WO2002068389A2

WO2002068389A2 - Indole derivatives with inhibitor effects on phenol oxidase

Info

Publication number: WO2002068389A2
Application number: PCT/EP2002/001920
Authority: WO
Inventors: Peter Mayser; Wolfgang Steglich; Hans-Joachim KRÄMER; Bernhard Irlinger
Original assignee: FREISTAAT BAYERN vertreten durch LUDWIG-MAXIMILIAN-UNIVERSITÄT MÜNCHEN
Priority date: 2001-02-23
Filing date: 2002-02-23
Publication date: 2002-09-06
Also published as: AU2002233359A1; WO2002068389A3; DE10109005A1

Abstract

The invention relates to indole derivatives having an inhibiting effect on phenol oxidases. Melanin-related hyperpigmentation of skin is a considerable medical and therapeutic problem. The current possibilities for therapeutic effects, in addition to invasive methods, particularly the insertion of decolorants, are limited. The invention also relates to the increased need for agents and substances for treating melanin-related hyperpigmentation. The amino acid tryptophane (as an L or D isomer or also as a racemate) is provided for a particular subpopulation of the yeast type Melassezia, particularly the yeast species Melassezia furfur, as a predominant nitrogen source, and compounds are isolated from the yeast conditioned in such a manner, said compounds represent potent inhibitors of phenol oxidases. The novel indole derivatives isolated from Melassezia is particularly suitable to be used as substances for producing medicaments for treating melanin-related hyperpigmentation.

Description

Indole derivatives with an inhibitory effect on phenol oxidases

The present invention relates to new indole derivatives with the pronounced property of effectively inhibiting phenol oxidase (tyrosinase). In particular, the invention relates to compounds which are suitable for the treatment of melanin-related hyperpigmentation.

Melanin-related hyperpigmentation of the skin is a major medical and therapeutic problem. Inborn hyperpigmentation includes, for example, the cafe-au-lait spots, i.e. milk coffee-colored rounded skin spots, which are often a partial symptom of phakomatosis, e.g. neurofibromatosis from Recklinghausen, and because of their number and extent they can be cosmetically disruptive. The acquired hypermelanoses include, for example, the melasma (chloasma), hyperpigmentation as a result of inflammatory processes, light dermatosis as a result of phototoxic or photoallergic reactions (e.g. Berloque dermatitis, Riehl melanosis), hyperpigmentation as a result of injuries, especially burns, circumscribed hyperpigmentation depending chronic or acute sun exposure (e.g. lentigo simplex, lentigo solaris, ephelids) and hyperpigmentation as a result of systemic or topical use of medication.

However, the possibilities for a therapeutic influence on hyperpigmentation are limited. In addition to eliminating the cause, which is not always possible, adjuvant use of potent UV filters can also be considered, since increased exposure to UV light can lead to an increase in melanin-related hyperpigmentation. Camouflage methods are also used. A large number of substances were used as so-called bleaching agents. Mercury and its compounds as well as peroxides and chlorates are largely of historical interest (H ₂ O ₂ , sodium hypochlorite). The phenolic formulations include hydroquinone. As a result of the inhibition of the enzymatic oxidation of tyrosine to dihydroxyphenylalanine (Dopa) and due to the inhibition of further metabolic reactions in melanocytes, locally applied hydroquinone causes depigmentation of pathological hyperpigmentation of the skin. Hydroquinone therefore acts as a competitive inhibitor of melanin synthesis.

Bleaching agents, especially hydroquinone, can, however, cause skin irritation and allergic reactions, or even cause exogenous ochronosis or pigmented colloid milly. The non-phenolic bleaching agents include vitamin A acid and its derivatives as well as azelaic acid. Vitamin A acid itself does not specifically intervene in melanin synthesis, but rather causes a certain bleaching of the skin via an increased cell proliferation in the epidermis.

Chemical peeling, cryotherapy and laser treatment are further methods of treating hyperpigmentation. However, these methods are associated with an invasive procedure.

