about 0.01 to 5 (w/v) cross links among its peptide chains and occurring in tissues % are preferably applied several times a day, several drops at with considerable extensibility such as the skin. Therefore, each time. inhibition of elastase in the skin would be useful for preven 0053 A medical drug containing the present compound tion of wrinkles and maintenance of the beauty of the skin, for may also contain other ingredients having the same or differ it would serve to maintain the extensibility and elasticity of ent pharmacological activity insofar as they do not act counter the skin. As evident from the test examples described below, to the purpose of the present invention. the present compound also has an elastase inhibiting activity. 0054 The compound of the present invention may be added as desired to creams, facial masking agents, powders, 0046. Thus, the present compound has, as its features, lotions, toilet waters for prophylaxis or treatment of blotches melanin production Suppressing activity and elastase inhibit and freckles or Suntan of the skin, or for whitening, skin ing activity. beatifying or anti-wrinkle activity. When the present com 0047. Therefore, the present compound is useful as a pro pound is added to cosmetic products, other additives conven phylactic or therapeutic agent for blotches and freckles or tionally used in cosmetic products may also be employed, Suntan of the skin, a whitening agent, a skin-beautifying agent Such as diluents, stabilizers, pigments, fragrant materials, and an anti-wrinkle agent. ultraviolet absorbents, antioxidants, preservatives, metal 0048. The present compound also exhibits anti-oxidation chelating agents and organic acids. activity and radical-suppressing activity (eliminating a stable 0055 When the present compound is used for cosmetic radical, 1,1-diphenyl-2-picrylhydroradical (DPPH)). That products, it is added usually at about 0.001 to 5 (w/w) %, preferably at about 0.005 to 2 (w/w)%, although this depends the present compound has such activity is demonstrated by on the employed species of the compound, the type of the the fact that one mole of the present compound is required to cosmetic product and the purpose of the addition. reduce and decolorize one mole of iodine (I2), which is con sistent with what is theoretically calculated, and the present EXAMPLES compound itself is thereby oxidized back to O-lipoylamino 0056. The present invention will be described below in acid (see the following scheme). further detail with reference to the reference examples, US 2010/0152288 A1 Jun. 17, 2010 examples and test examples. However, the scope of the nol gave 4.5 g of white crystals of the aimed compound. present invention is not limited by them. Melting point: over 300° C. TLC, Rf 0.47 (chloroform: methanol: water 4:1:2) Reference Example 1 Reference Example 6 N-C-lipoylaminoethanesulfonic acid sodium salt N-C-lipoyl-y-amino-n-butyric acid sodium salt 0057 To 6.2 g of DL-C.