EA044823B1 - MALASSESIN AND ITS ANALOGUES AS SKIN LIGHTENING AGENTS - Google Patents

Description

Cross reference to related applications

The present invention claims priority to U.S. provisional patent application no.

62/306468, filed March 10, 2016. The entire contents of this application are incorporated by reference.

Field of technology

The present invention relates to chemical analogues of compounds produced by Malassezia yeast. The invention includes compositions containing compounds produced by Malassezia yeast, as well as chemical analogues of compounds produced by Malassezia yeast. Methods of using the compounds (including analogues thereof) and compositions of the present invention are also discussed.

State of the art

People around the world use skin lightening agents to achieve several cosmetic purposes, including providing anti-aging benefits, correcting sun damage, and meeting certain cultural beauty standards. Many commercially available skin lightening products, while effective to varying degrees, contain harmful ingredients, some of which have been linked to cancer risk. Therefore, there is a need for new skin lightening agents and preparations that demonstrate higher levels of safety and/or effectiveness than agents currently available on the market.

Malassezia is a genus of lipophilic yeast commonly found in the normal flora of human skin. Malassezia is responsible for several skin diseases, including pityriasis versicolor (pityriasis versicolor), seborrheic dermatitis, and atopic dermatitis.

Tinea versicolor is a non-contagious skin disease caused by an overgrowth of Malassezia, which locally alters pigmentation levels. Yeast has two metabolic pathways for the synthesis of melanin and tryptophan-derived indole pigments. Indole pigments include malassezin, a tryptophan metabolite of Malassezia that can induce melanocyte apoptosis and contributes to the depigmentation characteristics of Malassezia overgrowth.

The invention described herein utilizes components produced by Malassezia yeast, including Malassezin and its chemical analogues, as the basis for safe and effective skin lightening compositions.

The essence of the invention

One embodiment of the present invention relates to a cosmetic composition for skin lightening containing a compound having the structure of formula (II)

(P) where R1, _R2 , _R3 , _R4 , R5, _R6 , R7, _R8 , _R9 , _R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or a cosmetically acceptable salt, and a cosmetically acceptable excipient, diluent or carrier, and further comprising one or more ingredients selected from the group consisting of fragrance, estrogen, vitamin A, vitamin C, vitamin E, pyruvic acid, lactic acid, glycolic acid, lanolin, petrolatum, aloe vera, methylparaben, propylparaben and pigments.

Another embodiment of the present invention relates to a composition that is a composition for topical or transdermal administration selected from the group consisting of powders, sprays, ointments, pastes, creams, lotions, gels, patches, emulsions, suspensions, aerosols and inhalers.

A further embodiment of the present invention relates to a cosmetic composition wherein the compound is selected from the group consisting of:

- 1 044823

or a cosmetically acceptable salt thereof.

An additional embodiment of the present invention relates to a composition in which the compound is

or a cosmetically acceptable salt thereof.

Another embodiment of the present invention relates to a method for lightening the skin of a human subject, comprising applying to the human subject a cosmetic skin lightening composition containing a compound having the structure of formula (II)

where R1, R2, _R3 , R4, _R5 , R6, _R7 , _R8 , _R9 , _R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or a cosmetically acceptable salt, and a cosmetically acceptable excipient, diluent or carrier, and further comprising one or more ingredients selected from the group consisting of fragrance, estrogen, vitamin A, vitamin C, vitamin E, pyruvic acid, lactic acid, glycolic acid, lanolin, petrolatum, aloe vera, methylparaben, propylparaben and pigments.

Another embodiment of the present invention relates to a method for lightening the skin of a human subject, comprising applying to the human subject a cosmetic skin lightening composition, which is a composition for topical or transdermal administration selected from the group consisting of powders, sprays, ointments, pastes , creams, lotions, gels, patches, emulsions, suspensions, aerosols and inhalers.

A further embodiment of the present invention relates to a method of lightening the skin of a human subject, comprising applying to the human subject a cosmetic skin lightening composition containing a compound that is selected from the group consisting of:

- 2 044823

and a cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention relates to a method of lightening the skin of a human subject, comprising applying to the human subject a cosmetic skin lightening composition containing a compound that is:

or a cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention relates to a method of lightening the skin of a human subject, comprising applying to the human subject a cosmetic skin lightening composition containing a compound that is:

or a cosmetically or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention relates to a pharmaceutical composition for skin lightening containing a compound having the structure of formula (II)

where R1, R2, _R3 , R4, R5, R6, _R7 , R8, R9, _R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or a pharmaceutically acceptable salt, and a pharmaceutically acceptable excipient, diluent or carrier, and further containing one or more ingredients selected from the group consisting of fragrances, estrogen, vitamin A, vitamin C, vitamin E, pyruvic acid, lactic acid, glycolic acid, lanolin, petrolatum, aloe vera, methylparaben, propylparaben and pigments.

A further embodiment of the present invention relates to a method for lightening the skin of a subject. The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

A further embodiment of the present invention relates to a pharmaceutical composition which is a composition for topical or transdermal administration selected from the group consisting of powders, sprays, ointments, pastes, creams, lotions, gels, patches, emulsions, suspensions, aerosols and inhalers.

A further embodiment of the present invention relates to a pharmaceutical composition that contains a compound selected from the group consisting of:

- 3 044823

or a pharmaceutically acceptable salt thereof.

An additional embodiment of the present invention relates to a pharmaceutical composition containing a compound that is:

or a cosmetically or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention relates to a cosmetic or pharmaceutical composition that is an emulsion or microemulsion.

A further embodiment of the present invention relates to a cosmetic or pharmaceutical composition that is a water-in-oil or oil-in-water emulsion or microemulsion.

A further embodiment of the present invention relates to a method of lightening the skin of a human subject, comprising applying to the human subject a skin lightening cosmetic composition that is an emulsion or microemulsion.

A further embodiment of the present invention relates to a method of lightening the skin of a human subject, comprising applying to the human subject a skin lightening cosmetic composition that is a water-in-oil or oil-in-water emulsion or microemulsion.

Brief description of graphic materials

The patent or application file contains at least one drawing in color. Copies of the publication of this patent or patent application with color graphics will be provided by the Patent Office upon request and the required payment.

In fig. 1A is a schematic diagram of the layers that make up the skin. The inserted diagram shows the cellular composition of the epidermis and dermis. In fig. 1B is a schematic diagram showing potential mechanisms of action of hypopigmentation-inducing agents.

In fig. Figure 2 shows a number of schemes for the synthesis of malassezin and malassezin derivatives: FIG. 2A: malassesin and indolo[3,2-b]carbazole; fig. 2B: compounds I and IV; fig. 2C: compound II.

In fig. 3A provides a summary table of EC50 values for Annexin V induction for certain compounds of the present invention in MeWo and WM115 cells. In fig. 3B-3M are line graphs showing the percentage of Annexin V-labeled MeWo (FIGS. 3B-3G) or WM115 (FIGS. 3H-3M) cells after exposure to various concentrations of the listed compounds.

In fig. 4A-4D are tables showing the levels of annexin V (%) in MeWo and WM115 cells after exposure to various concentrations of the listed compounds for 6, 24, 48 and 72 hours. FIG. 4E-4J are histograms showing the results of FIG. 4A-4D. In fig. 4K and 4L are histograms showing the percentage of Annexin V-labeled MeWo (Fig. 4K) and WM115 (Fig. 4L) cells after 6 hours of exposure to the listed compounds at the indicated concentrations.

In fig. 5A-5K are micrographs showing the morphology of MeWo cells after 6 hours of treatment with various concentrations of CV-8684, CV-8685, CV-8688, DMSO and staurosporine.

In fig. 6A-6K are micrographs showing the morphology of MeWo cells after 24 hours of treatment with various concentrations of CV-8684, CV-8685, CV-8688, DMSO and staurosporine.

In fig. 7A-7K are micrographs showing the morphology of MeWo cells after 48 hours of treatment with various concentrations of CV-8684, CV-8685, CV-8688, DMSO and staurosporine.

In fig. 8A-8K are micrographs showing the morphology of MeWo cells after 72 hours of treatment with various concentrations of CV-8684, CV-8685, CV-8688, DMSO and staurosporine.

