(12) United States Patent (10) Patent No.: US 8,097.590 B2 Pinel Et Al

USO08097590B2

(12) United States Patent (10) Patent No.: US 8,097.590 B2 Pinel et al. (45) Date of Patent: Jan. 17, 2012

(54) o-MSH-ANTAGONIST DIPEPTIDE FOREIGN PATENT DOCUMENTS CONUGATES EP O 389 95.0 A1 10, 1990 EP O 669938 B1 9, 1995 (75) Inventors: Anne-Marie Pinel, Toulouse (FR): E. : 29: R 1928 Pascal Verdie, Saint Mathieu de Treviers EP 1174 437 A1 1, 2002 (FR); Pascaline Dubs, Montpellier (FR): FR 1327363 5, 1962 Jean Martinez, Caux (FR); Gilles FR 2805744 A1 9, 2001 Subra, Juvignac (FR) FR 2810 323 A1 12/2001 s GB 1 OOO 897 8, 1965 (73) Assignees: Centre National de la Recherche W. W. 2.95. A. 3E Scientifique (CNRS), Paris (FR): WO WO 98.25584 A1 6, 1998 Institut Europeen de Biologie W. W. 9.3. . A. 358: Cellulaire, Ramonville St. Agne (FR): WO WO O2/O85925 A2 10, 2002 Universite de Montpellier I, WO WO 03/064.458 A2 8, 2003 Montpellier (FR); Universite de WO WOO3,O95474 A2 11/2003 Montpellier II, Montpellier (FR) WO WO 2004/099237 A1 11, 2004 WO WO 2004/110341 A2 12/2004 (*) Notice: Subject to any disclaimer, the term of this WO WO 2005,116068 A1 12/2005 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 1143 days. Stverteczky et al., Acta Chimica Academiae Scientiarum (21) Appl. No.: 11/596,286 Hungaricae, 1975, vol. 87, No. 3, pp. 269-283.* Baumann et al., “Specificity of O-chyrinotrypsin dipeptide Sub (22) PCT Filed: May 10, 2005 strates.” FEBS Letters, Jun. 1970, 8(5), 257-260. Beekman et al., “Synthetic peptide vaccines: palmitoylation of (86). PCT No.: PCT/FR2OOS/OO1164. peptide antigens by a thioester bond increases immunogenicityk, J. Peptide Res., 1997, 50: 357-364. S371 (c)(1), Chhajlani, Vijay, "Characterization of a Putative C-MSHAntagonist (2), (4) Date: May 23, 2007 153N-6 at Melanocortin Receptor Subtypes by Radioligand Bind ing.” Peptides, 1996, 17(2), 349-351. (87) PCT Pub. No.: WO2005/115174 Cone et al., “The Melanocortin Receptors: Agonists, Antagonists, and the Hormonal Control of Pigmentation.” Recent Progress in PCT Pub. Date: Dec. 8, 2005 Hormone Research, 1996, 51:287-317. Feliu et al., “Spiroimidazolidinone Library Derivatives on SynPhase (65) Prior Publication Data Lanterns. J. Comb. Chem., 2003, 4:356-361. US 2007/O231284 A1 Oct. 4, 2007 (Continued) (30) Foreign Application Priority Data Primary Examiner — Christopher R. Tate May 11, 2004 (FR) ...... O4 O5069 Assistant Examiner — Roy Teller Oct. 22, 2004 (FR) ...... O4 11279 (74) Attorney, Agent, or Firm — Foley & Lardner LLP (51) Int. Cl. C07C 229/00 (2006.01) (57) ABSTRACT (52) U.S. Cl...... 514/19:514/558; 424/62 The invention relates to a dipeptide conjugate having general (58) Field of Classification Search ...... None formula I, AA2-AA1-NH, wherein A represent the radical See application file for complete search history. corresponding to a monocarboxylic acid with general for mula II, HOOC R, in which: R represents a linear or (56) References Cited branched aliphatic radical at C-C which is optionally Sub stituted by a hydroxyl group and which can comprise one or U.S. PATENT DOCUMENTS more unsaturations, preferably between 1 and 6 unsatura 5,674,839 A 10/1997 Hruby et al. tions, and/or which can comprise a phenyl group or lipoic 5,714,576 A 2/1998 Hruby et al. acid or the reduced form thereof, dihydrolipoic acid or N-li 5,719, 126 A 2, 1998 Nordlund et al. poyllysine; and AA1 and AA2 represent identical or different 5,786.332 A 7, 1998 Girten et al. 5.830,994. A 1 1/1998 D'Hinterland et al. amino acids which are selected from the group containing 6,054,556 A 4/2000 Hruby et al. Ala, Asn. Cys, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser. Thr, Trp, 6,228,840 B1 5, 2001 Wei et al. Tyr, Val, Asp, Glu, Arg, His, Lys, Orn, Dap, Dab, the corre 6,245,342 B1 6/2001 Golz-Berner et al. sponding homo-amino acids and the corresponding beta 3.35 R 33 t et al. amino acids in the form of enantiomers or diastereoisomers 65.79848 B1 6/2003 Hearing, Jr. and mixtures thereof, including racemic mixtures. 2003/0194445 A1 10, 2003 Kuhner et al. 2004/0010010 A1 1/2004 Ebetino et al. 2005. O187164 A1 8, 2005 Pinel 9 Claims, No Drawings US 8,097.590 B2 Page 2

OTHER PUBLICATIONS Vogler et al., “Basic peptides containing fatty acids with an anti bacterial action.” 1964, 47:526-544, ZP002062066, and its English Haskell-Luevano et al., “Truncation Studies of O-Melanotropin translation. Peptied Identify TripeptideAnalogues Exhibiting Prolonged Agonist Office Action issued by the Examiner in U.S. Appl. No. 1 1/596,041 Bioactivity.” Peptides, 1996, 17(6), 995-1002. on Mar. 15, 2010. Holder et al., “Characterization of aliphatic, cyclic, and aromatic N-terminally 'capped His-D-Phe-Arg-Trp-NH tetrapeptides at the U.S. Appl. No. 1 1/596,041, filed Jun. 28, 2007, Martinez et al. melanocortin receptors.” European Journal of Pharmacology, 2003, Office Action issued by the Examiner in U.S. Appl. No. 1 1/596,041 462:41-52. on Sep. 29, 2009. Holder et al., “Structure-Activity Relationships of the Melanocortin Al-Obeidi et al., “Synthesis and Biological Activities of Fatty Acid Tetrapeptide Ac-His-D-Phe-Arg-Trp-NH at the Mouse Conjugates of a Cyclic Lactam O.