-lipoic acid, dissolved in 60 ml of 0065. Using 4.2 g of DL-C-lipoic acid and 2.3 g of chloroform, was added 3.2 g of triethylamine, and cooled at y-amino-n-butyric acid, the reaction was conducted in the -5°C. To this, 3.3 g of ethyl carbonyl chloride was slowly same manner as Reference Example 1. Recrystallization added dropwise and, 15 minutes after the completion of the from ethanol gave 4.0 g of sodium salt, the aimed compound. addition, 4.5 g of aminoethanesulfonic acid and 1.5 g of Melting point: gradual decomposition starting at about 235' sodium hydroxide dissolved in 60 ml of methanol were added C. TLC, Rf 0.76 (chloroform: methanol: water 4:1:2) at once, and stirred for 15 minutes at the same temperature and further one hour at room temperature. To this was added Reference Example 7 a solution of 1.5g of sodium hydroxide dissolved in 50 ml of methanol, the solvent was condensed under reduced pressure N-C-lipoylglycine sodium salt to /3 of the initial volume, 60 ml of ethanol was added, and 0066. Using 4.2 g of DL-C.-lipoic acid and 1.9 g of glycine, precipitated crystals were collected by filtration. Recrystal the reaction was conducted in the same manner as Reference lized of this from water-methanol gave 5.8g of white crystals Example 1. Recrystallization from methanol/ethanol gave 4.5 of aimed sodium salt. Melting point: 235-237°C. TLC, Rf 0. g of pale yellow crystals of the aimed compound. Melting 53 (n-butanol:acetic acid: water 4:1:2) point: 218-220° C. (decomp.) TLC, Rf-0.64 (chloroform: 0058 Elemental Analysis For CHNO.S.Na.HO methanol: water 4:1:2) 0059 Calculated: C, 34.08; H, 5.43; N, 3.97. 0060 Found C, 34.23; H, 5.54; N, 3.80. Reference Example 8 N-O-lipoylphenylalanine Reference Example 2 0067. Using 4.2 g of DL-C.-lipoic acid and 3.5g of phe N-C-lipoylaminoethanesulfonic acid potassium salt nylalanine, the reaction was conducted in the same manner as Reference Example 1. After evaporation of the solvent and 0061 The same procedure as Reference Example 1 was acidification with hydrochloric acid, extraction was carried followed except that sodium hydroxide in Reference out with ethyl acetate. Following washing with water, ethyl Example 1 was replaced with 4.0 g of potassium hydroxide. acetate was evaporated. The crystalline residue was recrys Recrystallization from water/methanol gave 6.5 g of white tallized from ethanol/isopropyl ether, giving 5.4 g of pale crystals of aimed potassium salt. Melting point: 240-242°C. yellow crystals. Melting point: 154-156°C. TLC, Rf 0.86 (n-butanol:acetic acid: water 4:1:2) Reference Example 3 Reference Example 9 N-C-lipoylaminoethanesulfonic acid calcium and magnesium salts N-O-lipoylanthranilic acid sodium salt 0068. Using 4.2 g of DL-C.-lipoic acid and 2.9 g of anthra 0062. The sodium salt obtained in Reference Example 1 nilic acid, the same procedure followed as Reference was dissolved in water, desalted with a sulfonic acid type Example 1 gave 3.6 g of white crystals. Melting point: over resin into a free acid form, and neutralized with calcium carbonate or basic magnesium carbonate to give soluble cal 300° C. TLC, Rf 0.89 (n-butanol:acetic acid:water 4:1:2) cium and magnesium salts, respectively. Melting point: over Reference Example 10 300° C., respectively. N-C-lipoylmethionine Reference Example 4 0069. Using 4.2 g of DL-C.-lipoic acid and 3.5g of L-me thionine, the same procedure followed as Reference Example N-C-lipoyl-6-aminohexanoic acid sodium salt 8 gave 4.0 g of pale yellow crystals. Melting point: 108-109 0063. Using 4.2 g of DL-C.