In fig. 9A-9K are micrographs showing the morphology of WM115 cells after 6 hours of treatment with various concentrations of CV-8684, CV-8685, CV-8688, DMSO and staurosporine.

In fig. 10A-10K are micrographs showing the morphology of WM115 cells after 24 hours of treatment with various concentrations of CV-8684, CV-8685, CV-8688, DMSO and staurosporine.

In fig. 11A-11K are micrographs showing the morphology of WM115 cells after 48 hours of treatment with various concentrations of CV-8684, CV-8685, CV-8688, DMSO and staurosporine.

In fig. 12A-12K are micrographs showing the morphology of WM115 cells after 72 hours of treatment with various concentrations of CV-8684, CV-8685, CV-8688, DMSO and staurosporine.

In fig. 13A-13D are tables showing the percentage of viable MeWo and WM115 cells remaining after treatment with various concentrations of CV-8684 (FIG. 13A), CV-8685 (FIG. 13B), CV-8688 (FIG. 13C), or staurosporine (FIG. 13D) for 6, 24, 48 and 72 hours. Cell viability was analyzed using CellTiter-Glo®. In fig. 13E-13J are histograms showing the results of FIG. 13A-13D. In fig. 13K provides a summary table comparing the percentage of viable MeWo and WM115 cells after exposure to the listed concentrations of Malassesin, indolocarbazole, Compound II, and staurosporine for 24, 48, and 72 hours.

In fig. 14A-14D are tables showing lactate dehydrogenase (LDH) release levels from MeWo and WM115 cells following treatment with various concentrations of CV-8684 (FIG. 14A), CV-8685 (FIG. 14B), CV-8688 (FIG. 14C), or staurosporine (Fig. 14D) for 6, 24, 48 and 72 hours. In FIG. 14E-14J are histograms showing the results of FIG. 14A-14D. In fig. 14K and 14L are histograms showing lactate dehydrogenase levels following exposure of MeWo (FIG. 14K) and WM115 (FIG. 14L) cells to the listed concentrations of malassesin, carbazole, Compound II, and staurosporine for 24 hours.

In fig. 15A-15E show raw data and line graphs of arylhydrocarbon receptor (AhR) activation in HepG2 cells stably transfected with an AhR-luciferase reporter gene plasmid following exposure to various concentrations of omeprazole (FIG. 15A), CV-8684 (FIG. 15B), CV-8685 (Fig. 15C), CV-8686 (Fig. 15D) and CV-8688 (Fig. 15E). In fig. 15F shows the EC ₅₀ values for each compound tested.

In fig. 16A-16K show photographs of MelanoDerm™ matrices on day 0 or day 7 without treatment (Fig. 16A), after exposure to sterile deionized water (Fig. 16B), 1% kojic acid (Fig. 16C), 0.2% DMSO ( Fig. 16D), 0.05% DMSO (Fig. 16E), 200 µM Cv-8684 (Fig. 16F), 50 µM CV-8684 (Fig. 16G), 200 µM CV-8686 (Fig. 16H), 50 µM CV-8686 (Fig. 16I), 200 µM CV-8688 (Fig. 16J), and 50 µM CV-8688 (Fig. 16K).

In fig. 17A-17K are micrographs at 15X magnification of MelanoDerm™ matrices on day 0 or day 7 without treatment (Fig. 17A), after exposure to sterile deionized water (Fig. 17B), 1% kojic acid (Fig. 17C), 0. 2% DMSO (Fig. 17D), 0.05% DMSO (Fig. 17E), 200 µM CV-8684 (Fig. 17F), 50 µM CV-8684 (Fig. 17G), 200 µM CV-8686 (Fig. 17H), 50 μM CV-8686 (Figure 17I), 200 μM CV-8688 (Figure 17J), and 50 μM CV-8688 (Figure 17K).

In fig. 18A-18F show photographs of zebrafish in the absence of treatment (Fig. 18A), after exposure to DMSO (Fig. 18B), phenylthiourea (PTU) (Fig. 18C) and compound II at 2.5 μM (Fig. 18D), 5 μM ( Fig. 18E) and 10 μM (Fig. 18F). Red arrows indicate normal melanocytes.

In fig. 19A-19F show photographs of zebrafish in the absence of treatment (Fig. 19A), after exposure to DMSO (Fig. 19B), phenylthiourea (PTU) (Fig. 19C) and compound II at 0.3 μM (Fig. 19D), 1 μM ( Fig. 19E) and 3 µM (Fig. 19F). Red arrows indicate normal melanocytes. Yellow arrows indicate abnormally small melanocytes.

In fig. Figure 20 presents a summary table showing the number and percentage of zebrafish with reduced skin pigmentation after exposure to the listed conditions. The last six rows show the effect of different concentrations of compound II.

In fig. 21A-21E are photographs of zebrafish untreated (FIG. 21A), after exposure to DMSO (FIG. 21B), FTM (FIG. 21C), 0.5 μM (FIG. 21D), and 1.5 μM (FIG. 21E) . The bottom panels include areas for changing the color scheme.

In fig. 22A and 22B are histograms showing pigmentation density as measured by the number of pigmented pixels/mm ³ (FIG. 22A) and the total number of pixels (FIG. 22B) in the exemplified photographs of zebrafish embryos in FIG. 21A-21E.

In fig. 23A-23C show mass spectra of CV-8684 in DMSO (FIG. 23A), RPMI (FIG. 23B), and DMEM (FIG. 23C). In fig. 23D-23F show the mass spectra of CV-8686 in DMSO (Figure 23d), RPMI (Figure 23E) and DMEM (Figure 23F). In fig. 23G-23I show mass spectra of CV-8688 in DMSO

- 5 044823 (Fig. 23G), RPMI medium (Fig. 23H) and DMEM (Fig. 23I). In fig. 23J is a summary table showing the percentage of test compound remaining in the indicated solvent after two hours of incubation.

Detailed Description of the Invention

One embodiment of the present invention relates to a skin lightening compound. The compound is a chemical analogue of a compound produced by Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

As used herein, the term compound refers to two or more atoms that are connected by one or more chemical bonds. In the present invention, chemical bonds include, but are not limited to, covalent bonds, ionic bonds, hydrogen bonds and van der Waals interactions. Covalent bonds according to the present invention include single, double and triple bonds. The compounds of the present invention include, but are not limited to, organic molecules.

The organic compounds/molecules of the present invention include linear, branched and cyclic hydrocarbons with or without functional groups. The term C _xy when used in combination with a chemical group such as an alkyl, alkenyl, alkynyl or alkoxy group is intended to include groups that contain x to y carbon atoms in the chain. For example, the term C _{x_y} alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight chain alkyl groups and branched chain alkyl groups, which contain from x to y carbon atoms in _the chain, including haloalkyl groups such as trifluoromethyl and 2. 2,2-trifluoroethyl, etc. The terms C _x _ _y alkenyl and C _x _ _y alkynyl refer to substituted or unsubstituted unsaturated aliphatic groups similar in length and possible substitutions to the alkyls described above, but containing at least one double or triple bond, respectively.

As used herein, the term aliphatic refers to a group consisting of carbon and hydrogen atoms that does not contain aromatic rings. Accordingly, aliphatic groups include alkyl, alkenyl, alkynyl and carbocyclyl groups.

The term alkyl refers to a radical of saturated aliphatic groups that does not have a ring structure, including straight chain alkyl groups and branched chain alkyl groups.

As used herein, the term alkenyl refers to an aliphatic group containing at least one double bond.

As used herein, the term alkynyl refers to an aliphatic group containing at least one triple bond.

As used herein, an aromatic compound, an aromatic substance, or a compound containing an aromatic ring is an aryl or heteroaryl compound. As used herein, the term aryl includes substituted and unsubstituted single-ring aromatic groups in which each ring atom is a carbon. Preferably the ring is a 3-8 membered ring, more preferably a 6 membered ring. The term aryl also includes polycyclic ring systems having two or more cyclic rings, in which two or more carbon atoms are common to two adjacent rings, at least one of the rings is aromatic, for example, the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc. The term heteroaryl includes substituted or unsubstituted single ring aromatic structures, preferably 3-8 membered rings, more preferably 5-7 membered rings, even more preferably 5-6 membered rings, whose ring structures contain at least one heteroatom, preferably from one to four heteroatoms, more preferably one or two heteroatoms. The term heteroaryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, at least one of the rings is heteroaromatic, for example, the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, indole, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, etc. Preferably, some of the compounds of the present invention contain at least one, preferably two, indole groups, as well as at least one aldehyde group.