-Melanotropin.” J. Med. Chem. Melanocortin Receptors. 4. Modifications at the Trp Position.” J. 1992, 34:118-123. Med. Chem., 2002, 45:5736-5744. Hadley et al., “Biological Activities of Melanotropic Peptide Fatty Holder et al., “Structure-Activity Relationships of the Melanocortin Acid Conjugates.” Pigment Cell Research, 1991, 4:180-185. Tetrapeptide Ac-His-D-Phe-Arg-Trp-NH2 at the Mouse Haskell-Luevano et al., “Characterization of Melanocortin NDP Melanocortic Receptors. 1. Modifications at the His Position.” J. MSHAgonist Peptide Fragments at the Mouse Central and Periph Med. Chem., 2002, 45:2801-2810. eral Melanocortin Receptors.” J. Med. Chem., 2001, 44, 2247-2252. Hruby et al., “Design, Synthesis, and Conformation of Superpotent Haskell-Luevano et al., “Discovery of Prototype Peptidomimetic and Prolonged Acting Melanotropins.” Ann. N.Y. Acad. Sci., 1993, Agonists at the Human Melanocortin Receptors MC1R and MC4R.” 680:51-63. J. Med. Chem., 1997, 40:2133-2139. Jacubovich et al., "Tumour-associated Antigens in Culture Medium Hiltz et al., “Alpha-MSH Peptides Inhibit Acute Inflammation and of Malignant Melanoma Cell Strains.” Cancer Immunol. contact Sensitivity.” Peptides, 1990, 11:979-982. Immunother, 1979, 7:59-64. Koikov et al., “Analogs of sub-nanomolar hMC1R agonist LK-184 Jayawickreme et al., “Discovery and Structure-Function Analysis of Ph(CH2)3CO-His-D-Phe-Arg-Trp-NH2). An additional binding O-Melanocyte-stimulating Hormone Antagonists. J. Biol. Chem. site within the human melanocortin receptor 12'. Bioorganic & Nov. 25, 1994, 269(47), 2.9846-298.54. Medicinal Chemistry Letters, 2004, 14:3997-4000. Lerner et al., “Effect of O- and 3-melanocyte stimulating hormones Koikov et al., "Sub-Nanomolar hMC1RAgonists by End-Capping of on the skin colour of man.” Nature, Jan. 21, 1961, 189: 176-179. Lopez-Fandino et al., “Protease-Catalyzed Synthesis of the Melanocortin Tetrapeptide His-D-Phe-Arg-Trp-NH2.” Oligopeptides in Heterogenous Substrate Mixtures.” Biotechnology Bioorganic & Medicinal Chemistry Letters, 2003, 13:2647-2650. and Bioengineering, 1994, 43:1024-1030. Lipton, James M.. “Modulation of Host Defense by the Neuropeptide Lu et al., “Agouti protein is an antagonist of the melanocyte-stimu C-MSH,”Yale Journal of Biology and Medicine, 1990, 63:173-182. lating-hormone receptor.” Nature, Oct. 27, 1994, 371:799-802. Tatro et al., “Specific Receptors for C-Melanocyte-Stimulating Hor Mountjoy et al., “The Cloning of a Family of Genes That Encode the moneAreWidely Distributed in Tissues of Rodents.” Endocrinology, Melanocortin Receptors.” Science, Aug. 28, 1992, 257:1248-1251. 1987, 121(5): 1900-1907. Nijenhuis et al., “Discovery and in vivo evaluation of new Todorovic et al., “N-Terminal Fatty Acylated His-DPhe-Arg-Trp melanocortin-4 receptor-selective peptides.” Peptides, 2003, 24:271 NH2 Tetrapeptides: Influence of Fatty Acid Chain Length of Potency 280. and Selectivity at the Mouse Melanocortin Receptors and Human Ollmann et al., “Antagonism of Central Melanocortin Receptors in Melanocytes.” J. Med. Chem., 2005, 48:3328-3336. Vitro and in Vivo by Agouti-Related Protein.” Science, Oct. 3, 1997. Yasumura et al., “Establishment of FourFunctional, Clonal Strains of 2.78:135-138. Animal Cells in Culture. Science, 1996, 154: 1186-1189. Quillan et al., “Combinatorial diffusion assay used to identify topi International Search Report issued in application No. PCT/FR2005/ cally active melanocyte-stimulating hormone receptor antagonists.” 001166 on Oct. 10, 2005. Proc. Natl. Acad. Sci. USA, Mar. 1995,92:2894-2898. International Search Report issued in application No. PCT/FR2005/ Shutter et al., “Hypothalamic expression of ART, a novel gene related 001165 on Apr. 21, 2006. to agouti, is up-regulated in obese and diabetic mutant mice.” Genes French Search Report issued in application No. FR 655954 on May & Development, 1997, 11:593-602. 11, 2005. Stverteczky et al., “Synthesis of N-acyl-oligopeptides of potential French Search Report issued in application No. FR 1056469 on Jan. antitubeculotic activity.” Acta Chim. Acad. Sci. Hungaricae, Tomus, 5, 2011. 1975, 87(3), 269-283. Campos et al., “The Conformational Versatility of DNA in the Pres Takahama, Motohide, "O-MSH discovered in primary root of sesame ence of Basic Peptides.” Studia Biophysica, vol. 81, No. 1, pp. 3-14, seeds, and trial on remelanization of gray hairs by their extract: May 1980. immunohistochemical study,” Journal of Dermatological Science, Notice of Allowance issued in U.S. Appl. No. 1 1/596,041 by the Apr. 2004, (34(2), p. 148, XP002302532 & 19' Annual meeting of Examiner on Dec. 17, 2010. the Japanese Society for Investigative Dermatology. Kyoto, Japan, Apr. 14-16, 2004, one page. * cited by examiner US 8,097,590 B2 1. 