-lipoic acid and 3.0 g of 6-ami C. TLC, Rf 0.81 (n-butanol:acetic acid: water 4:1:2) nohexanoic acid, the reaction was conducted in the same manner as Reference Example 1. Recrystallization from etha Reference Example 11 nol gave 3.0 g of yellowish white crystals of the aimed com N-C-lipoylcysteine sodium salt pound. Melting point: 200-202°C. (decomp.). TLC, Rf 0.84 (chloroform:methanol: water=5:4:1) 0070 4.2 g of DL-C-lipoic acid and 2.2 g of triethylamine were dissolved in 60 ml of tetrahydrofuran and cooled at -5° C. To this, 2.3 g of ethyl carbonyl chloride was slowly added Reference Example 5 dropwise and, 6 minutes after the completion of the addition, N-C-lipoylaspartic acid disodium salt a solution of 2.6 g of L-cysteine and 2.5g of triethylamine dissolved in 20 ml of water was added and reacted as in 0064. Using 4.2 g of DL-C.-lipoic acid and 2.9 g of L-as Reference Example 1. After acidification with hydrochloric partic acid, the reaction was conducted in the same manner as acid, extraction was carried out with ethyl acetate. Following Reference Example 1. Recrystallization from water/metha washing, ethyl acetate was evaporated, and the residue was US 2010/0152288 A1 Jun. 17, 2010 dissolved in ethanol. By gradual addition of sodium hydrox Example 4 ide in methanol to adjust the pH to 7, 4.3 g of precipitated white crystals were obtained. Melting point: gradual decom N-(6,8-dimercaptooctanoyl)aspartic acid sodium salt position starting at about 150° C. TLC, Rf 0.72 (n-butanol: Zinc chelate compound acetic acid: water 4:1:2). For a product where N-ethylmale I0084. Reduction was performed as in Example 1 using 3.0 imide was added, Rf 0.69 was found. g of the compound obtained in Reference Example 5. Pre cipitated white crystals were collected by filtration and sus Reference Example 12 pended in water, dissolved at pH 7-8 with sodium hydroxide, insoluble matter was filtered off, and filtrate concentrated. N-O-lipoyl-5-hydroxytryptophan Methanol was added and precipitated white crystals were collected by filtration, giving 2.3 g of the aimed compound. (0071 Using 4.2 g of DL-C-lipoic acid and 5.0 g of L-5- Melting point: decomposition starting at about 295°C. TLC, hydroxytryptophan, the same procedure followed as Refer Rf 0.53 (chloroform: methanol: water 5:4:1) ence Example 8 gave 6.4 g of white crystals. Melting point: I0085) Elemental Analysis For CHNO.S.NaZn.H.O 118-120°C. TLC, Rf 0.85 (n-butanol:acetic acid:water-4:1: I0086) Calculated: C, 32.11; H, 4.29; N, 3.12. 2) 0087. Found: C, 32.09: H, 4.44; N, 3.10. Example 1 Example 5 N-(6,8-dimercaptooctanoyl)aminoethanesulfonic N-(6,8-dimercaptooctanoyl)-6-aminohexanoic acid acid sodium salt zinc chelate compound sodium salt zinc chelate compound 0072 5.0 g of the compound in the form of sodium salt 10088 3.0 g of the compound obtained in Reference obtained in Reference Example 1 was dissolved in 100 ml of Example 4 was dissolved in 70 ml of 50% tetrahydrofuran water, and to this were added 10 ml of acetic acid and 1.3 g of and reduced as in Example 1. The solvent was evaporated and Zinc powder. After one-hour stirring at 50° C., unreacted zinc precipitated white crystals were collected by filtration. Melt was filtered off, the filtrate concentrated under reduced pres ing point: 215-217°C. These were suspended in water, and Sure, and ethanol was added. Precipitated white crystals were dissolved at pH 7–8 with sodium hydroxide. The solution was collected by filtration, dissolved in water, adjusted of the pH concentrated and methanol was added. Precipitated white to about 8 with sodium bicarbonate, and concentrated. crystals were collected by filtration, giving 2.0 g of the aimed Methanol then was added