The term substituted refers to groups containing at least one substituent that replaces a hydrogen atom on one or more backbone carbon atoms. It should be understood that substitution or substituted with implies that such substitution is made in accordance with the permissible valence of the substituted atom, and that the substitution results in a stable compound, for example, which does not undergo spontaneous transformation, for example, by rearrangement, cyclization, elimination, etc. d. Valid substituents can be one or more, as well as the same

- 6 044823 or various suitable organic compounds.

In the context of this document, the expression skin lightening and its grammatical variations generally refers to any objective or subjective decrease in skin pigmentation. Skin lightening treatments are used to reduce pigmentation in hyperpigmented areas of the skin due to age, sun exposure, or a hyperpigmentation disorder. Application of the compounds and compositions of the present invention, for example, to the skin of a subject may reduce pigmentation such that the skin appears lighter or whiter than before said application. Skin pigmentation can be assessed in several ways, including, but not limited to, visual assessment using, for example, the von Luschan chromatic scale, the Fitzpatrick skin type (Fitzpatrick et al., 1988) and the Taylor hyperpigmentation scale (Taylor et al., 2005). ), as well as reflectance spectrophotometry methods (Zonios, et al., 2001). For example, the Fitzpatrick skin type test includes six skin types (I-VI), and as used herein, if skin type VI becomes type V or less, the skin is said to be lightened. As further discussed below, skin lightening can occur as a result of a number of phenomena, including, but not limited to, modulation of melanocyte activity, induction of melanocyte apoptosis, aryl-hydrocarbon receptor (AhR) agonism or modulation of melanin production, melanosome biogenesis or melanosome trafficking.

Some of the compounds of the present invention are produced by or isolated from Malassezia yeast. Malassezia yeast is a yeast of the genus Malassezia and includes, but is not limited to, Malassezia globosa, Malassezia restricta, Malassezia furfur, Malassezia sympodialis, Malassezia slooffiae, Malassezia obtusa, Malassezia pachydermatis, Malassezia dermatis, Malassezia japonica, Malassezia nana, Malassezia yamatoensis , Malassezia equine , Malassezia caprae and Malassezia cuniculi. (Gueho, et al., 1996; Gaitanis, et al., 2013). Malassezia yeast is part of the normal human skin flora and is generally non-pathogenic. However, Malassezia yeast can cause a number of diseases, including, but not limited to, pityriasis versicolor (both hyperpigmented and hypopigmented varieties), seborrheic dermatitis, dandruff, atopic dermatitis, Malassezia folliculitis, psoriasis, and confluent papular papillomatosis. (Gaitanis, et al., 2013).

As used herein, the term chemical analogue refers to a compound that is structurally related to the parent compound and contains distinct functional groups or substituents. For example, the parent compound of the present invention is malassesin, and chemical analogs of malassesin contain certain functional groups and substituents that differ from malassesin. Chemical analogues of the present invention may have significant advantages over this parent compound, including a pharmacokinetic profile suitable for cosmetic use. In some embodiments, a chemical analogue is derived from a parent molecule through one or more chemical reactions. In other embodiments, alternative synthetic routes that do not rely on the parent compound may be used to create chemical analogues of the present invention.

A compound of the present invention is produced by Malassezia yeast if, during its life cycle, the Malassezia yeast synthesizes, secretes, accumulates or otherwise produces the compound under conditions suitable for growth. Malassezia yeast secretes different compounds depending on the additives in the growth medium. (Nazzaro-Porro, et al., 1978). The present invention includes any compound produced by Malassezia yeast under any growth conditions, but preferred compounds include, for example, Malassezin and its chemical analogues.

In one aspect of this embodiment, the compound produced by Malassezia yeast has the structure of formula (I)

(!).

In another aspect of this embodiment, the compound is a chemical analogue of Malassesin.

Malassezin is one example of a compound produced by the Malassezia yeast of the present invention. Malassezin, also known as 2-(1H-indol-3-ylmethyl)-1H-indole-3-carbaldehyde, is a tryptophan metabolite first isolated from Malassezia furfur. Malassesin is a known agonist of the aryl-hydrocarbon receptor (AhR), a receptor involved in cell growth, differentiation and gene expression. (Wille et al., 2001). Malassesin also

- 7 044823 induces apoptosis of primary human melanocytes. (Kramer, et al., 2005). Recently, some chemical analogues of malassesin were synthesized by Winston-McPherson and colleagues, who examined the activity of AhR agonist analogues. (Winston-McPherson, et al., 2014).

Another embodiment of the present invention relates to a compound for inducing apoptosis of melanocytes. The compound is a chemical analogue of a compound produced by Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

As used herein, the term melanocyte refers to a dendritic cell of the epidermis that typically synthesizes tyrosinase and, in melanosomes, the pigment melanin. Melanocytes of the present invention exhibit upregulation of several genes, including, but not limited to, the following genes: tyrosinase (oculocutaneous albinism IA), microphthalmia-associated transcription factor, alpha-2-macroglobulin, tyrosinase-dependent protein 1, solute transporter family 16, GS3955 protein, Hardy-Zuckerman V-set 4 feline sarcoma oncogene, ocular albinism 1, Rag D protein, glycogenin 2 G protein-coupled receptor family C, oculocutaneous albinism II, oesophageal cancer ablation 1, melan-A, SRY -box 10, ATPase, class V, type 10C, matrix metalloproteinase 1, latent transforming growth factor beta b, ATP-binding cassette, subfamily C, hydroxyprostaglandin dehydrogenase 15, transmembrane superfamily member 1 7, glutaminyl-peptide cyclotransferase and other genes identified Lee and colleagues. (Lee, et al., 2013).

Melanocytes, like many other cell types, are subject to programmed cell death or apoptosis. Melanocyte apoptotic pathways are known to those skilled in the art (Wang, et al., 2014), and a general review of apoptotic pathways has been made by Elmore (Elmore, 2007). The compound or composition of the present invention induces melanocyte apoptosis, for example by causing activation of certain pro-apoptotic signal transduction pathways or causing repression of certain anti-apoptotic pathways in melanocytes. It is understood that a compound or composition of the present invention can directly activate/repress an apoptosis-related pathway by directly interacting with a pathway signaling molecule or by indirectly interacting with a pathway molecule through direct interaction with one or more intermediate molecules that are not normally involved in the pathway.

In one aspect of this embodiment, the compound produced by Malassezia yeast has the structure of formula (I)

In another aspect of this embodiment, the compound is a chemical analogue of Malassesin.

An additional embodiment of the present invention relates to a compound for modulating melanocyte activity. The compound is a chemical analogue of a compound produced by Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

Melanocyte activity can be modulated in several ways contemplated by the present invention, including, but not limited to, inducing melanocyte apoptosis or altering melanocyte gene expression, cell motility, cell growth, melanin production, melanosome biogenesis, or melanosome trafficking.

In one aspect of this embodiment, the compound produced by Malassezia yeast has the structure of formula (I)

In another aspect of this embodiment, the compound is a chemical analogue of Malassesin.

A further embodiment of the present invention provides a compound for agonizing the aryl-hydrocarbon receptor (AhR). The compound is a chemical analogue of a compound produced by Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

As used herein, the terms agonist, agonist, and grammatical variations thereof refer to a molecule that initiates (eg, initiates or promotes), partially or fully enhances, stimulates, or activates one or more biological activities. Agonists according to the present invention include substances of natural origin as well as synthetic substances.

The aryl-hydrocarbon receptor (AhR) of the present invention is any aryl-hydrocarbon receptor that is naturally found in the body of a subject, as described herein. Aryl-hydrocarbon receptors are known to those skilled in the art. (Noakes, 2015). Aryl-hydrocarbon receptor agonists include, but are not limited to, tryptophan-related compounds such as kynurenine, kynurenic acid, cinnabaric acid, and 6-formylindolo[3,2-b]carbazole (FICZ). Malassesin is also known as an arylhydrocarbon receptor agonist. (Wille, et al., 2001).