2 C-MSH-ANTAGONST DIPEPTIDE Therefore, this invention relates to a dipeptide conjugate CONUGATES with general formula I below: This application is a National Stage application of PCT/ FR2005/001164, filed May 10, 2005, which claims priority 5 from French patent applications FR 0405069, filed May 11, in which 2004, and0411279, filed Oct. 22, 2004. The entire contents of A represents the radical corresponding to a monocarboxy each of the aforementioned applications are incorporated lic acid with the following general formula II: herein by reference. This invention relates to new alpha-MSH antagonist dipep 10 HOOC- R II tide conjugates and their use as a medicine or as a depigment in which R represents ing agent. a linear or branched aliphatic radical in C-C optionally Melanocortine receptors belong to the superfamily of Substituted by a hydroxyl group, which can comprise one or seventransmembrane receptors coupled to protein G and they more unsaturations, preferably between 1 and 6 unsatura stimulate the AMPc signal transduction channel (Cone et al. 15 tions, and/or which can comprise a phenyl group, Recent Prog. Horm. Res. 1996, 51, pages 287-317). The melanocortine system is involved in many physiological or lipoic acid or the reduced form thereof, dihydrolipoic channels including pigmentation, inflammation, the erectile acid or N-lipoyllysine. function, food behavior, energy homeostasis, weight homeo AA1 and AA2 represent identical or different amino acids Stasis and the exocrine glands function. The endogenic ago chosen from the group consisting of Ala, ASn, Cys, Gln, Gly, nist ligands for these melanocortine receptors are derived by Ile, Leu, Met, Phe, Pro, Ser. Thr, Trp, Tyr, Val, Asp, Glu, Arg, post-translational modification of the transcript of the proo H is, Lys, Orn, Dap, Dab, the corresponding homo-amino piomelanocortine gene, that during differential treatment acids and the corresponding beta-amino acids, causes generation of C, B and Y hormones stimulating mel in the form of enantiomers or diastereoisomers and mixtures anocytes (MSH) and corticotrophine (ACTH). Subtypes of 25 thereof including racemic mixtures. melanocortine receptors are activated by all endogenic mel Amino acids in the dipeptide conjugate with formula (I) anocortine peptides, except for the melanocortine MC recep may have a D. L. or DL configuration if it is not specified tor that is only stimulated by corticotrophine. The family of otherwise. melanocortine receptors also has two endogenic antagonists, Thus, dipeptide conjugates with formula (I) may comprise namely agouti and protein related to agouti (AGRP) (Lu et al. 30 one or more asymmetric carbon atoms. Therefore, they may Nature 1994, 371, pages 799-802, Ollmann et al., Science exist in the form of enantiomers or diastereoisomers. The 1997, pages 135-138, Shulter et al., Genes Dev. 1997, 11, invention includes these enantiomers, diastereoisomers and pages 593-602) that are the only known antagonists discov mixtures thereof, including racemic mixtures. ered at the moment existing in the natural state of these Within the framework of this invention, the following receptors coupled with protein G. These are polypeptides of 35 abbreviations have the following meanings: 132 and 49 amino acid residues respectively. The most stud Ala, Alanine, ied melanocortine receptor ligands are MC, receptor ligands ASn, Asparagine, of melanocortine of the skin that are involved in pigmentation Cys, Cystein, and coloring of animal hair coat (Hruby et al. Ann. N.Y. Acad. Gln, Glutamine, Sci. 1993, 680, pages 51-63; Lerner et al. Nature 1961, pages 40 Gly, Glycine, 189, 176; Mountjoy et al. Science 1992, 257, pages 1248 Ile, Isoleucine, 1251). Leu, Leucine, Nonapepdide 153 N-6 (Jayawickreme et al., J. Biol. Chem. Met, Methionine, 1994, 269, pages 2.9846-29854) (H-Met-Pro-D-Phe-Arg-d- Phe, Phenylalanine or similar, particularly a halogenated Trp-Phe-Lys-Pro-Val-NH2: Ki-11 nM) is a synthetic antago 45 derivative, and particularly para-fluoro-Phe, Homo-Phe, nist of the receptor MC. However, this compound has a high para-nitro-Phe or phenylglycine molecular weight and therefore a very limited therapeutic or Pro, Proline, cosmetic activity. Its size makes it difficult to optimize and its Ser, Serine, bioavailability is limited. It is also expensive and difficult to Thr, Threonine, prepare. 50 Trp, Tryptophan, Tripeptide D-Trp-Arg-Leu-NH. (Proc. Natl. Acad. Sci. Tyr, Tyrosine, (1995), 92, pages 2894-2898) also has an antagonist activity. Val, Valine, However, it contains tryptophan that is an unstable amino acid Asp, Aspartic acid, and therefore can cause stability problems during storage. Glu, Glutamic acid, Patent EP 1 174437 describes di- or tripeptides comprising 55 Arg, Arginine, a naphthyl group and in particular a naphthylalanyl group. H is, Histidine, However, the presence of the naphthyl group increases the Lys, Lysine, fabrication price of the product. Furthermore in some coun Orn, Ornithine, tries such as Japan, unnatural amino acid based peptides Dap, Diaminopropionic acid, cannot be sold for cosmetic applications. Furthermore, no 60 Dab, Diaminobutyric acid. dipeptide activity is indicated. Note also that the dipeptide conjugates mentioned above Surprisingly, the inventors discovered that dipeptides con according to this invention are obtained in the terminal form jugated at the C-terminal with carboxylic acids have an NH (in other words they present an amide function). antagonist activity of the MSH alpha. These antagonists have Dipeptide conjugates according to this invention are a very low molecular weight