and precipitated white crystals compound. Melting point: decomposition starting at about were collected by filtration. Recrystallization from water/ 295°C. TLC, Rf 0.84 (chloroform:methanol: water=5:4:1) methanol gave 4.3 g of the aimed compound. Melting point: I0089 Elemental Analysis For CHNO.S.NaZn.H.O decomposition starting at about 293°C. TLC, Rf 0.51 (n-bu (0090 Calculated: C, 39.58; H, 6.17; N, 3.30. tanol: acetic acid:water-: 4:1:2) 0.091 Found: C, 39.38: H, 6.02: N, 3.13. (0073 Elemental Analysis For CHNO.S.NaZn.H.O 0074 Calculated: C, 28.75; H, 4.58; N, 3.35. Example 6 0075 Found: C, 28.56; H, 4.69; N, 3.13. N-(6,8-dimercaptooctanoyl)-Y-amino-n-butyric acid Example 2 sodium salt zinc chelate compound 0092 4.0 g of the compound obtained in Reference N-(6,8-dimercaptooctanoyl)aminoethanesulfonic Example 6 was reduced and worked out as in Example 1, acid potassium salt zinc chelate compound giving 2.1 g of white crystals of the aimed compound. Melt ing point: decomposition starting at about 2.97° C. TLC, 0076. The same procedure as in Example 1 was followed Rf 0.70 (chloroform: methanol: water 5:4:1) using 6.5g of the compound obtained in Reference Example I0093 Elemental Analysis For CHNO.S.NaZn.HO 2, giving 5.0 g of the aimed compound. (0094 Calculated: C, 36.32; H, 5.59; N, 3.53. I0077. Elemental Analysis For CHNOSKZn./2HO 0.095 Found: C, 36.08; H, 5.81; N, 3.29. 0078 Calculated: C, 28.27; H, 4.27; N, 3.30. 007.9 Found C, 28.38: H, 4.52: N, 3.10. Example 7 Example 3 N-(6,8-dimercaptooctanoyl)phenylalanine sodium salt Zinc chelate compound N-(6,8-dimercaptooctanoyl)glycine sodium salt zinc 0096) To 5.4 g of the compound obtained in Reference chelate compound Example 8 were added 80 ml of 30% methanol, 2.0 g of zinc powder, 10 ml of acetic acid and 20 ml of 2 NHC1. After 0080. The same procedure as in Example 1 was followed 3-hour stirring at 50° C., unreacted zinc was filtered off, and using 4.5g of the compound obtained in Reference Example the filtrate was concentrated. Following addition of water, 7, giving 3.9 g of the aimed compound. Melting point: decom precipitated oily matter was collected. For conversion to a position starting at about 2.97°C. TLC, Rf 0.64 (chloroform: Sodium salt, this was dissolved in methanol and pH adjusted methanol: water 4:1:2) to 7 with sodium hydroxide/methanol. Precipitated crystals I0081 Elemental Analysis For CHNO.S.NaZn.HO were collected by filtration, giving 3.9 g, Melting point: 0082 Calculated: C, 32.57; H, 4.92: N, 3.80. gradual decomposition starting at about 270° C. TLC, Rf 0. 0083. Found: C, 32.43; H, 4.83; N, 3.74. 82 (n-butanol:acetic acid: water 4:1:2) US 2010/0152288 A1 Jun. 17, 2010
0097 Elemental Analysis: For CHNO.S.NaZn. 0114. A test compound added to 100LLM tyrosine contain /2HO ing L-3,5-H tyrosine (0.5 Ci/assay), 1 mg of tyrosine 0.098 Calculated: C, 45.39; H, 5.15; N, 3.11. hydroxylase, 0.2 mM of (6R)-5,6,7,8-tetrahydro-L-biopterin, 0099 Found: C, 45.55; H, 5.33: N, 3.23. 1.8 mg/ml catalase, 5 mM dithiothreitol (in MES buffer, pH 6.0) was incubated at 37°C. for 40 minutes. The reaction was Example 8 terminated by addition of 7.5% charcoal in 1 M hydrochloric N-(6,8-dimercaptooctanoyl)anthranilic acid sodium acid. salt Zinc chelate compound I0115 HO derived from 3,4-dihydroxyphenylalanine 0100. The same procedure as Example 7 was followed (DOPA) was determined by liquid scintillation. using 3.6 g of the compound obtained in Reference Example 9, giving 2.1 g of white crystals. Melting point: decomposi (Test Compound) tion starting at about 290°C. TLC, Rf 0.88 (n-butanol: acetic 0116. The compounds of Reference Example 1. Example acid: water 4:1:2) 0101 Elemental Analysis For CHNO.S.NaZn.HO 1, and O-lipoic acid (final concentration: 0.1 mM, 1 mM). 