There is therefore still a considerable need for agents and substances for the treatment of melanin-related hyperpigmentation. The invention is therefore based on the object of providing substances for the treatment of such melanin-related hyperpigmentations which do not have the disadvantages of the few means and methods known from the prior art described above and with the aid of which within a short treatment period (a few weeks) visible healing success can be achieved.

According to the invention, it has surprisingly been found that a specific subpopulation of the yeast genus Malassezia, in particular the yeast species Malassezia furfur, is able, with a specific nutrient supply, to synthesize potent, novel inhibitors of phenol oxidase. Is Malassezia considered Mainly nitrogen source the amino acid tryptophan (as L- or D-isomer or also as racemate) can be isolated from such conditioned yeast compounds with which the phenol oxidase reaction could be inhibited in different models in vitro and in volunteers with topical ones Application after a few days a lightening of pre-existing melanin-related hyperpigmentation could be achieved.

In particular, it has also been found that when tryptophan is substituted at the 4-, 5-, 6- and / or 7-position of the indole ring, these substituents are incorporated almost unchanged in the indole derivatives synthesized by Malassezia and on which the invention is based. This is of particular interest if the relatively hydrophobic indole derivatives synthesized from pure tryptophan, e.g. should be made more hydrophilic by means of hydroxy substituents.

The compounds isolated from Malassezia have the following general structure,

in which mean:

Y = H or ^ C = O. Z = H or OH; M = NH, OH or Y

under the conditions when X ^: and Z = H

and there is a ring closure over X and Y, and if X = / -γ OOH then M = OH or ^ NH> ^{unci if M =} OH then

M and if M = _/ NH, Y = ^ -C = O and is cyclized together with ^ NH to form an azepine ring system, with the indole ring systems at the 4-, 5-, 6- and / or 7-position each individually or in combination with substituents selected from the group OH, F, Cl, Br, NO ₂ , NH ₂ , COOH, HSO ₃ can be substituted or form aza compounds.

The compounds with the following structures are preferred:

C ₂₀ H ₁₈ NA MW: 334.37

The compounds of the two upper structures with the empirical formulas C ₂ ιH, ₇ N ₃ O _{4 and} C ₂ ιH ₁₇ N ₃ O ₃ are particularly preferred. The first three links contain an asymmetry center. It was found that both the pure stereoisomers and the racemate are able to effectively inhibit phenol oxidase.

Another indole derivative structurally related to the above compounds with pronounced phenol oxidase inhibitor properties, which can also be isolated from Malassezia, has the following structure:

C ₂₀ H [ ₁₂ N ₂ O ₃

MG: 328.33

With this compound too, the indole ring systems at the 4-, 5-, 6- and / or 7-position can each be substituted individually or in combination with the groups indicated above.

The recovery of the above compound with the empirical formula C ₂₁ H ₁₇ N ₃ O ₃ and the designation KO27 is described below as an example:

A yeast subpopulation of the genus Malassezia, in particular the species Malassezia furfur, is offered the amino acid tryptophan (L, D isomer or racemate) as the sole nitrogen source. From the pigments and fluorochromes formed under these conditions by Malassezia, the above isolate the phenol oxidase inhibitors mentioned and derivatives thereof (oxidation products) which likewise have an inhibitory effect on phenol oxidase.

As a suitable nutrient medium, 30 ml of Tween ^® 80 ultra (Sigma, St. Louis, USA) and 20 g of purest agar (Merck), made up to 1L with water, are autoclaved. After cooling to 50 ° C., sterile-filtered D- or L-tryptophan or DL-tryptophan (Trp; Sigma) is added in a concentration of, for example, 0.3% by weight. The pH is adjusted to 5.5. 10 ml of the medium are poured into sterile petri dishes (10 cm in diameter) and a corresponding population of Malassezia furfur (CBS 1878) is spread on them. The substances can also be obtained in liquid medium (without the agar portion).