and are therefore easy to opti 65 bonded to acid with formula II in the form of salts or esters. mize, they have good bioavailability and are very easy to The conjugations according to this invention may be made by prepare. making the acid function of the amino acid react with the acid US 8,097,590 B2 3 4 function of the formula II acid, or it is even possible to take ing a dipeptide conjugate according to this invention and advantage of the presence of a hydroxyl function on the possibly a cosmetically or pharmaceutically acceptable formula II acid. excipient. This invention relates to all these conjugations and non Dipeptide conjugates can be administered for their cos functional conjugates. Conjugations may be physical or 5 metic orpharmaceutical use by topical route. They can also be chemical. used orally in food Supplements, in other words in the nutra Advantageously, at least one of the amino acids AA2 or ceutical domain. AA1, and advantageously both of them, represent a basic Dipeptide conjugates according to the invention are pref amino acid, advantageously selected from the group consist erably administered topically. ing of Arg, H is, Dap, Dab, Ornor Lys, advantageously it will 10 The cosmetic, pharmaceutical or dermatological composi be Arg. tion according to this invention intended for topical adminis Advantageously, AA2 represents a basic amino acid tration may be presented informs that are normally known for advantageously selected from the group consisting of Arg, H this type of administration, in other words particularly is, Lys, Orn, Dap, Dab, advantageously it is Arg. lotions, foams, gels, dispersions, sprays, serums, masks, body 15 milk, pomades, solutions, emulsions, gels, or creams for Advantageously, AA1 and/or AA2 do not represent Trp. example with excipients particularly for skin penetration in Advantageously, AA1 and/or AA2 do not represent Cys. order to improve the properties and accessibility of the active Advantageously, at least one of the amino acids AA1 or ingredient. These compositions usually also contain the AA2 is selected from the group consisting of Ser and Pro. dipeptide conjugate according to this invention and usually Advantageously AA1 represents Pro. also a physiologically acceptable medium, usually based on Advantageously AA2 represents Ser. water or solvent, for example alcohols, ethers or glycols. Advantageously, the acid with formula (II) is a polyunsatu They can also contain Surface active agents, preservatives, rated fatty acid, in other words it comprises between 1 and 6 stabilizers, emulsifiers, thickeners, other active constituents unsaturations. Even more advantageously, it is an omega-3 leading to a complementary or possibly synergic effect, trace acid. 25 elements, essential oils, perfumes, coloring agents, collagen, Among these omega-3 acids, there is particularly O-lino chemical or mineral filters, moisturizers or thermal water. lenic acid, cervonic acid, timnodonic acid and pinolenic acid. In the composition according to this invention, the dipep Cervonic, timnodonic and pinolenic acids are also known tide conjugate according to the invention may be present at a under the names 4,7,10,13,16,19-docosahexaenoic acid concentration of between 10 M and 10 M, advanta (DHA), 5,8,11,14, 17-eicosapentaenoic acid (EPA) and 5.9, 30 geously between 107M and 10 M. 12-octodecatrienoic acid, respectively. This invention also relates to a dipeptide conjugate accord When A represents a monocarboxylic acid radical with ing to this invention or a pharmaceutical composition accord general formula (II), it may advantageously be selected from ing to this invention for its use as a medicine, advantageously among acetic acid, myristic acid, palmitic acid, hydroxyde designed to prevent, improve or treat immunitary abnormali cenoic and decenoic acid, and particularly trans-10-hydroxy 35 ties, immunodeficiency, to regulate the body weight by con A2-decenoic acid and trans-OXO-9-decene-2-oic acid. trolling the appetite, to treat disorders of the central nervous Advantageously, acid with formula (II) is an acid selected system, to regulate Satiety, to treat anorexia or some skin from among lipoic acid (Lip) or its reduced form dihydroli CaCCS. poic acid, N-lipoyllysine or phenylbutyric acid (Pbu). This invention also relates to the use of a cosmetic compo Advantageously. A represents the radical corresponding to 40 sition according to this invention as a depigmenting agent to palmitic acid (Palm). lighten or whiten the epidermis, to eliminate skin spots, par The dipeptide conjugates of the invention include the ticularly age spots or freckles, or to prevent pigmentation of dipeptide conjugates selected from among the group consist the epidermis. ing of Finally, this invention relates to a cosmetic treatment pro a) A-Arg-His-NH2, 45 cess to lighten, depigment or whiten the epidermis, to elimi b) A-Arg-Arg-NH2, nate skin spots and particularly age spots or freckles, or c) A-Arg-Pro-NH. prevent pigmentation of the epidermis including application d) A-Arg-Lys-NH2, of a cosmetic composition according to this invention to the e) A-Ser-Pro-NH. skin. f) A-DPhe-Arg-NH2, 50 The following examples are given for non-limitative guid in which the definition of A is as given above. aCC. In particular, the dipeptide conjugates in the invention may be selected from among the group consisting of EXAMPLE 1. 