Melanocortin-1-receptor agonists

Abstract

The present invention relates to novel selective melanocortin-1 receptor (MC1R) agonists as well as the use thereof as skin tanning agents.

Claims

1. A compound or a cosmetically acceptable salt thereof, wherein the compound is selected from the group consisting of: ##STR00015## ##STR00016## ##STR00017##

2. The compound according to claim 1, wherein the cosmetically acceptable salt is an acetate or a trifluoroacetate thereof.

3. A cosmetic composition comprising the compound of formulas (I-a) through (I-i) according to claim 1 or the cosmetically acceptable salt thereof and a cosmetically acceptable carrier.

4. The cosmetic composition according to claim 3, wherein the compound of formulas (I-a) through (I-i) or the cosmetically acceptable salt thereof is present in a total amount of about 0.00001 to 0.5 wt. %, based on the total weight of the cosmetic composition.

5. The cosmetic composition according to claim 3, wherein the compound of formulas (I-a) through (I-i) or the cosmetically acceptable salt thereof is present in a total amount of about 0.0001 to 0.25 wt. %, based on the total weight of the cosmetic composition.

6. The cosmetic composition according to claim 3, wherein the compound of formulas (I-a) through (I-i) or the cosmetically acceptable salt thereof is present in a total amount of about 0.0001 to 0.1 wt. %, based on the total weight of the cosmetic composition.

7. The cosmetic composition according to claim 3, wherein the cosmetic composition comprises at least one further ingredient selected from the group consisting of self-tanning agents, UV-filters, agents for the treatment of hyperpigmentation, agents for the prevention or reduction of inflammation, firming agents, moisturizing agents, soothing agents, energizing agents, agents to improve elasticity and agents to improve skin barrier properties.

8. The cosmetic composition according to claim 3, wherein the cosmetic composition comprises at least one further ingredient selected from the group consisting of polysilicones-15, phenylbenzimidazol sulfonic acid, 3-benzylidene camphor, octocrylene, ethylhexyl methoxycinnamate, ethyl hexylsalicylate, homosalate, zinc oxide, bis-ethylhexyloxyphenol methoxyphenyl triazine, methylene bis-benzotriazolyl tetramethylbutylphenol, titanium dioxide, butyl methoxydibenzoylmethane, erythrulose, potassium cetyl phosphate, tocopherol and tocopherol acetate.

9. A self-tanning or artificial/sunless tanning agent which comprises the compound of formulas (I-a) through (I-i) according to claim 1 or a cosmetically acceptable salt thereof.

10. A method for artificial/sunless tanning of human skin, enhancement of the natural glow of the skin, protection of skin against UV-radiation and/or prevention of photoage-induced skin structure defects, wherein the method comprises topically applying the cosmetic composition according to claim 3 to the skin of a subject in need thereof.

Description

EXPERIMENTAL PART

General Information

Abbreviations

(1) AA amino acid Ad adamantyl Boc tert-butyloxycarbonyl DCM dichloromethane DIPEA N,N-diisopropylethylamine DMAP N,N-dimethylaminopyridine DMF dimethylformamide Fmoc fluorenylmethoxycarbonyl HPLC High Pressure Liquid Chromatography IndEt 2-(1H-indol-3-yl)ethyl- MeCN acetonitril Naphala naphtylalanine Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl Phe Phenylalanine Pro proline TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborat TCTU 2-(2-Pyridon-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TFA trifluoroacetic acid TIPS triisopropylsilane Trp Tryptophan

(2) Preparative HPLC purifications: performed on a Waters High Performance Liquid Chromatography LC-2525 equipped with a Waters 2767 Sample Manager and a Waters FCII automated fraction collector, using a Grom Saphir 110 C18 10 μm 50×300 mm.sup.2 preparative column and a Waters 2487 double wavelength UV-Vis detector operating at 220 and 254 nm.

(3) H.sub.2O+0.07% TFA (A″ phase) and MeCN+0.07% TFA (B″ phase) were used as eluents, with a flow of 55 mL/min.

(4) 1. Synthetic Strategies

(5) Unsubstituted amides were prepared on rink linker amide resin using solid phase synthesis approach. After simultaneous cleavage of the side chain protecting groups as well as the attachment to the resin, the crude peptide is purified by preparative HPLC.