In one aspect of this embodiment, the compound produced by Malassezia yeast has the structure of formula (I)

In another aspect of this embodiment, the compound is a chemical analogue of Malassesin.

Another embodiment of the present invention provides a compound for improving hyperpigmentation caused by a hyperpigmentation disorder. The compound is a chemical analogue of a compound produced by Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

As used herein, the compounds, compositions and methods of the present invention can be used to reduce hyperpigmentation caused by a hyperpigmentation disorder, for example, by reducing the level of hyperpigmentation in areas affected by the hyperpigmentation disorder, delaying further hyperpigmentation, or preventing the appearance of further hyperpigmentation. However, since not every subject may demonstrate a response to a particular dosing protocol, regimen, or process, achieving the desired physiological response or outcome in all subjects or in the entire population of subjects is not necessary to reduce hyperpigmentation caused by a hyperpigmentation disorder. Accordingly, a given subject or a given population of subjects may not demonstrate a response or demonstrate an inadequate response to dosing, but other subjects and populations of subjects may demonstrate a response and therefore experience improvement in their hyperpigmentation disorder.

As used herein, the term hyperpigmentation refers to an objective or subjective skin disorder or darkening. The skin disorder may be objective, such as related to age, overexposure to sunlight, or a disease or condition that results in dark areas of the skin. Dark areas of skin may take the form of patches or relatively large areas of dark color. The skin disorder may also be subjective, for example related to the individual's opinion that his/her skin is too dark. An individual may wish to lighten their skin tone cosmetically.

Hyperpigmentation disorders are disorders in which hyperpigmentation is the primary symptom, as well as disorders in which hyperpigmentation occurs as a secondary symptom. Hyperpigmentation disorders according to the present invention include, but are not limited to, hereditary hyperpigmentation disorders and acquired hyperpigmentation disorders. The hereditary hyperpigmentation disorders of the present invention include, but are not limited to, those associated with epidermal hyperpigmentation (nevocytic nevus, Spitz nevus and macular nevus), dermal hyperpigmentation (blue nevus, nevus of Ota, dermal melanosis, nevus of Ito and Mongolian spots), freckles, reticular acropigmentation, Spitzenpigment/acropigmentation and lentiginosis (generalized lentiginosis, leopard syndrome, hereditary patterned lentiginosis, Carney syndrome, Peutz-Jeghers syndrome, Laugier-Hunziker-Baran syndrome and Cronkhite-Canada syndrome). (Yamaguchi, et al., 2014). Acquired hyperpigmentation disorders of the present invention include, but are not limited to, senile lentiginosis/lentigo, melasma/chloasma, Riehl melanosis, labial melanotic macules, penile/vulvovaginal melanosis, Kitamura follicular erythromelanosis of the face and neck, UV-induced pigmentation ( tanning and petaloides actinica pigmentation), post-inflammatory pigmentation (friction melanosis and ashy dermatosis), chemical/drug induced pigmentation (polychlorinated biphenyl, arsenic, 5-FU, bleomycin, cyclophosphamide, methotrexate, chlorpromazine, phenytoin, tetracycline and chloroquine), pigmentary boundary lines and deposits of foreign materials (such as carotene, silver, gold, mercury, bismuth and tattoos). Hyperpigmentation associated with systemic disorders includes metabolic/enzyme disorders (hemochromatosis, Wilson's disease, Gaucher's disease, Niemann-Pick disease, amyloidosis, ochronosis, acanthokeratoderma and cutaneous porphyria tarda), endocrine disorders (Addison's disease, Cushing's syndrome and hyperthyroidism), nutritional disorders (pellagra, vitamin B12 deficiency, folic acid deficiency, hobo's disease and pruritus pigmentosa), mastocytosis, collagen diseases, liver failure and renal failure. Hyperpigmentation may also be associated with infectious diseases (measles, syphilis, and Malassezia dandruff) and syndromes (Recklinghausen disease, Sotos syndrome, POEMS syndrome, Nägeli syndrome, Cantu syndrome, McCune-Albright syndrome, Watson syndrome, and Bloom syndrome). (Yamaguchi, et al., 2014).

In one aspect of this embodiment, the compound produced by Malassezia yeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogue of Malassezin.

An additional embodiment of the present invention relates to a compound for modulating melanin production. The compound is a chemical analogue of a compound produced by Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

Melanin is a naturally produced pigment that gives color to skin and hair. A schematic diagram of skin is shown in Fig. 1A. Melanin is produced by melanocytes in organelles known as melanosomes. The compound or composition of the present invention modulates melanin production in a subject, for example, by modulating melanosome biogenesis and directly or indirectly inhibiting melanin synthesis at the enzymatic level.

Melanosome biogenesis occurs in four stages: Stage I is characterized by the presence of premelanosomes, which are predominantly unpigmented vacuoles. At stage II, pre-melanosomes develop striations, in which melanin is deposited at stage III. Stage IV leads to the appearance of mature melanosomes having a high melanin content. The compounds or compositions of the present invention modulate melanosome biogenesis by inhibiting or attenuating the biological processes that typically promote any or all of these steps. (Wasmeier, et al., 2008).

Melanin synthesis mainly involves three enzymes: tyrosinase, tyrosine-dependent protein 1 and dopachrome tautomerase. Additional factors that influence the intracellular migration of these enzymes include, but are not limited to, BLOC-1, OA1, and SLC4A2. The compounds or compositions of the present invention may modulate melanin production, for example, by inhibiting or reducing the activity of any of these enzymes or factors. (Yamaguchi, et al., 2014).

After melanosome formation and melanin synthesis, melanosomes must be transferred from epidermal melanocytes to keratinocytes of the skin and hair. Melanosomes appear near the nucleus of melanocytes and are transported to the periphery of melanocytes along microtubules and actin filaments. The compounds or compositions of the present invention modulate melanosome trafficking by interfering with any of the biological processes that result in the transfer of melanosomes from the perinuclear region to the periphery of the melanocyte and into adjacent keratinocytes. A schematic diagram of melanin synthesis, melanin transport and melanocyte apoptosis is shown in Fig. 1B.

In one aspect of this embodiment, the compound produced by Malassezia yeast has the structure of formula (I)

- 10 044823

In another aspect of this embodiment, the compound is a chemical analogue of Malassesin.

An additional embodiment of the present invention relates to a compound for modulating melanosome biogenesis. The compound is a chemical analogue of a compound produced by Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound produced by Malassezia yeast has the structure of formula (I)

In another aspect of this embodiment, the compound is a chemical analogue of Malassesin.

Another embodiment of the present invention relates to a compound for modulating melanosome trafficking. The compound is a chemical analogue of a compound produced by Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound produced by Malassezia yeast has the structure of formula (I):

In another aspect of this embodiment, the compound is a chemical analogue of Malassesin.

An additional embodiment of the present invention relates to a composition. The composition contains Malassezia yeast and a cosmetically or pharmaceutically acceptable excipient, diluent or carrier.

An additional embodiment of the present invention relates to a composition. The composition contains a compound isolated or recoverable from the yeast Malassezia, and a cosmetically or pharmaceutically acceptable excipient, diluent or carrier.

The compound isolated from the Malassezia yeast according to the present invention is necessarily present before the isolation in the Malassezia yeast or is produced by the Malassezia yeast. Therefore, the compound isolated from Malassezia yeast is derived from actual yeast cells. Standard protocols for extracting compounds from cellular material are known to those skilled in the art.

A compound that can be isolated from Malassezia yeast does not necessarily need to be derived from actual yeast cells. Instead, synthesis reactions can be used to create compounds produced in yeast without involving actual yeast cells. Organic synthesis reactions are well known to those skilled in the art and can be used in this regard.

Another embodiment of the present invention relates to a composition. The composition contains any of the compounds described herein, including analogs, and a cosmetically or pharmaceutically acceptable excipient, diluent or carrier.

