ANTIMICROBIAL TETRAPEPTIDES

Abstract

The present invention relates to novel tetrapeptides as well as the use thereof as antimicrobial agents.

Claims

1. A compound of formula (I) ##STR00016## wherein R.sup.1 is selected from H, a C.sub.1-C.sub.6alkyl group or a C.sub.1-C.sub.6alkoxy group, R.sup.2 is an amino acid side chain of a basic amino acid, R.sup.3 is an arylC.sub.1-C.sub.6alkyl group or a heteroarylC.sub.1-C.sub.6alkyl group, R.sup.4 and R.sup.5 are, independently of each other H, an arylC.sub.1-C.sub.6alkyl group or a C.sub.1-C.sub.10alkyl group, wherein the alkyl group is optionally substituted with up to three hydroxy groups, and n is an integer selected from 0 to 3, with the proviso that if R.sup.2 is the amino acid side chain of arginine and R.sup.3 is (1H-indol-3-yl)methyl then (1) if R.sup.4 and R.sup.5 are H and n is an integer from 0 to 3, then R.sup.1 is not H, or (2) if R.sup.4 and R.sup.5 are H and n is 0, then R.sup.1 is not butoxy, or (3) if R.sup.1 and R.sup.4 are H and n is 3, then R.sup.5 is not methyl or ethyl, or a cosmetically acceptable salt thereof.

2. The compound according to claim 1, which is a compound of formula (I-A) ##STR00017##

3. The compound according to claim 1, wherein R.sup.1 is selected from H, C.sub.1-C.sub.2alkyl group or a C.sub.1-C.sub.2alkoxy group, preferably from H or methoxy.

4. The compound according to claim 1, wherein R.sup.2 is the amino acid side chain of arginine or diaminobutyric acid.

5. The compound according to claim 1, wherein R.sup.3 is an aryl(m)ethyl group or an heteroaryl(m)ethyl group, preferably phenyl(m)ethyl, naphthyl(m)ethyl or (1H-indol-3-yl)(m)ethyl.

6. The compound according to claim 1, wherein R.sup.4 and R.sup.5 are independently of each other selected from the group of H, benzyl and an unbranched C.sub.1-C.sub.10alkyl group, wherein the alkyl group may be substituted with up to two hydroxyl groups, preferably from the group of H, benzyl, propyl, butyl, octyl and 2,3-hydroxypropyl.

7. The compound according to claim 1, wherein n is 2 or 3, preferably 3.

8. The compound of formula (I) according to claim 1, which is a compound of formula ##STR00018## ##STR00019## or a cosmetically acceptable salt thereof.

9. A cosmetic or pharmaceutical composition comprising at least one compound according to claim 1 and a cosmetically acceptable carrier.

10. The cosmetic or pharmaceutical composition according to claim 9, wherein the total amount of the at least one compound of formula (I) is selected in the range of about 0.00001 to 0.5 wt.-%, more preferably in the range of 0.0001 to 0.25 wt.-%, most preferably in the range of 0.0001 to 0.1 wt.-% based on the total weight of the cosmetic composition.

11. The cosmetic or pharmaceutical composition according to claim 9, wherein the 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, moisturizing, soothing, and/or energizing agents as well as agents to improve elasticity and skin barrier.

12. The cosmetic or pharmaceutical composition according to claim 9, wherein the composition further comprises at least on 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/or tocopherol acetate as well as mixtures thereof.

13. A method of treating the skin and/or the scalp, said method comprising the steps of contacting the skin and/or scalp with a composition according to claim 9.

14. A method according to claim 13 for maintaining a healthy skin homeostasis and/or for maintaining skin microbiome balance.

15. Use of a composition according to claim 9 for maintaining a healthy skin homeostasis and/or for maintaining skin microbiome balance.

16. Use of a compound according to claim 1 as antimicrobial agent against Bacillus subtilis, and optionally Propionibacterium acnes and/or Staphylococcus aureus.

Description

EXPERIMENTAL PART

[0115] General Information

[0116] Abbreviations: [0117] AA Amino acid [0118] Arg arginine [0119] Boc tert-butyloxycarbonyl [0120] Dab 2,4 diaminobutyric acid [0121] DCM dichloromethane [0122] DIPEA N,N-diisopropylethylamine [0123] DMAP N,N-dimethylaminopyridine [0124] DMF dimethylformamide [0125] Fmoc fluorenylmethoxycarbonyl [0126] Gly Glycine [0127] His Histidine [0128] HPLC High Pressure Liquid Chromatography [0129] IPE Di iso propyl ether [0130] NaphAla Naphthylalanin [0131] PhBu 4-phenyl butyric acid [0132] Phe phenylalanin [0133] TBTU O-(Benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborat [0134] TCTU 2-(2-Pyridon-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate [0135] TFA trifluoroacetic acid [0136] TIPS triisopropylsilane [0137] Trp Tryptophan [0138] Trt Trityl

[0139] Preparative HPLC Purifications:

[0140] 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 m 50300 mm.sup.2 preparative column and a Waters 2487 double wavelength UV-Vis detector operating at 220 and 254 nm.