(6) Substituted amides were prepared on 2-chloro trityl resin side as chain protected peptides with free acid and coupled with the corresponding amine to give the side chain protected test item in solution, which is deprotected and thoroughly purified by HPLC.

(7) a. Preparation

(8) Typical Procedure for the Preparation of the Free Amides

(9) Approximately 2.5 g Fmoc-ramage-resin (loading approx. 0.5 mmol/g) is placed in a peptide synthesizer reaction tube and the sequence is assembled on a peptide synthesizer. 1.25 eq of the respective Fmoc-amino acids (side-chain functional groups may be Boc/Pbf/Trt) protected if present) are coupled to the growing peptide chain with 1.25 eq TBTU and 3 eq DIPEA. Fmoc protection group is removed with 4 methyl piperidine. The N-terminal Bz group is attached using 1.1 eq benzoyl chloride with a 5-fold excess of DIPEA.

(10) The fully assembled peptide is cleaved from the resin with 25.8 ml of a TFA/TIPS/DCM=22.5/0.8/2.5 mixture (v/v). The crude peptide is precipitated by adding the solution to a 200 ml of IPE/Hexan=1/1 (v/v). The precipitate is directly purified by preparative HPLC resulting in the yields as given in table 2.

(11) TABLE-US-00002 TABLE 2 Free amide peptides Entry Sequence Amount, yield I-a Bz-Gly-His-D-Phe-Arg-Trp-NH.sub.2 *2TFA 196 mg (19%) I-b Bz-Gly-His-D-Phe-Arg-D-2-NaphAla-NH.sub.2 *2TFA 351 mg (32%) I-c Bz-Gly-His-D-Phe-Arg-L-2-NaphAla-NH.sub.2 *2TFA 413 mg (39%) I-g Bz-Gly-His-D-Phe-Arg-D-Phe-NH.sub.2 *2TFA 218 mg (22%) I-h Bz-Gly-His-D-Phe-Dab-Trp-NH.sub.2 *2TFA 170 mg (14%) I-i Bz-Gly-His-D-Phe-Arg-(N-IndEt)Gly-NH.sub.2 *2TFA 135 mg, (13%)
Typical Procedure for the Preparation of the Substituted Amides

(12) Approximately 2 g 2-chloro-trityl-resin (loaded with the first amino acid approx. 0.5 mmol/g) is placed in a peptide synthesizer reaction tube and the sequence is assembled on a peptide synthesizer. 1.25 eq of the respective Fmoc-amino acids (side-chain functional groups are Boc/Pbf/Trt) protected if present) are coupled to the growing peptide chain with 1.25 eq TBTU and 3 eq DIPEA. Fmoc protection group is removed with 4 methyl piperidine. The N-terminal Bz group is attached using 1.1 eq benzoyl chloride with a 5-fold excess of DIPEA.

(13) The fully assembled peptide is cleaved from the resin with three times 20 ml of DCM containing 0.1% TFA. The combined DCM portions are combined in a separatory funnel and washed neutral. Organic phase is dried over Na.sub.2SO.sub.4 and all volatile compounds removed in vacuum. Crude peptide is carefully coupled using 3 eq of 2,4,6-trimethyl-pyridine 1 eq of TPTU and 1.1 eq amine at 0° C. Regular aqueous workup (NaHCO.sub.3, KHSO.sub.4, NaCl) is followed by removal of all side chain protecting groups with TFA/TIPS/DCM=22.5/0.8/2.5 mixture (v/v) and precipitation by adding the solution to IPE/Hexan=1/1 (v/v). The precipitate is directly purified by preparative HPLC resulting in the yields as given in table 3.