An additional embodiment of the present invention relates to a method for lightening the skin of a subject. The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

As used herein, the term bringing into contact and its grammatical variations refer to bringing two or more materials into sufficient proximity so that they can interact. Thus, for illustrative purposes only, a compound of the present invention may contact a melanocyte, for example, by interacting with a receptor on the surface of the melanocyte. Likewise, the composition of the present invention can contact a melanocyte, for example, when directly applied to the skin of a subject.

As used herein, subject means a cell, tissue, mammalian organism or population of mammals. Subjects of the present invention are preferably humans, including human cells, tissues and human beings, but otherwise include primates, farm animals, pets, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of pets include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.

An additional embodiment of the present invention relates to a method of inducing apoptosis of melanocytes in a subject. The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

Another embodiment of the present invention relates to a method of modulating melanocyte activity in a subject. The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

An additional embodiment of the present invention relates to a method of agonizing the aryl-hydrocarbon receptor (AhR). The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

A further embodiment of the present invention provides a method for reducing hyperpigmentation caused by a hyperpigmentation disorder in a subject in need thereof. The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

As used herein, a subject in need of reduction of hyperpigmentation caused by a hyperpigmentation disorder includes subjects with a real or subjective need for improvement.

Another embodiment of the present invention relates to a method of modulating melanin production in a subject. The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

An additional embodiment of the present invention relates to a method of modulating melanosome biogenesis in a subject. The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

An additional embodiment of the present invention relates to a method of modulating melanosome transfer in a subject. The method includes bringing a subject into contact with any of the compounds or any of the compositions described herein.

Another embodiment of the present invention relates to a connection. The compound has the structure of formula (II) (P) where R1, R2, _R3 , R4, R5, R6, _R7 , _R8 , _R9 , _R10 and R11 are independently selected from the group consisting of hydrogen and methyl, and at least one of R1, R2, R3, _R4 , R5, _R6 , _R7 , _Rg , R9, R1o and R11 is methyl or a crystalline form, hydrate or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

- 12 044823

An additional embodiment of the present invention relates to a connection. The compound has the structure of formula (III)

where R1, R2, _R3 , _R4 , R5, R6 _{, R7, R8, R9 and R10} are independently selected from the group consisting of hydrogen and methyl, and at least one of R1, R2, _R3 , R ₄ , R5, _R6 , R7, _R8 , _R9 and _R10 is methyl or its crystalline form, hydrate or cosmetically or pharmaceutically acceptable salt.

In one aspect of this embodiment, the connection is

A further embodiment of the present invention relates to a skin lightening compound. The compound has the structure of formula (II):

where R1, R2, _R3 , R4, R5, _R6 , R7, _R8 , _R9 , _R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

- 13 044823

Another embodiment of the present invention relates to a skin lightening compound. The compound has the structure of formula (III)

where R1, R2, _R3 , R4, R5, _R6 , _R7 , _R8 , _R9 and _R10 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

An additional embodiment of the present invention relates to a compound for inducing melanocyte apoptosis. The compound has the structure of formula (II)

where R1, R2, _R3 , R4, R5, _R6 , _R7 , _R8 , _R9 , _R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

- 14 044823

An additional embodiment of the present invention relates to a compound for inducing melanocyte apoptosis. The compound has the structure of formula (III)

where R1, R2, _R3 , R4, R5, R6, _R7 , _R8 , _R9 and _R10 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

Another embodiment of the present invention relates to a compound for agonizing the arylhydrocarbon receptor (AhR). The compound has the structure of formula (II)

where R1, R2, _R3 , _R4 , R5, R6, _R7 , _R8 , _R9 , _R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

- 15 044823

An additional embodiment of the present invention provides a compound for agonizing the aryl-hydrocarbon receptor (AhR). The compound has the structure of formula (III)

where R1, R2, _R3 , R4, R5, R6, _R7 , _R8 , _R9 and _R10 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

An additional embodiment of the present invention relates to a composition. The composition contains a compound having the structure of formula (II)

where R1, R2, _R3 , R4, R5, _R6 , _R7 , _R8 , _R9 , R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or representing a crystalline form, hydrate or cosmetically or pharmaceutically acceptable salt thereof, and a cosmetically or pharmaceutically acceptable excipient, diluent or carrier.

In one aspect of this embodiment, the connection is selected from the group consisting of:

- 16 044823

Another embodiment of the present invention relates to a composition. The composition contains

where R1, R2, _R3 , R4, R5, R6, _R7 , _R8 , _R9 and _R10 are independently selected from the group consisting of hydrogen and methyl;

or representing a crystalline form, hydrate or cosmetically or pharmaceutically acceptable salt thereof, and a cosmetically or pharmaceutically acceptable excipient, diluent or carrier.

In one aspect of this embodiment, the connection is selected from the group consisting of:

A further embodiment of the present invention relates to a method for lightening the skin of a subject.

The method involves bringing a subject into contact with a compound having the structure of formula (II)

where R1, R2, _R3 , R4, R5, R6, _R7 , _R8 , _R9 , R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

- 17 044823

A further embodiment of the present invention relates to a method for lightening the skin of a subject.

The method involves bringing a subject into contact with a compound having the structure of formula (III)

where R1, R2, R3, R4, R5, R6, R7, _R8 , R9 and R10 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

Another embodiment of the present invention relates to a method of inducing apoptosis of melanocytes in a subject. The method involves bringing a subject into contact with a compound having the structure of formula (II)

where R1, R2, R3, _R4 , R5, R6, R7, _R8 , R9, _R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

- 18 044823

An additional embodiment of the present invention relates to a method of inducing apoptosis of melanocytes in a subject. The method involves bringing a subject into contact with a compound having the structure of formula (III)

where R1, R2, _R3 , R4, R5, _R6 , R7, _R8 , R9 and _R10 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

An additional embodiment of the present invention relates to a method of agonizing the aryl-hydrocarbon receptor (AhR) in a subject. The method involves bringing a subject into contact with a compound having the structure of formula (II)

where R1, R2, _R3 , R4, R5, _R6 , R7, _R8 , R9, _R10 and R11 are independently selected from the group consisting of hydrogen and methyl;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

- 19 044823

Another embodiment of the present invention relates to a method of agonizing the arylhydrocarbon receptor (AhR) in a subject. The method involves contacting a subject with a compound having the structure of formula (III) wherein R1, R2, R3, R4, R5, _R6 , _R7 , _R8 , _R9 and _R10 are independently selected from the group consisting of hydrogen and methyl ;

or is a crystalline form, a hydrate, or a cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the connection is selected from the group consisting of:

As used herein, the term composition means a substance containing a compound of the present invention, as well as a substance that is obtained, directly or indirectly, from combinations of a compound of the present invention with other ingredients. The compositions of the present invention can be used, for example, as in vitro or in vivo research reagents. The compositions of the present invention can also be applied directly to the skin of a human or non-human subject to provide a cosmetic effect.

The compositions of the present invention can be administered by any necessary and effective route: for oral administration or for parenteral or other administration by any suitable route, such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal or intralymphatic. In addition, the compositions of the present invention can be administered in combination with other compositions. If necessary, the compositions of the present invention may be encapsulated or otherwise protected from gastric or other juices.

The compositions of the invention contain one or more active ingredients in admixture with one or more cosmetically or pharmaceutically acceptable carriers and, optionally, one or more other compounds, ingredients and/or materials. Regardless of the chosen route of administration, the compounds and compositions of the present invention are prepared in cosmetically or pharmaceutically acceptable dosage forms by conventional methods known in the art.

- 20 044823 stam in this area.

Cosmetically or pharmaceutically acceptable excipients, diluents and carriers are well known in the art and include materials suitable for contact with the tissues of humans and non-human beings without exhibiting undue toxicity, incompatibility, instability, irritation, allergic reaction, etc. Cosmetically or pharmaceutically acceptable excipients, diluents and carriers include any non-toxic substance conventionally used, for example, for topical, oral, peritoneal or subcutaneous administration of cosmetics or pharmaceuticals, in which the compounds and compositions of the present invention will remain stable and bioavailable upon application, ingestion , injection or any other administration to a human or non-human subject. Cosmetically or pharmaceutically acceptable carriers suitable for topical use are known to those skilled in the art and include cosmetically or pharmaceutically acceptable liquids, creams, oils, lotions, ointments, gels or solids, such as conventional cosmetic night creams, base creams, lotions tanning products, sunscreens, hand lotions, makeup and foundations, masks, etc. Carriers suitable for the selected dosage form and intended route of administration are well known in the art, and those skilled in the art can determine suitable carriers for the selected dosage form and route of administration.