[0141] 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.

[0142] Synthetic Strategies

[0143] 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. 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.

[0144] Preparation

[0145] 1. Free Amides:

[0146] Typical procedure: approx. 2.0 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 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 phenylbutyric acid or its derivative is coupled on the peptide using the standard peptide coupling procedure.

[0147] 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 200 ml of IPE/Hexan=1/1 (v/v). The precipitate is directly purified by preparative HPLC. The acidic modifier of the liquid phase matches the desired salt form (acetic acid for I-c, TFA for all others)

TABLE-US-00002 TABLE 2 Entry Sequence Amount, yield I-a PhBu-His-D-Phe-Arg-L-2NaphAla-NH2 *2TFA 425 mg (41%) I-b PhBu-His-D-Phe-Arg-D-2NaphAla-NH2 *2TFA 307 mg (31%) I-c 4-MeOPhBu-His-D-Phe-Arg-Trp-NH2 2 AcOH 386 mg (13%) I-e PhBu-His-D-Phe-Dab-Trp-NH2 *2TFA 374 mg (37%)

[0148] Substituted Amides

[0149] Typical procedure: approx. 2 g 2-chloro-trityl-resin (loaded with the fist 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 phenylbutyric acid is coupled on the peptide using the standard peptide coupling procedure.

[0150] 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.

TABLE-US-00003 TABLE 3 Entry Sequence Amount, yield I-d PhBu-His-D-Phe-Arg-Trp-N(Propyl).sub.2 *2TFA 262 mg (33%) I-f PhBu-His-D-Phe-Arg-Trp-NH-Bn *2TFA 209 mg (11%) I-g PhBu-His-D-Phe-Arg-Trp-NH-Octyl *2TFA 279 mg (28%) I-h PhBu-His-D-Phe-Arg-Trp-NHCH.sub.2CH(OH)CH.sub.2OH *2TFA 227 mg (18%)

[0151] 3. Antimicrobial Activity

[0152] The peptides are dissolved in culture medium at 400 ppm. A control in the microbiological medium alone and a control consisting of Phenonip prepared to 5% (v/v) directly in the microbiological medium was used.

[0153] The determination of minimal inhibitory concentration of each product is performed according to a liquid media method adapted in 96-wells plates.

[0154] To determine the minimum inhibitory concentration (MIC), the peptides are tested in serial dilutions ( to for 8 dilutions) directly in the liquid culture medium to favor the growth of bacteria in microplate 96 wells. Then, each point of dilution is contaminated with each strain at about 710.sup.5 cfu/ml for S. aureus (ATCC 6538), 5.510.sup.5 cfu/ml for P. acnes (ATCC 11827) and 1,210.sup.5 cfu/ml for B. subtilis (ATCC 6633) per well. Finally, plates are incubated 48 hours at 32, 5 C.2.5 C., taking care to respect the respiratory type of each strain.

[0155] At the end of the incubation time of 48 hours, the presence or absence of turbidity reveals the state of microbial growth. The last dilution corresponding to the absence of bacterial growth is taken as minimum inhibitory concentration (MIC) for the microbial strain considered. The results of the study are presented in the following table:

TABLE-US-00004 TABLE 4 MIC values # Peptide B. subtilis S. aureus P. acnes I-a PhBu-His-D-Phe-Arg-L-2NaphAla-NH.sub.2 62.5 ppm 125 ppm 500 ppm *2TFA I-b PhBu-His-D-Phe-Arg-D-2NaphAla-NH.sub.2 100 ppm 400 ppm *2TFA I-c 4-MeOPhBu-His-D-Phe-Arg-Trp-NH.sub.2 2 200 ppm AcOH I-d PhBu-His-D-Phe-Arg-Trp-N(Propyl.sub.)2*2TFA 15.6 ppm 31.3 ppm 125 ppm I-e PhBu-His-D-Phe-Dab-Trp-NH.sub.2 *2TFA 400 ppm I-f PhBu-His-D-Phe-Arg-Trp-NH-Bn*2TFA 252 ppm 400 ppm I-g PhBu-His-D-Phe-Arg-Trp-NH-Octyl *2TFA 252 ppm 200 ppm I-h PhBu-His-D-Phe-Arg-Trp-NHCH.sub.2CH(OH)CH.sub.2OH 317 ppm *2TFA Phenonip 790 ppm 630 ppm 630 ppm

[0156] As can be seen, all peptides exhibited a significant antimicrobial activity against B. subtilis, which is even better than the one of Phenonip, a well-known cosmetic antimicrobial agent. Some of the peptides also inhibited (selectively) the growth of P. acnes and/or S. aureus.

[0157] Cosmetic Composition

[0158] Table 5 outlines exemplary O/W emulsions, wherein one compound selected from the group of (I-h) as outlined in table 1, is incorporated in the indicated amount.

TABLE-US-00005 TABLE 5 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-h) 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 Erythrulose 1 1 Dihydroxyacetophenone 1 0.5 0.5 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