(14) TABLE-US-00003 TABLE 3 Substituted amide peptides Entry Sequence Amount, yield I-d Bz-Gly-His-D-Phe-Arg-D-Trp-N(Propyl).sub.2 *2TFA 131 mg (12%) I-e Bz-Gly-His-D-Phe-Arg-Trp-N(Propyl).sub.2 *2TFA 196 mg (36%) I-f Bz-Gly-His-D-Phe-Arg-Trp-NHOctyl *2TFA 278 mg (28%) R1 Bz-Gly-D-His-D-Phe-Arg-Trp-N(Propyl).sub.2 *2TFA 113 mg (10%) R2 Bz-Gly-His-Phe-Arg-Trp-N(Propyl)2 *2TFA 176 mg (15%) R3 Bz-Gly-His-D-Phe-D-Arg-Trp-N(Propyl).sub.2 *2TFA 214 mg (18%) R4 Bz-Gly-His-D-Phe-Arg-Tryptamide *2TFA 40 mg (4%)

(15) 3. MC1R Stimulation

(16) Human MC1 Receptor Using cAMP HTRF Functional Assay:

(17) Twenty seven (27) compounds were tested for agonist activity at the human MC1 receptor using cAMP HTRF (homogeneous time-resolved fluorescence) assays at eight (8) concentrations and in duplicate.

(18) On the day of experimentation, compounds were tested at several concentrations in duplicate wells (n=2) to obtain a dose-response curve and an estimated EC50 value. Values obtained for the reference compound were compared to historical values obtained from the same receptor and used to validate the experimental session. For replicate determinations, the maximum variability tolerated in the test was of +/−20% around the average of the replicates.

(19) Assay: After cultivation Chinese Hamster Ovary (CHO) cells were detached and incubated at a specific cell density in assay buffer. At this stage the CHO cells express the human MC1R at their surface. Agonist compounds were added at determined concentration and incubated for 30 min. Cells were lysed in detection buffer in order to extract cAMP. The amount of cAMP within the cells correlates with GPCR-activity, meaning that in this assay it correlates with MC1R-activity. cAMP was detected in a competitive HTRF assay for binding to an anti-cAMP-antibody.

(20) TABLE-US-00004 TABLE 4 Results of the MC1R stimulation assay # Name EC50 [nM] (I-a) Bz-Gly-His-D-Phe-Arg-Trp-NH.sub.2 *2TFA 0.041 (I-c) Bz-Gly-His-D-Phe-Arg-L-2-NaphAla-NH.sub.2 *2TFA 0.11 (I-b) Bz-Gly-His-D-Phe-Arg-D-2-NaphAla-NH.sub.2 *2TFA 0.25 (I-d) Bz-Gly-His-D-Phe-Arg-D-Trp-N(Propyl).sub.2 *2TFA 0.42 (I-e) Bz-Gly-His-D-Phe-Arg-Trp-N(Propyl).sub.2 *2TFA 0.88 (I-f) Bz-Gly-His-D-Phe-Arg-Trp-NHOctyl *2TFA 1.06 (I-g) Bz-Gly-His-D-Phe-Arg-D-Phe-NH.sub.2 *2TFA 1.79 (I-h) Bz-Gly-His-D-Phe-Dab-Trp-NH.sub.2 *2TFA 7.51 (I-i) Bz-Gly-His-D-Phe-Arg-(N-Trp)Gly-NH.sub.2 *2TFA 4.4

REFERENCES

(21) As references, various analogues of compounds formula (I) as outlined in table 3 have been prepared tested in the assay. As can be retrieved from table 5, these compounds showed significant lower activity.

(22) TABLE-US-00005 TABLE 5 # Name EC50 [nM] R1 Bz-Gly-D-His-D-Phe-Arg-Trp-N(Propyl).sub.2 *2TFA 36 R2 Bz-Gly-His-Phe-Arg-Trp-N(Propyl).sub.2 *2TFA 51.4 R3 Bz-Gly-His-D-Phe-D-Arg-Trp-N(Propyl).sub.2 *2TFA 70.1 R4 Bz-Gly-His-D-Phe-Arg-Tryptamide *2TFA 42.9