01.02 Calculated: C, 41.81; H, 4.68; N, 3.25. (0103 Found: C, 41.98; H, 4.64; N, 3.26. (Test Results) Example 9 0117. The results are shown in Table 1. N-(6,8-dimercaptooctanoyl)methionine zinc chelate TABLE 1 compound Tyrosine Hydroxylase Inhibiting 0104. The same procedure was followed as Example 7 Activity of The Present Compound using 4.0 g of the compound obtained in Reference Example Concentration (%) O.1 mM 1 mM 10, giving 2.8 g of the aimed compound, free acid. Melting Compound of Reference Example 1 11 10 point: gradual decomposition starting at about 260° C. TLC, Compound of Example 1 15 53 Rf 0.82 (n-butanol:acetic acid:water 4:1:2) C-lipoic acid 2 1 0105 Elemental Analysis: For CHNOSZn. 1.5H2O 01.06 Calculated: C, 36.32; H, 6.10: N, 3.26. Values are inhibition rates (%) 01.07 Found: C, 36.17; H, 5.78; N, 3.34. 0118. As evident from Table 1, tyrosine hydroxylase Example 10 inhibiting activity was noted with the present compound. In contrast, C-lipoic acid exhibited no tyrosine hydroxylase N-(6,8-dimercaptooctanoyl)cysteine Zinc chelate inhibiting activity. Thus, the present compound was found to compound inhibit tyrosinase, an enzyme regulating melanin production. 0108. To 5.7 g of the compound obtained in Reference Example 11 were added 100 ml of 50% methanol, 3.5g of Test Example 2 Zinc powder, 10 ml of acetic acid and 40 ml of 2 N hydro chloric acid. After 3-hour stirring at 50° C., unreacted Zinc Melanin Production Suppressing Activity of The was filtered off, the filtrate concentrated to about /2. The pH Present Compound of this was adjusted to 3-4 with 2 N sodium hydroxide and precipitated white crystals were collected by filtration and Test Method washed with 3% acetic acid and water. They were dissolved in 1% sodium hydroxide, acidified with acetic acid, and precipi 0119 B16-F0 melanoma cell line of mouse origin, pur tated crystals was collected by filtration, washed with water chased from Dainippon Pharmaceutical Co., Ltd. was used in and methanol, and dried. Melting point: decomposition start the experiment. In 60-mm petri dish, 200,000 cells were ing at about 280°C. TLC (after dissolving by neutralization incubated for five days in a medium (D-MEM*+10% FBS*) with ammonium water), Rf 0.71 (chloroform:methanol: wa supplemented with 0.1% D-glucosamine hydrochloride, an ter=5:4:1) inhibitor of carbohydrate synthesis, to halt melanin synthesis 0109 Elemental Analysis For CHNOSZn.3H2O and turn the culture white. 0110 Calculated: C, 31.79; H, 4.61; N, 3.37. 0.120. Following the 5-day culture, the cells were washed 0111 Found: C, 31.98; H, 4.77; N, 3.14. with PBS(-)* to remove D-glucosamine hydrochloride, and 2 mM theophylline (250-fold concentrated solution/distilled Test Example 1 water), a phosphodiesterase inhibitor, was added to raise intracellular cAMP and thereby promote the recovery of tyro Tyrosine Hydroxylase Inhibiting Activity of The sinase synthesis. At the same time, a test compound (250-fold Present Compound concentrated solution/PBS(-)) was added. Three days after 0112 The present