The compositions of the present invention may contain other ingredients traditional in cosmetology, including fragrances, estrogen, vitamins A, C and E, alpha hydroxy and alpha keto acids such as pyruvic, lactic or glycolic acids, lanolin, petrolatum, aloe vera, methyl or propylparaben, pigments, etc. Non-limiting cosmetically or pharmaceutically acceptable excipients, diluents and carriers according to the present invention include sugars (for example, lactose, sucrose, mannitol and sorbitol), starches, cellulose preparations, calcium phosphates (for example, dicalcium phosphate, tricalcium phosphate and dicalcium phosphate), sodium citrate, water, aqueous solutions (eg, saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (eg, ethyl alcohol, propyl alcohol and benzyl alcohol), polyols ( e.g. glycerin, propylene glycol and polyethylene glycol), organic esters (e.g. ethyl oleate and triglycerides), biodegradable polymers (e.g. polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils ( e.g. corn, wheat germ, olive, castor, sesame, cottonseed and peanut), coconut oil, wax (e.g. suppository wax), paraffin, silicone, talc, silicate, etc.

The compositions of the invention may optionally contain additional ingredients and/or materials commonly used in cosmetic compositions. These ingredients and materials are well known in the art and include, for example, (1) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; (2) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropylmethylcellulose, sucrose and acacia; (3) humectants such as glycerin; (4) disintegrants such as agaragar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates, sodium starch glycolate, cross-linked sodium carboxymethylcellulose and sodium carbonate; (5) solution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols and sodium lauryl sulfate; (10) suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonin, agaragar and tragacanth; (11) buffering agents; (12) excipients such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, coconut oil, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, oxide zinc, aluminum hydroxide, calcium silicates and polyamide powder; (13) inert diluents such as water or other solvents; (14) preservatives; (15) surfactants; (16) dispersing agents; (17) release modulating or absorption retarding agents such as hydroxypropyl methylcellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin and waxes; (18) opacifiers; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3butylene glycol, oils (especially cottonseed, peanut, corn, wheat germ, olive, castor and sesame oil), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and sorbitol fatty acid esters; (23) propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane; (24) antioxidants; (25) agents rendering the composition isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26)

- 21 044823 thickeners; (27) coating materials such as lecithin; and (28) sweeteners, flavors, colors, flavors and preservatives. Each such ingredient or material must be acceptable in the sense of being compatible with the other ingredients of the formulation and not be harmful to the subject. Ingredients and materials suitable for the selected dosage form and intended route of administration are well known in the art, and one skilled in the art can determine suitable ingredients and materials for the selected dosage form and route of administration.

Compositions of the present invention suitable for oral administration may take the form of capsules, sachets, pills, tablets, powders, granules, solution or suspension in an aqueous or non-aqueous liquid, oil-in-water or water-in-oil emulsion, elixir or syrup, lozenge , bolus, electuary or paste. These compositions can be prepared by methods known in the art, for example, using traditional pre-coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.) can be prepared, for example, by mixing the active ingredient(s) with one or more cosmetically or pharmaceutically active carriers and, optionally one or more fillers, extenders, binders, humectants, disintegrants, retarders, absorption enhancers, wetting agents, absorbents, lubricants and/or colorants. Solid compositions of a similar type can be used as fillers in soft and solid-filled gelatin capsules using a suitable excipient. Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surfactant or dispersant. Molded tablets can be produced by molding on a suitable machine. Tablets and other solid dosage forms, such as capsules, pills and granules, may optionally have a break line or be formulated with a coating and coating, such as enteric coating and other coatings well known in the cosmetics art. They can also be formulated to provide a slow or controlled release of the active ingredient they contain. They can be sterilized, for example, by filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifiers and may be of a type of composition in which only the active ingredient is released or, preferably, the release occurs in a specific part of the gastrointestinal tract, optionally in a delayed manner. The active ingredient may also be in microencapsulated form.

Liquid dosage forms for oral administration include cosmetically or pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms may contain suitable inert diluents commonly used in the art. In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, colors, flavoring agents and preservatives. Suspensions may contain suspending agents.

The compositions of the present invention for rectal or vaginal administration may be presented in the form of a suppository, which can be prepared by mixing one or more active ingredients with one or more suitable non-irritating carriers that are solid at room temperature but liquid at body temperature and therefore , melt in the rectal or vaginal cavity to release the active compound. Compositions of the present invention that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or sprays containing such cosmetically or pharmaceutically acceptable carriers as are known in the art as appropriate.

Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops, emulsions, suspensions, aerosols, and inhalers. Any traditional fillers, excipients and, optionally, additional active ingredients can be added to the composition.

Preferred excipients belong to the group consisting of preservatives, antioxidants, stabilizers, solubilizers, vitamins, colors, flavors, film formers, thickeners and humectants.

Solutions and emulsions may contain conventional excipients such as solvents, solubilizers and emulsifiers, for example water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol, oils, in particular cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol fatty acid esters, polyethylene glycols and sorbitol fatty acid esters, or mixtures of these substances.

Emulsions can exist in various forms. Thus, they may be, for example, an emulsion or microemulsion of the water-in-oil (W/O) or oil-in-water (O/W) type, or

- 22 044823 multicomponent emulsion, for example, water in oil in water (W/O/W).

The compositions according to the invention may also be in the form of non-emulsion particulate preparations. For example, they may be hydrodispersions or Pickering emulsions.

Suspensions may contain conventional excipients such as liquid diluents such as water, ethanol or propylene glycol, suspension vehicles such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.

Pastes, ointments, gels and creams may contain traditional excipients, for example, animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures of these substances.

Face and body oils may contain traditional excipients such as synthetic oils such as fatty acid esters, fatty alcohols, silicone oils, natural oils such as vegetable oils and fatty plant extracts, paraffin oils, lanolin oils, or mixtures of these substances. .

Sprays may contain traditional propellants such as CFCs, propane/butane or dimethyl ether.

Compositions of the present invention suitable for parenteral administration contain one or more compounds in combination with one or more cosmetically or pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted in sterile solutions before use or injectable dispersions, which may contain suitable antioxidants, buffers, solutes that render the composition isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper flow can be maintained, for example, by the use of coating materials, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants such as wetting agents, emulsifying agents and dispersing agents. There may also be a need to include isotonic agents. In addition, prolonged absorption of the injectable cosmetic form can be achieved by the inclusion of agents that delay absorption.

In some cases, to prolong the effect it is necessary to slow down the absorption by subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material having poor solubility in water.

The rate of absorption of the active agent/drug will then depend on the rate of its dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered composition can be achieved by dissolving or suspending the active composition in an oil vehicle. Injectable depot forms can be prepared by creating microencapsulated matrices of the active ingredient in biodegradable polymers. Depending on the ratio between the active ingredient and the polymer and the nature of the particular polymer used, the rate of release of the active ingredient can be controlled. Depot injectable formulations are also prepared by incorporating the drug into liposomes or microemulsions that are compatible with body tissues. Injection materials can be sterilized, for example, by filtration through a bacteria-retaining filter.

The compositions of the present invention may be presented in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a lyophilized state, requiring only the addition of a sterile aqueous vehicle, such as water for injection, immediately prior to use. Injectable solutions and suspensions prepared for immediate administration can be prepared from sterile powders, granules and tablets of the type described above.

In the present invention, the term crystalline form means that a compound has a crystalline structure. A compound may exist in one or more crystalline forms, which may have different structural, physical, pharmacological or chemical characteristics. Different crystalline forms can be obtained by variations in nucleation, growth kinetics, agglomeration and breakdown. The formation of crystallization centers occurs when the energy barrier of the phase transition is exceeded, thus allowing the formation of particles from a supersaturated solution. Crystal growth is the enlargement of crystal particles caused by the deposition of a chemical compound on an existing crystal surface. The relative rate of formation of crystallization centers determines the size distribution of the resulting crystals.