(23) 4. Ex-Vivo Skin Pigmentation Trial

(24) Skin samples (from abdominal plastic surgery) have been cut in pieces of approx. 8×3 mm (Øx thickness) and were cultured up to day 6 in an air-liquid interface in a perforated ring of stainless steel in contact with a culture medium (modified Williams'E medium), the culture medium has been renewed every three days. Six skin specimens have been used for each treatment. Each test sample (4 μl/200 μM of active) has been applied topically on top of each piece after gentle cleaning of the surface with a cotton pad followed by covering with a 6 ø mm delivery membrane, which procedure has been repeated daily. After 6 days of incubation, the melanin amount was assessed on twelve skin sections for each treatment by staining using a modified Fontana-Masson stain. The amount of melanin present in each slide has been evaluated by estimating the intensity and the distribution of grey tone: 8-bit grey-scale images for each treatment have been captured at the microscope. Then the colour space of the images have been transformed from RGB into L*a*b* images and each pixel of the picture has been evaluated according to its L* values. The obtained results have been transformed in ranks of L* and then normalized on the ratio between the selected area and the area of the slide. The result is indicated as % increase versus untreated at day 6.

(25) TABLE-US-00006 TABLE 5a skin pigmentation increase versus untreated p- # Name increase SEM.sup.+ value* (I-a) Bz-Gly-His-D-Phe-Arg-Trp-NH.sub.2 *2TFA +18% 3% <0.01 (I-d) Bz-Gly-His-D-Phe-Arg-D-Trp-N(Propyl).sub.2 +21% 6% <0.05 *2TFA .sup.+standard error of the mean *calculated by Student’s t-test for unpaired samples
Cosmetic Composition

(26) Table 6 outlines exemplary O/W emulsions, wherein one compound selected from the group of (l-h) as outlined in table 1, is incorporated in the indicated amount.

(27) TABLE-US-00007 TABLE 6 Exemplary O/W emulsion O/W Emulsions 1 2 3 4 5 6 7 8 Glyceryl Stearate 2.5 2 1.2 1 1 1  PEG-40 Stearate 1 PEG-100 Stearate 2.5 1 Ceteareth-20 1 Glyceryl Stearate Citrate 0.5 Potassium Cetyl Phosphate 3 1.5 Stearic Acid 2.5 3 Cetearyl Alcohol 4 2 2 Stearyl Alcohol 2 1 Cetyl Alcohol 1 1 0.5 Acrylates/C.sub.10-30 Alkyl Acrylate 0.2 0.2 0.4 0.2 Crosspolymer Carbomer 0.1 0.2 Xanthan Gum 0.3 0.3 C.sub.12-15 Alkyl Benzoate 5 2 5 5 10 5 Petrolatum 5 3 Butylene Glycol Dicaprylate/Dicaprate 4 2 9 9 Hydrogenated Polydecene 3 2 2 Caprylic/Capric Triglyceride 1 3 5 5 5 Cyclomethicone 5 2 10 Methylpropanediol 2 3 3 Glycerine 4 7 3 4 3 5 3 Glyceryl Glucoside 3.5 3 1 1 2 2 Alcohol denat. 1 3 0.5 10 4 8 4 Butylene Glycol 3 Ascorbylglucoside 0.5 1.0 1.5 0.1 Ubiquinone (Coenzyme 10) 0.1 0.05 0.01 Hyaluronic acid 0.2 Bisabolol 0.5 0.2 Isotridecylsalicylate 1 3 5 2 3 5 Compound selected from the group of 0.001 0.25 0.0001 0.05 0.1 0.0003 0.03 0.002 (I-a) to (I-i) Dibutyl Adipate 1.5 3 Diisopropyl sebacate 1 1 2 3 Ethylhexyl Benzoate 0.75 1.5 1 Titanium Dioxide (PARSOL TX) 0.5 2 Methylene Bis-Benztriazoyl 0.5 4 6 2 Tetramethylbutylphenol Ethylhexyl methoxycinnamate 2 Phenylbenzimidazole Sulfonic Acid 2 2 2 Butyl Methoxydibenzoylmethane 1 2 2 3 3 3 Methylbenzylidene Camphor 2 3 Octocrylene 5 2 10 Polysilicone-15 2 3 Ethylhexyl Salicylate 5 Homosalate 4 2 Bis-Ethylhexyloxyphenol 1.5 2 Methoxyphenyltriazine Silica 1 2.5 0.5 Silica & Methicone 4 1 2.5 Methyl Methacrylate Crosspolymer 1 2 Disodium EDTA 0.1 0.5 Fragrance, Preservatives q.s. Sodium Hydroxide q.s. Water Ad 100