compound was examined for tyrosine the addition of theophylline and the test compound, the cells hydroxylase inhibiting activity. were harvested by trypsin treatment. The cell pellets were resuspended in 1 ml of PBS(-), 0.1 ml of which was used for counting cells, and 0.9 ml for melanin determination. Deter (Test Method) mination of melanin was carried out by washing the cell 0113 Tyrosine hydroxylase from rat brain was used in the pellets once with 5% trichloroacetic acid, ethanol/diethyl teSt. ether (3:1), and then diethyl ether, adding 1 ml of 2N NaOH US 2010/0152288 A1 Jun. 17, 2010 to lyse the cells, measuring optical density at 420 nm, and potent Suppressive action on melanin production, which was comparing the result with a calibration curve produced with significantly more potent than arbutin or kojic acid, com reference standard. pounds so far used as whitening agent. *(NB) Test Example 3 D-MEW: Dulbecco's Modified EAGLE MEDIUM “Nissui Elastase Inhibiting Activity of The Present Com (2) pound FBS: Fetal Bovine Serum Certified, Origin: United States Test Method PBS(-): Dulbecco's PBS(-) “Nissui” I0123 96-well plates were used. 1 mM of Suc(Ome)-Ala 0121. The results are shown in Table 2. Ala-Pro-Val-MCA, 30 ul of a test sample, 210 ul of Tris-NaCl
TABLE 2 Melanin Production Suppressing Activity of The Present Compound Melanin amount (% of control) Control 100.00 Kojic acid 0.1 mM 92.70 Kojic acid 0.3 mM 88.04 Kojic acid 1 mM 68.8O Kojic acid 10 mM 18.73 Arbutin 0.1 mM 97.29 Arbutin 0.3 mM 88.86 Arbutin 1 mM 83.02 N-(6,8-dimerca ptooctanoyl)aminoethanesulfonic acid Na–Zn 0.03 mM 78.33 N-(6,8-dimerca ptooctanoyl)aminoethanesulfonic acid Na Zn 0.1 mM 30.01 N-(6,8-dimerca ptooctanoyl)aminoethanesulfonic acid Na–Zn 0.3 mM 12.28 N-(6,8-dimerca ptooctanoyl)-6-aminohexanoic acid Na–Zn 0.03 mM 64.43 N-(6,8-dimerca ptooctanoyl)-6-aminohexanoic acid Na Zn 0.1 mM 27.69 N-(6,8-dimerca ptooctanoyl)-6-aminohexanoic acid Na Zn 0.3 mM 9.85 N-(6,8-dimercaptooctanoyl)-y-amino-n-butyric acid Na Zn 0.03 mM 61.18 N-(6,8-dimercaptooctanoyl)-y-amino-n-butyric acid Na Zn 0.1 mM 29.20 N-(6,8-dimercaptooctanoyl)-y-amino-n-butyric acid Na Zn 0.3 mM 9.73 N-(6,8-dimercaptooctanoyl)methionine Zn 0.03 mM 63.05 N-(6,8-dimercaptooctanoyl)methionine Zn 0.1 mM SO.18 N-(6,8-dimercaptooctanoyl)methionine Zn 0.3 mM 16.41 N-(6,8-dimercaptooctanoyl)phenylalanine Na-Zn 0.03 mM 71.43 N-(6,8-dimercaptooctanoyl)phenylalanine Na-Zn 0.1 mM 37.78 N-(6,8-dimercaptooctanoyl)anthranilic acid Na–Zn 0.03 mM 72.47 N-(6,8-dimercaptooctanoyl)anthranilic acid Na–Zn 0.1 mM 47.64 N-(6,8-dimercaptooctanoyl)anthranilic acid Na–Zn 0.3 mM 16.74 In the table, Zn denotes zinc chelate, and Na-Zinzinc chelate of sodium salt (the same applies below),
0122. As evident from Table 2, N-(6,8-dimercaptooc Buffer (25°C., pH 7.5) and 30 ul of 1 unit/ml elastase were tanoyl)aminoethanesulfonic acid Na-Zn, N-(6,8-dimercap tooctanoyl)aminohexanoic acid Na—Zn, N-(6,8-dimercap added and fluorescence was measured every minute (EX. tooctanoyl)-y-amino-n-butyric acid Na Zn, N-(6.8- 360/40 nm, Em. 460/40 nm). Inhibition rates of elastase activ dimercaptooctanoyl)-methionine Zn, N-(6.8- ity were determined based on a calibration curve produced dimercaptooctanoyl)phenylalanine Na—Zn, and N-(6.8- with AMC (7-amino-4-methyl-coumarin) reference standard. dimercaptooctanoyl)anthranilic acid Na Zn exhibited 0.124. The results are shown in Table 3.