39) Palm-Arg-His-NH. 41) Palm-Arg-Arg-NH2, 55 Preparation of 361 Dipeptides According to the 49) Palm-Arg-Pro-NH. Invention 50) Palm-Arg-Lys-NH2, 125) Palm-Ser-Pro-NH. A bank of acylated dipeptides with 361 members was 269) Palm-DPhe-Arg-NH. synthesized using SynPhaseTM Lanterns and a “split and 362) Pbu-DPhe-Arg-NH. 60 pool' strategy for color marking as described in the article by 363) Lip-DPhe-Arg-NH Feliu et al. (J. Comb. Chem., 2003, 5, pages 256-361). Dipeptide conjugates according to this invention may be Thus, these 361 compounds were synthesized on series D obtained either advantageously by classical chemical synthe SynPhaseTM Lanterns with Rink amide PS resin using the sis, or by enzymatic synthesis using any processes known to standard Fmoc (9-fluorenyl-methoxycarbonyl) synthesis those skilled in the art. 65 strategy in the Solid phase using a Multipin 96 arrangement This invention also relates to a cosmetic, dermatological or format. Component blocks AA1 and AA2 were selected from pharmaceutical composition or a food Supplement compris a chemical assembly of 19 D and L amino acids including US 8,097,590 B2 5 6 several types of lateral chains (alkyl, aromatic, acid, Volumi acetonitrile/water (50/50, V/v) containing 0.1% of TFA were nous, basic) to produce 19x19–361 combinations. distributed on the freeze-dried compounds. 10ul of each tube The following chemical products were used: was sampled for analysis of CLHP and CL/SM ESI+. Amino acids protected at the N-terminal end by an The CLHP analyses were carried out on a CLHP Waters C.-Fmoc, Fmoc-Ala-OH, Fmoc-D-Ala-OH, Fmoc-Arg(Pbf)- 5 Alliance 2690 system and a Waters 996 photodiode strip OH, Fmoc-D-Arg-(Pbf)-OH, Fmoc-His(Trt)-OH, Fmoc-D- detector and a 50x4.6 mm Merck Chromolith Speed ROD His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-D- C18 column. A flow rate of 5 ml/min and a gradient from Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-D-Trp(Boc)-OH, 100% of B to 100% of C was used over 3 minutes (Eluant B, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, water/0.1% of TFA: Eluant C, acetronitrile/0.1% of TFA). Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc 10 Estimates of the purity are based on the percent of the area of Pro-OH, or Fmoc-ASn(trt)-OH group, were purchased from peaks detected at 214 nm. SENN chemicals and Advanced Chemtech. The CL/SM system was composed of a Waters Alliance The coupling agent, HBTU (hexafluorophosphate of 2-(1- 2690 CLEHP coupled to a Micromass Platform II spectrometer H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium), WaS (ionization by electronebulization mode: ESI+). All analyses purchased from SENN chemicals. 15 were done using a 2.1x30 mm Waters Symmetry C18 col N,N-dimethylformamide (DMF), dichloromethane, umn, 3.5 um. A 600 ul/min flow rate and a gradient from methanol, acetonitrile, ethyl ether, trifluoroacetic acid (TFA), 100% of B to 100% of Cover 3 minutes were used (Eluant B, piperidine were purchased from Riedel de Haen, Carlo Erba water/0.1% of TFA: Eluant C, acetonitrile/0.1% of TFA). or Acros organics and used without purification. Mass spectra by positive ionic electronebulization were N,N-diisopropylethylamine (DIEA), triisopropyl-silane, acquired at a solvent flow rate of 100 ml/min. Nitrogen was palmitic acid were purchased from Aldrich or Avocado. All used both for the nebulizing gas and for the drying gas. Data reagents and chemical products were of analytic quality and were acquired in read mode m/z, 400 to 1400 at intervals of they were used without any other purification. 0.1; 10 readings were added to produce the final spectrum. D series Synphase Polystyrene Rink amide Lanterns were The molecular weights of all compounds were calculated supplied by Mimotopes, Pty. 25 using mono-isotopic masses (C=12.000, H=1.007. The standard procedure for manufacturing these dipeptides N=14.003, O=15.994, S=31.972). includes the following steps: Table 1 below contains the analysis results. 1—Fmoc Standard Deprotection Protocol Fmoc deprotection steps were done by immersing lanterns TABLE 1 immobilized on a support of 96 rods in a mix of dimethylsul 30 foxide (DMF)/piperidine (80/20, v/v) for 30 minutes. Rect Analytic results of the Palm-dipeptides bank angular polypropylene receptacles of the same size as a stan Dipeptide dard plate with 96 wells were used. The excess deprotection conjugate Solution was simply eliminated by stirring the rod Support number vigorously. 35 according 2—Standard Washing Protocol to the Sequence % Molecular After the coupling or deprotection step, washing steps were invention A AA2 AA1 purity weight carried out by dipping the lanterns arranged in a Multipin 1 Palm His His NH, 97 529.3 format into polypropylene receptacles containing DMF (3x5 2 Palm His Phe NH, OO 539.4 min), methanol (2x5min) and dichloromethane (DCM) (1x5 40 3 Palm His Arg NH, OO 548.4 min), all in sequence. The lanterns were dried in air for 5 4 Palm His Trp NH, 89 S78.4 minutes under a vaporhood after the last washing with DCM. 5 Palm His Glu NH, OO S21.3 6 Palm His Ala NH, OO 463.3 3—Standard Coupling Protocol 7 Palm His Ser NH, OO 479.3 0.4 M solutions of each Fmoc amino acid, HBTU and 8 Palm His Leu NH, OO SOS.4 DIEA were prepared in DMF and were kept at 4°C. through 45 9 Palm His Tyr NH, OO 555.3 10 Palm His Gly NH, 96 449.3 out the synthesis. 