After an incubation period of about 14 days at 30-37 ° C, the culture medium or the liquid culture medium is extracted with ethyl acetate and the phenol oxidase inhibitors are isolated by means of column chromatography, thin-layer chromatography and preparative high-performance liquid chromatography (HPLC).

The R _f value of compound K027 with the eluent toluene-ethyl formate-formic acid (10: 5: 3) on silica gel 60 plates (Merck) is approximately 0.38.

The retention time of this compound determined by HPLC in acetonitrile / water 2: 3 (VN) on a Merck-Hitachi system, equipped with an Rpl8 column, 4 mm ² diameter at a flow of 1 ml / min and a pressure of 140-160 bar , a sensitivity of 0.3 (mVolt) and a measuring frequency of the detector of 220 nm and a linear gradient of water with increasing proportion of acetonitrile (from 0 to 100% in steps of 1% / min) is 27 min.

Inhibition of phenol oxidase activity in vitro: A) Enzyme kinetic measurements

The inhibitory effect was determined using the following approach:

2.5 mg dopa (L-3,4-dihydroxyphenylalanine) in 2 ml 0.05M phosphate buffer,

150 U phenol oxidase from fungi (EC 1.14.18.1) in 320 ml phosphate buffer,

1.4 ml 0.05M phosphate buffer,

Connection in 100 μl DMSO.

The concentration of the dopa (MW .: 197.2) was 2.94 "10 ^{" 3} mol.

The measurement was carried out in cuvettes with a layer thickness of 1 cm using a Beckmann DU-68 spectrophotometer. The increase in the optical density at 475 nm of the dopachrome formed by the phenol oxidase from dopa was measured. The pH was adjusted to 7.0 and the incubation temperature was 20 ° C. The blank consisted of phosphate buffer and DMSO.

An assay according to Shin et al. Can be used as another enzyme kinetic measurement method. (Biochem. Biophys. Res. Comm. 243, 801-803, 1998), which is composed as follows.

40 μl of 25 mM L-dopa, 80 μl of phosphate buffer (67 mM, pH 6.8), 40 μl of phosphate buffer with or without test substance are placed in a microtiter plate and mixed. Then 40 μl tyrosinase solution (EC 1.14.18.1 in phosphate buffer; 125 U / ml, i.e. 5 U / well in the assay) are added to each well with an 8-channel pipette. The measurement is then carried out at 492 nm using an Elisa reader every 30 seconds for about 40 minutes.

From the increase in extinction, the maximum speed of the reaction was determined according to the Lineweaver-Burk evaluation.

The compound K027 proved to be a competitive inhibitor of mushroom tyrosinase with a Ki of 1.1 x 10 ^' \ which can be seen from the figure. B) Inhibition of Phenol Oxidase Activity in Human Skin:

Freshly removed human skin was first incubated in 2N Na bromide solution at 37 ° C. over a period of 45 minutes in order to detach the epidermis from the dermis. After separation, the epidermis was incubated at 37 ° C in a 0.1% solution of dopa in 0.05M PBS at pH 7.4. The solution was renewed every hour (over a period of 2-5 hours in total) until dopa-positive melanocytes were recognizable. Then it was fixed in 3% formaldehyde.

The dopa-phenol oxidase reaction was effectively suppressed by adding the above compound. With a 10 ^"4 molar solution (final concentration) of the above compound a complete inhibition of phenoloxidase reaction was observed in human melanocytes.

C) Lightening of melanin-related hyperpigmentation

4 volunteers with melanin-related hyperpigmentation were given a 0.1% (w / w) distribution of the above compound in Unguenta emulsificans topically. After four weeks of treatment, a clear lightening of the hyperpigmentation was observed.

The phenol oxidase inhibitors according to the invention can be used for the production of medicaments with a pronounced action against melanin-induced hypere pigmentations. The brightening effect is equally aimed at congenital or genetic as well as acquired hypo pigmentation.