TABLE 3 Esterase Inhibiting Activity of The Present Compound Elastase inhibition rate (%) Elastinal 0.03 mM 25.29 Elastinal 0.1 mM 56.87 Elastinal 0.3 mM 77.45 Elastinal 1 mM 93.01 C-lipoic acid 0.03 mM -2.94 C-lipoic acid 0.1 mM 11.76 US 2010/0152288 A1 Jun. 17, 2010
TABLE 3-continued Esterase Inhibiting Activity of The Present Compound Elastase inhibition rate (%) C-lipoic acid 0.3 mM 10.29 N-(6,8-dimercaptooctanoyl)-6-aminohexanoic acid Na–Zn 0.03 mM 94.74 N-(6,8-dimercaptooctanoyl)-y-amino-n-butyric acid Na Zn 0.03 mM 78.95 N-(6,8-dimercaptooctanoyl)methionine Zn 0.03 mM 7.41 N-(6,8-dimercaptooctanoyl)methionine Zn 0.1 mM 68.52 N-(6,8-dimercaptooctanoyl)methionine Zn 0.3 mM 88.89 N-(6,8-dimercaptooctanoyl)phenylalanine Na-Zn 0.03 mM 83.33 N-(6,8-dimercaptooctanoyl)anthranilic acid Na–Zn 0.03 mM 24.07 N-(6,8-dimercaptooctanoyl)anthranilic acid Na–Zn 0.1 mM 48.15 N-(6,8-dimercaptooctanoyl)anthranilic acid Na–Zn 0.3 mM 72.22
0.125. As evident from Table 3, elastase inhibiting activity Preparation Example 3 comparable to Elastinal was noted with N-O-(6,8-dimercap Tablets tooctanoyl)-6-aminohexanoic acid Na-Zn, N-O-(6.8- 0131) dimercaptooctanoyl)-y-amino-n-butyric acid Na-Zn, N-C- (6,8-dimercaptooctanoyl)methionine Zn, N-O-(6.8- dimercaptooctanoyl)phenylalanine Na-Zn, N-O-(6.8- The compound of Example 1 50 mg dimercaptooctanoyl)anthranilic acid Na Zn. On the other Lactose 80 mg hand, O-lipoic acid did not show any elastase inhibiting activ Potato starch 17 mg ity. Polyethylene glycol 3 mg 0126 From the results, the present compound was found 0.132. Using the above ingredients as the materials for one to be useful as an anti-wrinkle agent. tablet, tables are prepared by a conventional method. Preparation Example 4 Preparation Example 1 Cosmetic Cream Injection 0.133 O127 The compound of Example 7, 8 or 9 0.5g Stearic acid 2.0 g Stearyl alcohol 7.0 g Squalane 5.0 g The compound of Example 1 2.0 mg Octyldecanol 6.0 g D-mannitol 5.0 g Polyoxyethylene cetyl ether 3.0 g Distilled water for injection Total amount 100 ml Glycerol monostearate 2.0 g Propylene glycol 5.0 g Methyl p-hydroxybenzoate 0.2g 0128. Using the above ingredients, an injection is prepared Propyl p-hydroxybenzoate 0.1 g by a conventional method. Sterile purified water 73.7g Preparation Example 2 I0134. The above ingredients are mixed to form a cosmetic CCa: Eye Drops INDUSTRIAL APPLICABILITY 0.135 The metal chelate compound of the present inven tion or a pharmacologically acceptable salt thereof has tyro 0129 sinase inhibiting activity, melanin production Suppressing activity and elastase inhibiting activity, and therefore is useful as an agent for prophylaxis and treatment of blotches and The compound of Example 2 0.5 g. freckles and Suntan of the skin, a whitening agent, a skin Sodium chloride 0.5 g. beautifying agent and an anti-wrinkle agent. Boric acid 0.7 g Borax 0.3 g 0.136 Furthermore, the metal chelate compound of the Methyl p-hydroxybenzoate 0.026 g present invention is also useful for prophylaxis and treatment Propyl p-hydroxybenzoate 0.014 g of a variety of disorders induced by oxidative stress, such as Sterile purified water Total amount 100 ml ischemic cardiovascular disease, cerebral ischemia, arterio Sclerosis, diabetes mellitus and cataract. 0.137 Some of the embodiments of the present invention 0130. Using the above ingredients, eye drops are prepared are described in detail above. However, as various modifica by a conventional method. tions and changes can be made by those who skilled in the art US 2010/0152288 A1 Jun. 17, 2010 to the specific embodiments without Substantially departing from the novel disclosure and benefit of the present invention, all of Such modifications and changes also fall within the (I) spirit and scope of the present invention defined by the fol lowing claims. 0.138. The present application is based on Japanese patent application No. 2001-078571, filed in Japan, and all the con tents thereof are included in the present application.