200 ul of amino acid solution were distrib 11 Palm His Pro NH, OO 489.3 uted in plates with 96 deep wells. 200ul of DIEA solution and 12 Palm His Lys NH, OO 520.4 200 ul of HBTU solution were then added and finally, the 13 Palm His ASn NH, OO SO6.3 Support of the rods Supporting the lanterns was adapted to the 14 Palm His Met NH, 88 523.3 deep wells plate for 2 hours. 50 15 Palm His DPhe NH 81 539.4 16 Palm His DTrp NH, 88 S78.4 4—Cleavage 17 Palm His DArg NH2 OO 548.4 500 ul of TFA/water/triisopropylsilane (95/2.5/2.5. V/v/v) 18 Palm His DHis NH, 97 529.3 solution was distributed in individual polypropylene tubes 19 Palm His DAla NH, 88 463.3 arranged in Micronic plates with 96 wells. Cleavage was done 2O Palm Phe His NH, OO 539.4 for 3 hours. The cleavage cocktail was concentrated directly 55 from plates using a Jouan RC 1010 vacuum centrifuge. Com 22 Palm Phe Arg NH, OO SS8.4 pounds were precipitated with dry diethyl ether, and were 23 Palm Phe Trp NH, OO S88.4 centrifuged and settled one by one. Precipitation, centrifug 24 Palm Phe Glu NH, OO 531.3 25 Palm Phe Ala NH, OO 473.3 ing and settlement operations were repeated twice. 500 ul of 26 Palm Phe Ser N OO acetonitrile/water (50/50, V/v) containing 0.1% of TFA were 60 distributed in each tube to solubilize the samples. The samples were then frozen at -80° C. and freeze dried. This Palm Phe Tyr NH, OO 565.4 operation was repeated twice to completely eliminate the Palm Phe Gly NH, OO 459.3 Palm Phe Pro NH, 23 499.35 triisopropylsilane purification group. Palm Phe Lys NH, OO S304 5—Preparation and Analysis of Samples 65 Palm Phe ASn NH, 8O S16.3 Complete banks and simple re-synthesized peptides were Palm Phe Met NH, OO 533.3 analyzed by CLHP in inverse phase and CL/SM. 500 ul of US 8,097,590 B2 7 8 TABLE 1-continued TABLE 1-continued Analytic results of the Palm-dipeptides bank Analytic results of the Palm-dipeptides bank Dipeptide Dipeptide conjugate 5 conjugate number number according according to the Sequence % Molecular to the Sequence % Molecular invention A AA2 AA1 purity weight invention A AA2 AA1 purity weight 10 34 OO 549.4 98 Palm Ala Arg NH, OO 482.4 99 Palm Ala Trp NH, 98 S123 & OO Palm Ala Glu NH, OO 455.3 36 Palm Phe DArg OO SS8.4 O1 Palm Ala Ala NH, OO 397.3 37 Palm Phe DHis 96 539.4 O2 Palm Ala Ser NH, OO 41.3.3 38 Palm Phe DAla OO 473.3 15 O3 Palm Ala Leu NH, OO 439.3 39 Palm Arg His 94 548.4 O4 Palm Ala Tyr NH, OO 489.3 40 Palm Arg Phe OO SS8.4 05 Palm Ala Gly NH, OO 383.3 41 Palm Arg Arg OO S67.4 O6 Palm Ala Pro NH, OO 423.3 42 Palm Arg Trp 95 597.4 O7 Palm Ala Lys NH, OO 454.4 43 Palm Arg Glu OO 540.4 O8 Palm Ala ASn NH, OO 440.3 44 Palm Arg Ala 94 482.4 2O 09 Palm Ala Met NH, OO 457.3 45 Palm Arg Ser OO 498.4 10 Palm Ala DPhe NH, OO 473.3 46 Palm Arg Leu OO 524.4 11 Palm Ala DTrp NH, 96 S123 47 Palm Arg Tyr 93 S744 12 Palm Ala DArg NH2 OO 482.4 48 Palm Arg Gly OO 468.3 13 Palm Ala DHis NH, 84 463.3 49 Palm Arg Pro 87 SO8.4 14 Palm Ala DAla NH, OO 397.3 50 Palm Arg Lys OO 539.4 15 Palm Ser His NH, 95 479.3 51 Palm Arg ASn 91 S25.4 25 16 Palm Ser Phe NH, OO 489.3 52 Palm Arg Met OO 542.4 17 Palm Ser Arg NH, OO 498.4 53 Palm Arg DPhe OO SS8.4 18 Palm Ser Trp NH, 96 S28.3 S4 Palm Arg DTrp 60 597.4 19 Palm Ser Glu NH, OO 471.3 55 Palm Arg DArg OO S67.4 2O Palm Ser Ala NH, OO 41.3.3 56 Palm Arg DHis OO 548.4 21 Palm Ser Ser NH, OO 429.3 57 Palm Arg DAla OO 482.4 30 22 Palm Ser Leu NH, OO 455.3 58 Palm Trip His 93 S78.4 23 Palm Ser Tyr NH, 97 505.3 59 Palm Trip Phe OO S88.4 24 Palm Ser Gly NH OO 399.3 60 Palm Trip Arg OO 597.4 25 Palm Ser Pro NH, OO 439.3 & 26 Palm Ser Lys NH, OO 470.4 27 Palm Ser ASn NH, 32 456.3 35 28 Palm Ser Met NH, 50 473.3 29 Palm Ser DPhe NH, OO 489.3 30 Palm Ser DTrp NH, 93 S28.3 31 Palm Ser DArg NH, O 498.4 32 Palm Ser DHis NH, 91 479.3 l 33 Palm Ser DAla NH, OO 41.3.3 l 40 34 Palm Cl His NH, 92 SOS.4 l 35 Palm Cl Phe NH, OO S15.4 l 36 Palm Cl Arg NH, OO 524.4 l 37 Palm Cl Trp NH, OO SS4.4 l 38 Palm Cl Glu NH, OO 4974 39 Palm Cl Ala NH, OO 439.3 XXYY YY 3 40 Palm Cl Ser NH, OO 455.3 73 Palm Trip DTrp NH 76 627.4 45 41 Palm Cl Leu NH, OO 4814 42 Palm Cl Tyr NH, OO 531.4 74 Palm DArg NH, 93 597.4 43 Palm el Gly NH OO 425.3 75 Palm Trip DHis NH, 79 S78.4 2 76 Palm Trip DAla NH, OO S12.3 77 Palm Glu His NH, 94 S21.3 78 Palm Glu Phe NH OO 531.3 45 Palm Leu Lys NH, OO 496.4 79 Palm Glu Arg NH, OO 540.4 50 46 Palm Cl ASn NH, 15 482.4 8O Palm Glu Trp NH, 82 570.3 47 can Cl Met NH, OO 499.3 81 Palm Glu Glu NH, OO S13.3 48 8 Cl DPhe NH, OO S15.4 82 Palm Glu Ala NH OO 455.3 49 Palm Leu DTrp NH 87 SS4.4 83 Palm Glu Ser NH, OO 471.3 50 Palm Leu DArg NH, OO 524.4 84 Palm Glu Lll NH, OO 497.4 51 Palm Cl DHis NH, 86 SOS.4 85 Palm Glu Tyr NH, OO 547.3 55 52 Palm Cl DAla NH, OO 439.3 86 Palm Glu Gly NH, OO 441.3 53 Palm Tyr His NH, 87 Palm Glu Pro NH, 10 481.32 88 Palm Glu LyS NH, OO 512.4 89 Palm Glu ASn NH, 7 498.31 55 Palm Tyr Arg NH, 90 Palm Glu Met NH, OO 515.3 56 Palm Tyr Trp NH, 604.4 91 Palm Glu DPhe NH, OO 531.3 60 57 Palm Tyr Glu NH, 100 547.3 58 Palm Tyr Ala NH, 100 489.3 59 Palm Tyr Ser NH, 100 505.3 93 Palm Glu DArg NH, OO 540.4 60 Palm Tyr Leu NH, 100 531.4 94 Palm Glu DHis NH, OO S21.3 61 Palm Tyr Tyr NH, 98 5814 95 Palm Glu DAla NH, OO 455.3 62 Palm Tyr Gly NH, 100 475.3 96 Palm Ala His NH, 97 463.3 65 63 Palm Tyr Pro NH, 93 515.3 97 Palm Ala Phe NH, 94 473.3 64 Palm Tyr Lys NH, 100 546.4 US 8,097,590 B2