The thermodynamic driving force for both nucleation and growth is supersaturation, which by definition is a deviation from thermodynamic equilibrium. Agglomeration is the process of formation of larger particles due to the adhesion of two or more particles (such as crystals) and the formation of a larger crystalline structure.

As used herein, the term hydrate refers to a solid or semi-solid form of a chemical compound containing water in a molecular complex. Water is generally present in stoichiometric amounts relative to the chemical compound.

As used herein, a cosmetically or pharmaceutically acceptable salt refers to a derivative of the compounds described herein, wherein the compounds are modified by converting them into acidic or basic salts. Examples of cosmetically or pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic moieties such as amines; alkaline or organic salts of acidic residues such as carboxylic acids; etc. For example, such salts include those derived from ammonia, L-arginine, betaine, benetamine, benzathine, calcium hydroxide, choline, dinol, diethanolamine (2,2'-iminobis(ethanol)), diethylamine, 2-(diethylamino)-ethanol , 2-aminoethanol, ethylenediamine, N-ethylglucamine, hydrabamine, 1-Himidazole, lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, 1(2-hydroxyethyl)-pyrrolidine, sodium hydroxide, triethanolamine (2 ,2',2-nitrilotris(ethanol)), tromethamine, zinc hydroxide, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 2,5 -dihydroxybenzoic acid, 4-acetamidobenzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, decanoic acid, dodecylsulfuric acid, ethane-1,2 -disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethylenediamonotetraacetic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, D-glucoheptonic acid, D-gluconic acid, D-glucuronic acid, glutamic acid, glutamic acid, glutaric acid, 2 -oxoglutaric acid, glycerophosphoric acid, glycine, glycolic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, DL-lactic acid, lactobionic acid, lauric acid, lysine, maleic acid, (-)-L-malic acid, malonic acid, DLmandelic acid, methanesulfonic acid, galactaric acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, octanoic acid, oleic acid , orotic acid, oxalic acid, palmitic acid, pamoic acid (embonic acid), phosphoric acid, propionic acid, (-)-L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid and undecylenic acid. Additional cosmetically or pharmaceutically acceptable salts may be formed with metal cations such as aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, etc.

Cosmetically or pharmaceutically acceptable salts of the present invention can be synthesized from a compound described herein that contains a basic or acidic component by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or an organic diluent such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile, or a mixture thereof.

It is understood that the compounds and compositions of the present invention may be included in cosmetic or pharmaceutical compositions.

The terminology used herein is used to describe specific embodiments only and is not intended to be limiting. The use of the singular in this specification and in the accompanying claims includes reference to the plural unless the context clearly indicates otherwise.

Where numerical ranges are specified herein, each intervening number within them is expressly implied to the same degree of precision. For example, the range 6-9 implies the numbers 7 and 8 in addition to 6 and 9, and the range 6.0-7.0 explicitly implies the numbers 6.0, 6.1, 6.2, 6.3, 6 ,4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are in no way intended to limit the scope of the invention.

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Examples

Example 1. Materials and methods.

Isolation of compounds produced by Malassezia.

Malassesin is isolated using, for example, the procedures described in Wille et al., 2001. The protocol is briefly described below.

Wednesday.

A medium consisting of Tween 80 (30 ml), cycloheximide (0.5 g), chloramphenicol (0.05 g), agar (20 g) and a volume of water sufficient for 1000 ml of the mixture is sterilized and mixed with 0.3% sterile filtered L-tryptophan at a concentration of 0.3 g% at 50°C, 10 ml portions are poured into 10 cm Petri dishes and the pH is adjusted to 5.5 with 0.1 M HCl.

Cultivation of Malassezia furfur and isolation of compounds produced by M. furfur.

Malassezia furfur is applied to the medium described above and incubated for 14 days at 30°C. The contents of the Petri dish are pureed and extracted with ethyl acetate for 12 hours. The extract is filtered through glass wool, evaporated to dryness and dissolved in methanol. The extract is then fractionated on Sephadex LH-20 with methanol as eluent. Additional separation is carried out using preparative thin layer chromatography using toluene:ethyl formate:formic acid (10:5:3). The main zones are divided between water and ethyl acetate. Fractions are analyzed for the activity of interest. Compounds from the fractions of interest are isolated by HPLC.

Synthesis of malassezin and chemical analogues of malassezin.

Malassesin is synthesized according to the protocol described in Wille et al., 2001. Chemical analogues of Malassesin are synthesized according to new synthesis protocols, as well as those described in Winston-McPherson, et al., 2014.

Screening protocols.

Effective skin lightening compounds are evaluated using both screening protocols known to those skilled in the art and new screening methods. For example, malassesin and its chemical analogues are assessed using the tyrosinase assay as described above. Other screening protocols are also used, involving both in vitro cell and in vivo tissue models, including the aryl-hydrocarbon receptor (AhR) binding assay.

Bioassay with tyrosinase.

The tyrosinase bioassay is performed as described in Wille et al., 2001. Briefly, L-DOPA is mixed with the tyrosinase enzyme. Extinction is measured within 1 min, indicating the formation of dopaquinone. Using, for example, the fractions described above, these fractions are dissolved in DMSO and added directly to the tyrosinase reaction, with pure DMSO serving as a control. Tyrosinase inhibitory activity is measured as a decrease in the increase in extinction compared to the control.

Aryl-hydrocarbon receptor binding assay.

The AhR binding assay is performed according to the protocol described, for example, in Song, et al., 2002. Briefly, human and mouse AhR are expressed in vitro using, for example, the TnT reticulocyte lysate rapid coupling system reaction (Promega, Madison, WI) . Receptor ligand binding studies use rapid sedimentation on a sucrose gradient as described in Karchner, et al., 1999.

EROD analysis.

The compounds, compositions and formulations of the present invention are also evaluated using an ethoxyresorufin-O-deethylase (EROD) assay known to those skilled in the art. (Donato, et al., 1993; Whyte, et al., 2000; Wille et al., 2001).

Melanocyte apoptosis assay.

The candidate compounds are evaluated for apoptosis-inducing activity in melanocytes. Human epidermal melanocytes were cultured in 254 medium supplemented with human melanocyte growth supplement (HMGS) (Thermo-Fisher Scientific, Waltham, MA) or dermal cell basal medium (ATCC, Manassas, VA). Additional components of human melanocyte growth media may include, but are not limited to, insulin (5 μg/ml), ascorbic acid (50 μg/ml), L-glutamine (6 mM), epinephrine (1.0 μM), and calcium chloride (0.2 mM). Human melanocyte cultures are maintained at 37°C in 5% CO2.

Candidate compounds are diluted in DMSO and mixed directly with melanocyte cultures. Equivalent volumes of pure DMSO are used as controls. Cytotoxicity assays known to those skilled in the art are performed in accordance with the manufacturer's instructions. Cytotoxicity assay options useful in the present invention include, but are not limited to, the CellTox™ Green Dye Cytotoxicity Assay, the Apo-ONE Fluorescent Caspase Assay, the ApoTox-Glo™ Assay, and the Caspase-Glo® Assay (Promega, Madison, WI). Fluorescence detection is performed using standard FACS or microscopic analysis known to those skilled in the art, including those described in Kramer, et al., 2005.

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Additional tools are used to assess apoptosis, including FACS analysis for annexin

V and Western blot analysis for caspase-9 expression. Western blotting is carried out in accordance with methods known to those skilled in the art.

Analysis of mouse xenografts.

Mouse xenograft models of human skin are generated according to protocols known in the art. (Black, et al., 1985; Manning et al., 1973; Reed, et al., 1973; Plenat, et al., 1992; Scott et al., 1998; Otulakowski, et al., 1994). After engraftment, murine xenograft models are exposed to the compounds of the present invention and changes in pigmentation compared to controls are observed. Changes in skin pigmentation are assessed using various pigmentation scales known to those skilled in the art, including, but not limited to, the Fitzpatrick Skin Type Test and the Taylor Hyperpigmentation Scale. (Taylor, et al., 2005).

Analysis on humans.