1. An N-(6,8-dimercaptooctanoyl)amino acid metal che wherein M denotes a Zinc, and Adenotes an amino acid which late compound represented by the following formula (I), is bound via the amino acid nitrogen; or a pharmacologically acceptable salt thereof, wherein the amino acid is selected from the group consisting of aminomethylcyclohexanoic (I) acid, anthranilic acid, aminoethanesulfonic acid, glycine, ala nine, Valine, leucine, isoleucine, serine, threonine, tyrosine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, tryptophan, B-alanine, Y-amino-n-butyric acid, carnitine, 5-aminolevulinic acid, 5-aminovaleric acid, and 6-aminohex anoic acid. 4. The composition of claim 3 comprising a cream base; and wherein the composition is a cosmetic preparation. wherein M denotes a Zinc, and A denotes an amino acid 5. The N-(6,8-dimercaptooctanoyl)amino acid metal che which is bound via the amino acid nitrogen; or a phar late compound of claim 1, wherein the N-(6,8-dimercaptooc macologically acceptable salt thereof, wherein the tanoyl)amino acid metal chelate compound is selected from amino acid is selected from the group consisting of the group consisting of N-(6,8-dimercaptooctanoyl)-6-ami aminomethylcyclohexanoic acid, anthranilic acid, ami nohexanoic acid Zinc chelate, N-(6,8-dimercaptooctanoyl) noethanesulfonic acid, glycine, alanine, Valine, leucine, aminoethanesulfonic acid Zinc chelate and N-(6,8-dimercap isoleucine, serine, threonine, tyrosine, cysteine, tooctanoyl) phenylalanine Zinc chelate. methionine, aspartic acid, asparagine, glutamic acid, 6. The N-(6,8-dimercaptooctanoyl)amino sulfonic acid glutamine, arginine, lysine, histidine, phenylalanine, metal chelate of claim 1, wherein the N-(6,8-dimercaptooc tryptophan, B-alanine, Y-amino-n-butyric acid, car tanoyl)amino Sulfonic acid metal chelate compound is N-(6. nitine, 5-aminolevulinic acid, 5-aminovaleric acid, and 8-dimercaptooctanoyl)aminoethanesulfonic acid Zinc che 6-aminohexanoic acid. late. 2. (The N-(6,8-dimercaptooctanoyl)amino acid metal che 7. An N-(6,8-dimercaptooctanoyl)amino sulfonic acid late compound of claim 1, wherein the N-(6,8-dimercaptooc metal chelate compound represented by the following for tanoyl)amino acid metal chelate compound is selected from mula (I), the group consisting of N-(6,8-dimercaptooctanoyl)amino ethanesulfonic acid zinc chelate, N-(6,8-dimercaptooc tanoyl)glycine Zinc chelate, N-(6,8-dimercaptooctanoyl)as (I) partic acid Zinc chelate, N-(6,8-dimercaptooctanoyl)-6- aminohexanoic acid Zinc chelate, N-(6.8- dimercaptooctanoyl)-y-amino-n-butyric acid Zinc chelate, N-(6,8-dimercaptooctanoyl)phenylalanine Zinc chelate, N-(6,8-dimercaptooctanoyl)-anthranilic acid Zinc-chelate, N-(6,8-dimercaptooctanoyl)methionine Zinc chelate, and N-(6,8-dimercaptooctanoyl)cysteine Zinc chelate. wherein M denotes a Zinc, and A denotes an aminoethane 3. A composition comprising a pharmaceutically accept sulfonic acid which is bound via the amino sulfonic acid able vehicle or a cream base; and nitrogen; or a pharmacologically acceptable salt thereof. an N-(6,8-dimercaptooctanoyl)amino acid metal chelate compound represented by the following formula (I), c c c c c