The effect is based on a competitive inhibition of phenol oxidase, which causes the enzymatic oxidation of tyrosine to dihydroxyphenylalanine (dopa) and the further reaction via dopaquinone to dopachrome is prevented. The effective concentration is about 10 ^" " ¹ to 10 ^{~ 6} M. The inhibitory effect of the inhibitors according to the invention also extends to other metabolic processes in melanocytes and to the inhibition of pigment transport of melanocytes in keratocytes. The phenol oxidase inhibitors according to the invention can therefore also be used for the production of medicaments which are used for the treatment of benign, semimalignant and malignant changes in the melanocytes, in particular also for influencing their growth.

The phenol oxidase inhibitors according to the invention can be administered topically distributed in an ointment. However, other compositions, formulations and bases are also possible. In particular, they can be used together with active ingredients known in the art, such as e.g. Stabilizers, antioxidants (e.g. tocopherol), light stabilizers, glucocorticosteroids, vitamin A acid and their derivatives can be used in any combination and in different proportions.

Claims

claims

1. phenol oxidase inhibitor, characterized in that it is an indole derivative and can be isolated from the yeast genus Malassezia, which tryptophan has been administered as a predominant nitrogen source.

2. phenol oxidase inhibitor according to claim 1, characterized in that it has the general formula (I),

in which mean: COOH COOH

X = or

M M

Y = H or ^ C = O. Z = H or OH; M = NH, OH or Y

under the conditions when X ^: Y ^: _/ C = O and Z = H

and there is a ring closure over X and Y,

COOH and if X = then M = OH or ^> H _> and if M = OH then Y = H

M and if M

and is cyclized together with / NH to form an azepine ring system, the indole ring systems at the 4-, 5-, 6- and / or 7-position in each case individually or in combination with substituents selected from the group OH, F, Cl, Br, NO ₂ , NH ₂ , COOH, HSO ₃ can be substituted or form aza compounds.

3. phenol oxidase inhibitor according to claim 1, characterized in that it has the general formula (II),

in which the indole ring systems at the 4-, 5-, 6- and / or 7-position each individually or in combination with substituents selected from the group OH, F, Cl, Br, NO ₂ , NH ₂ , COOH, HSO ₃ may be substituted or form aza compounds.

4. phenol oxidase inhibitor according to claim 1 and 2, characterized in that it has the empirical formula C ₂ ιH | N ₃ O and has the following structure:

5. phenol oxidase inhibitor according to claim 1 and 2, characterized in that it has the empirical formula C ₂ jHι ₇ N ₃ O and has the following structure:

6. phenol oxidase inhibitor according to claim 1 and 2, characterized in that it has the empirical formula C ₂ 0H ₁ 8N ₂ O ₃ and has the following structure:

7. phenol oxidase inhibitor according to claim 1 and 2, characterized in that it has the empirical formula C ₂₁ H ₁ 7N ₂ O3 and has the following structure:

8. phenol oxidase inhibitor according to claim 1 and 3, characterized in that it has the empirical formula C ₂₀ H ₁₂ N ₂ O3 and has the following structure:

9. phenol oxidase inhibitor according to claim 1 and 3, characterized in that it has the empirical formula C | H ₍ 2N2θ and has the following structure:

10. A process for the preparation of a phenol oxidase inhibitor according to one of claims 1 to 9, characterized in that a population of the yeast genus Malassezia is propagated in a nutrient medium which is tryptophan and / or at the 4-, 5-, 6- as the predominant nitrogen source. and / or 7-position each individually or in combination with substituents selected from the group OH, F, Cl, Br, NO ₂ , NH ₂ , COOH, HSO ₃ substituted or present as an aza compound tryptophan, after the incubation Culture medium is extracted together with the yeast population with a suitable solvent and the inhibitor is isolated from the extract by means of suitable chromatographic methods.

11. Use of a phenol oxidase inhibitor according to any one of claims 1 to 9 for the manufacture of a medicament for the treatment of melanin-related hypo pigmentations, for the treatment of benign, semimalignant and malignant changes in melanocytes and for the inhibition of growth of melanocytes.