TABLE 1-continued TABLE 1-continued Analytic results of the Palm-dipeptides bank Analytic results of the Palm-dipeptides bank Dipeptide Dipeptide conjugate 5 conjugate number number according according to the Sequence % Molecular to the Sequence % Molecular invention A AA2 AA1 weight invention A AA2 AA1 purity weight 10 65 Palm Tyr ASn 225 Palm Lys DTrp NH, 92 569.40 226 Palm Lys DArg NH- 94 539.42 M 227 Palm Lys DHis NH, 94 S2O38 Palm Tyr DPhe 228 Palm Lys DAla NH, 83 454.36 229 Palm ASn His NH, 28 SO6.33 XXYY 15 Palm Tyr DArg NH, Palm Tyr DHis NH,

Palm Tyr DAla NH, Palm Gly His NH, Palm Gly Phe NH, Palm Gly Arg NH, 2O Palm Gly Trp NH, Palm Gly Glu NH, Palm Gly Ala NH, Palm Gly Ser NH,

Palm Gly Tyr NH, 25 Palm Gly Gly NH, OO 369.3 Palm Gly Pro NH, OO 409.3 Palm Gly Lys NH, 81 440.3 Palm Gly ASn NH, 57 426.3 Palm Gly Met NH, OO 443.3 Palm Gly DPhe NH OO 459.3 30 Palm Gly DTrp NH, 68 498.3 Palm Gly DArg NH- OO 468.3 Palm Gly DHis NH, 90 449.3 Palm Gly DAla NH, OO 383.3 C N E. E. NE 97 489.34 35 247 Palm ASn DAla NH2 2 248 Palm Me His NH, 85 523.33 249 Palm Me Phe NH, 84 533.33 250 Palm Me Arg NH, 76 S42.37 251 Palm Me Trp NH, 85 572.34 252 Palm Me Glu NH, 87 515.31 253 Palm Me Ala NH, 99 457.31 40 254 Palm Me Ser NH, 55 473.30 255 Palm Me Leu NH, 78 499.35 Y 256 Palm Me Tyr NH, 8O 549.33 197 Palm Pro Ser NH, 257 Palm Me Gly NH, 63 443.29 198 Palm Pro Leu NH, 258 Palm Me Pro NH, 85 483.32 199 Palm Pro Tyr NH, 259 Palm Me Lys NH, 88 S14.36 2OO Palm Pro Gly NH, 45 260 Palm Me ASn NH, 92 SOO.31 2O1 Palm Pro Pro NH, 261 Palm Me Met NH, 85 517.31 2O2 Palm Pro Lys NH, 262 Palm Me DPhe NH, 91 533.33 2O3 Palm Pro ASn NH, 263 Palm Me DTrp NH, 78 572.34 204 Palm Pro Met NH, 264 Palm Me DArg NH, 57 S42.37 205 Palm Pro DPhe NH, 26S Palm Me DHis NH, 86 523.33 l NH, 50 266 Palm Me DAla NH, 31 457.31 l NH, 267 Palm DPhe His NH, 95 539.35 l NH, 268 Palm DPhe Ple NH, 76 549.36 l NH, 269 Palm DPhe Arg NH, 85 558.39 l NH, 270 Palm DPhe Trp NH, 70 588.37 271 Palm DPhe Glu NH, 33 531.34 YYYY 55 272 Palm DPhe Ala NH, 97 473.33 212 Palm N 273 Palm DPhe Ser NH, 83 489.33 213 Palm N 274 Palm DPhe Leu NH, 79 515.38 214 Palm N 275 Palm DPhe Tyr NH, 93 S65.36 215 Palm N 276 Palm DPhe Gly NH, 88 459.32 216 Palm N 277 Palm DPhe Pro NH, 50 499.35 217 Palm N 278 Palm DPhe Lys NH, 65 S30.39 218 Palm N 60 279 Palm DPhe ASn NH, 77 S16.34 219 Palm N 28O Palm DPhe Met NH, 97 533.33 220 Palm N 281 Palm DPhe DPhe NH 76 549.36 Palm N 282 Palm DPhe DTrp NH, 87 588.37 283 Palm DPhe DArg NH- 92 558.39 284 Palm DPhe DHis NH, 96 539.35 223 2 65 285 Palm DPhe DAla NH 78 473.33 224 LyS 2 286 Palm DTrp His NH, 69 578.36 US 8,097,590 B2

TABLE 1-continued TABLE 1-continued Analytic results of the Palm-dipeptides bank Analytic results of the Palm-dipeptides bank Dipeptide Dipeptide conjugate 5 conjugate number number according according to the Sequence % Molecular to the Sequence % Molecular invention A AA2 AA1 weight invention A AA2 AA1 purity weight 10 287 Palm DTrb Phe NH, 578.36 348 Palm DAla Ala NH, 53 455.31 288 Palm DTrb Arg NH, 588.37 349 Palm DAla Ser NH, 11 397.30 289 Palm DTrb Trp NH, 597.40 350 Palm DAla Leu NH, 78 413.30 290 Palm DTrb Glu NH, 627.38 351 Palm DAla Tyr NH, 439.35 291 Palm DT Ala NH, 570.35

& t 15 353 Palm DAla Pro NH, Palm DTrb Leu NH, S28.34 3S4 Palm DAla Lys NH, Palm DTrb Tyr NH, SS439 355 Palm DAla Asn NH, Palm DTrb Gly NH, 604.37 356 Palm DAla Met NH, Palm DTrb Pro NH, 72 498.33 357 Palm DAla DPhe NH, Palm DTrb Lys NH, 96 S38.36 2O 358 Palm DAla DTrp Palm DTrb ASn NH, 84 569.40 359 Palm DAla DArg Palm DTrb Met NH, 60 555.35 360 Palm DAla DHis Palm DTrb DPhe NH 70 572.34 Palm DTrb DTrp NH, 52 588.37 Palm DTrb DArg NH- 86 627.38 l DT DHis NH, 95 597.40 8 25 Except for 35 members of the bank (shown in grey), all compounds were detected by a CL/SM ES+ analysis. The average purity determined based on the percent of the area of Palm DArg Phe NH, 42 S48.3 the expected peak detected at 214 nm on the chromatogram is Palm DArg Arg NH, 60 558.39 greater than 83%. Palm DArg Trp NH, 42 567.42 30 Palm DArg Glu NH, 73 597.40 Palm DArg Ala NH 82 540.37 EXAMPLE 2 Palm DArg Ser NH, 74 482.36 Palm DArg Leu NH, 34 498.36 Biological Properties of Five Dipeptide Conjugates Palm DArg Tyr NH, 27 524.41 A ding to this I ti Palm DArg Gly NH, 32 574.39 35 ccord1ng to un1S Invenuon Palm DArg Pro NH, 40 468.35 Experiments on inhibition of AMPc production were car ried out on the M4Behuman cell line with a concentration of 318317 Palm DA.DAir ASnMet NHN 9 5x10M of C-MSH. Five Palm-dipeptides according to this 319 Palm DArg DPhe NH 82 40 invention were introduced at different concentrations and 32O Palm DArg DTrp NH 30 each measurement was made in triplicate. Two or three series 321 Palm DArg DArg NH2 2O f iment d 322 Palm DArg DHis NH2 70 oI experiments were made. 323 Palm DArg DAla NH2 83 The tests were made as follows: 324 Palm DHis His NH, 75 The M4Be human cell line (Jacubovich et al. Cancer 325 C alm DHis Phe NH, 66 45 Immunol. Immunother. 1979, 7, 59-64), a melanocytes cell 327326 Palm DHisDHis TrpArg NH, 53 539.35 li1ne capableble of producingproduci melanines,lani was usedd inin this studystud to determine the values of CIso X The cells were maintained in the Dulbecco modified Eagle 329 Palm DHis Ala NH, 63 S21.33 medium with 10% of foetal calf serum (FCS), 1 mM of 3. t Ris St. NE 3. is: 50 glutamine, 100 U/ml of penicillin and 10 g/ml of strepto 332 Palm DHis Tyr NH, 79 505.37 myc1ne. 333 Palm DHis Gly NH, 89 555.35 All cell lines were maintained at 37°C. in an atmosphere 3. Palm PHis Pro NH, 96 449.31 with 5% of CO and cell culture media were renewed every S. N Ris R N: s two days. The cells were applied in contact with a plate with 337 Palm DHis Met NH, 76 so.33 55 96 wells (Nunc, Roskilde) 24 hours before contact of dipep 338 Palm DHis DPhe NH, 96 523.33 tides according to the invention.

339 Palm DHis DTrp NH, 66 539.35 AMPc was measured as follows: & Cells applied in contact the day before with 8x10" cells per well were put in the presence of one of the five dipeptide 60 conjugates according to the invention at various concentra tions for 10 minutes at 37°C. with 5x10 Mofo-MSH. After this time, the lysis of the cells was made and the AMPc content was measured using a connection test box by compe tition (RPN225, Amersham Pharmacia Biotech). Each inde 65 pendent experiment was carried out at least twice in triplicate. The peptidic activity was determined with reference to the AMPc content synthesized by untreated cells and the produc US 8,097,590 B2 13 14 tion of AMPc induced by C.-MSH alone. The curves were AA1 and AA2 represent identical or different amino acids adjusted and the values of CIs were determined with non- Selected from the group consisting of Pro, Arg, His, LyS, linear regression in the GraphPad Prism (GraphPad software, the corresponding homo-amino acids, and the corre San Diego, Calif., United States). sponding beta-amino acids, Table 2 below contains the results. 5 in the form of enantiomers or diastereoisomers and mix tures thereof including racemic mixtures, TABLE 2 with the proviso that at least one of the amino acids AA2 or AA1 is Arg and with the exception of the dipeptide Experiments on inhibition of AMPc production on M4Be conjugates Palm-Orn-Arg-NH and Palm-Arg-Arg cells 10 NH. CISO CISO CISO CISO 2. Dipeptide conjugate according to claim 1, wherein AA2 Purity (IM) (M) (IM) (M) represents a basic amino acid. Compound Sequence (%) expl exp2 exp3 aver. 3. Dipeptide conjugate according to claim 1 wherein it is 39 Palm-Arg-His-NH2 94 29 4.6 4.4 13 selected from among the group consisting of: 41 Palm-Arg-Arg-NH2 100 36 17 – 26 15 a) A-Arg-His-NH2, : ENEN . . . . b) A-Arg-Pro-NH, and 125 RSS 100 45 5.5 1.0 17 c) A-Arg-Lys-NH2, in which the definition of A is as given in claim 1. 4. Dipeptide conjugate according to claim 1, wherein it is Thus, unexpectedly, the tested Palm-dipeptides appeared 20 selected from among the group consisting of to be antagonists of the human MC receptor of melanocort- 39) Palm-Arg-His-NH. ine using M4Be melanoma cell lines. These dipeptide conju- 49) Palm-Arg-Pro-NH2, and gates have a CIso within a micromolar range. These dipep- 50) Palm-Arg-Lys-NH. tides are the first example of short synthesis antagonist 5. Cosmetic or pharmaceutical composition comprising a compounds binding to the MC receptor and they open up the 25 dipeptide conjugate according to claim 1, and possibly a field for small molecule non-peptidic antagonists of C-MSH. cosmetically or pharmaceutically acceptable excipient. In particular, these compounds comprise an arginine resi- 6. Method for lightening or whitening the epidermis, for due in position AA2. Interestingly, compound 125 (Palm-Ser- eliminating skin spots, or for preventing pigmentation of the Pro-NH2) does not have any basic residue in its sequence, and epidermis comprising the administration of an effective has a value of Clso equal to 174M. This result showed that the 30 amount of a cosmetic composition according to claim 5 or a bond to the MC, receptor does not necessarily require a posi- cosmetic composition comprising the dipeptide Palm-Arg tively charged radical. Arg-NH2 to a patient in need thereof. With only two amino acid residues, these palmitoyled com- 7. Method according to claim 6, wherein the cosmetic pounds can be considered as being leader compounds useful composition is applied onto the skin. for design of non-peptide antagonists of C-MSH. 35 8. Dipeptide conjugate according to claim 2, wherein AA2 The invention claimed is: is Arg. 1. Dipeptide conjugate with general formula I below: 9. Dipeptide conjugate according to claim 6, wherein the A-AA2-AA1-NH2, I skin spots are age spots or freckles. in which A represents the radical corresponding to palmitic acid, k . . . .