The compounds, compositions and formulations of the present invention are applied to humans, for example, to human skin, and compared with control substances. Changes in skin pigmentation are assessed using various pigmentation scales known to those skilled in the art, including, but not limited to, the Fitzpatrick Skin Type Test and the Taylor Hyperpigmentation Scale.

Example 2.

Biochemical purpose of malassezin and its analogues.

It is expected that the compounds and compositions of the present invention will demonstrate, for example, tyrosinase inhibition and AhR agonist activity comparable to Malassesin. It is expected that the compounds and compositions of the present invention will demonstrate, for example, more effective tyrosinase inhibition and stronger AhR agonism compared to Malassesin. Likewise, some compounds and compositions of the present invention are expected to be less effective tyrosinase inhibitors and AhR agonists compared to Malassesin. Such compounds, compositions and formulations may have preferential toxicity profiles compared to more effective compounds.

Example 3.

In Vitro Efficiency.

The compounds and compositions of the present invention are expected to induce melanocyte apoptosis and modulate melanocyte activity, melanin production, melanosome biogenesis and/or melanosome trafficking at least as effectively as malassesin. It is also expected that certain compounds and compositions of the present invention will act less effectively on these biological processes than Malassesin. Such compounds and compositions may have preferential toxicity profiles compared to more effective compounds.

Example 4.

In Vivo effectiveness.

The compounds and compositions of the present invention are expected to be at least as effective as Malassesin in lightening skin and reducing hyperpigmentation caused by hyperpigmentation disorders. Additionally, it is expected that the compounds and compositions of the present invention will exhibit favorable pharmacokinetic profiles, for example, in terms of half-life and absorption. Some compounds will exhibit a longer half-life, while others will exhibit a shorter half-life. Likewise, certain compounds will exhibit different absorption profiles, with some compounds taking longer to be fully absorbed and others taking less time to be fully absorbed.

Example 5.

Synthesis of malassesin and malassesin derivatives.

Malassesin (CV-8684) and its cyclized derivative indolo[3,2-b]carbazole (CV-8685) were synthesized according to the scheme shown in FIG. 2A.

Synthesis of tert-butyl (2-iodophenyl)carbamate, compound 1.

To a solution of 2-iodoaniline (25.0 g, 0.114 mol) in tetrahydrofuran (250 ml) at 0°C, LiHMDS (251.0 ml, 1 M in THF, 0.251 mol) was slowly added, maintaining the internal temperature below 5°C at for 40 minutes. After 30 minutes of stirring at 0°C, a solution of BOC anhydride (27.0 g, 0.125 mol) in THF (50 ml) was slowly added, maintaining the internal temperature below 5°C for 40 minutes. The reaction mixture was heated to ambient temperature and stirred for 1 hour. A saturated solution of NH ₄ Cl (250 ml) was added to quench the reaction. The organic layer was separated and washed with water (150 ml). The combined aqueous layer was extracted with ethyl acetate (2x150 ml) and the layers were separated. The ethyl acetate layer was combined with the original organic layer and concentrated in vacuo to give a brown oil. The crude product was purified by column chromatography (0-5% ethyl acetate/hexanes). Compound 1 was obtained as a light yellow liquid (29.0 g, 80%).

Synthesis of compound 2.

Copper iodide (0.95 g, 10 mol percent) and PdCl ₂ (PPh ₃ ) ₄ (1/75 g, 5 mol percent) were added to a degassed solution of compound 1 (16.0 g, 0.05 mol), propargyl methyl ether (4.25 g, 0.06 mol) in triethylamine (200 ml) at ambient temperature. After stirring at ambient temperature for 2 hours, the reaction was complete (by TLC using 10% ethyl acetate/hexanes). The reaction mixture was diluted with ethyl acetate (300 ml), washed with water, saturated NaCl solution and dried over Na ₂ SO ₄ . The solvent was filtered and concentrated in vacuo to give a brown oil. The crude product was purified by column chromatography (10% ethyl acetate/hexane). Compound 2 was obtained as a light yellow liquid (13.0 g, 99%).

Synthesis of compound 3.

PtCl ₂ (0.26 g, 0.001 mol), Na ₂ CO ₃ (1.6 g, 0.015 mol), indole (2.32 g, 0.02 mol) and compound 2 (2 .6 g, 0.01 mol) in dioxane (120 ml). The flask was degassed with nitrogen, sealed and heated to 100°C overnight. The reaction was then complete (as determined by TLC using 10% ethyl acetate/hexanes). The solvent was evaporated under reduced pressure. The reaction mixture was diluted with ethyl acetate (200 ml), washed with water, saturated NaCl solution and dried over Na ₂ SO ₄ . The solvent was filtered and concentrated in vacuo to give a brown oil.

This reaction was repeated using compound 2 (2.6 g, 0.01 mol) from another batch. Crude compounds from both batches were combined and purified by column chromatography (10% ethyl acetate/hexane). Compound 3 was obtained as a light brown solid (3.8 g, 55%).

Synthesis of compound 4.

Potassium carbonate (4.6 g, 0.0329 mol) was added to a solution of compound 3 (3.8 g, 0.0109 mol) in a mixture of methanol (150 ml) and water (50 ml) at ambient temperature. The resulting suspension was heated to reflux overnight. The reaction was then complete (as determined by TLC using 20% ethyl acetate/hexanes). The reaction mixture was cooled to ambient temperature and concentrated with the solvent under vacuum. The residue was dissolved in ethyl acetate (200 ml) and washed with water and brine, then dried (sodium sulfate), filtered, concentrated with the solvent in vacuo to give a brown solid. The crude compound was purified by column chromatography (20% ethyl acetate/hexane). Compound 4 was obtained as an orange solid (2.2 g, 81%).

Synthesis of the compound malassesin (CV-8684).

Dimethylformamide (20 mL) was added to a dry 100 mL two-neck round bottom flask under argon at 0°C. POCl ₃ (0.75 g, 0.0048 mol) was added slowly while maintaining the internal temperature below 5°C for 10 minutes. After 30 min of stirring at 0°C, a solution of compound 4 (1.0 g, 0.004 mol) in dimethylformamide (5 ml) was slowly added, maintaining the internal temperature below 5°C for 10 min. The resulting mixture was stirred at ambient temperature overnight. The reaction was then complete (as determined by TLC using 20% ethyl acetate/hexanes). The reaction mixture was poured into saturated aqueous sodium bicarbonate (150 ml) and stirred for 1 hour. The resulting mixture was extracted with ethyl acetate (2x100 ml). The organic layers were combined and washed with water, saturated NaCl solution and dried over Na ₂ SO ₄ . The solvent was filtered and concentrated in vacuo to obtain a brown solid. The crude product was purified by column chromatography (0-20% ethyl acetate/hexanes). The compound Malassesin (CV-8684) was obtained as a light pink solid (0.82 g, 74%).

HPLC purification: 97.8% (area%). ¹ H-NMR, ¹³ C spectrum corresponding to the structure. IER-MS: Calc. for C ₁₈ H ₁₅ N ₂ O (M+H)+: 275, obtained value: 275.2.

Synthesis of the indolo[3,2-b]carbazole compound (CV-8685).

Concentrated HCl (0.25 ml) was added to a solution of malassesin (0.75 g) in tetrahydrofuran (120 ml) at ambient temperature. The resulting mixture was heated to reflux overnight. The reaction was then complete (as determined by TLC using 40% ethyl acetate/hexanes). The reaction mixture was cooled to ambient temperature and stirred for 1 hour. The solid was filtered, washed with tetrahydrofuran (20 ml) and dried to give indolo[3,2-b]carbazole (CV-8685) as a light yellow solid ( 0.55 g, 78%).

HPLC purification: 96.22% (area%). ¹ H-NMR, ¹³ C spectrum corresponding to the structure. IERMS: Calc. for C ₁₈ H ₁₃ N ₂ (M+H)+: 257, obtained value: 257.5.

Compound I (CV-8686) and Compound IV (CV-8687) were synthesized according to the scheme shown in FIG. 2B.

Synthesis of compound 5.

PtCl ₂ (1.0 g, 0.0038 mol), Na ₂ CO ₃ (6.1 g, 0.057 mol), 6-methylindole (10.0 g, 0.076 mol) and compound 2 were added to the oven-dried flask (10.0 g, 0.038 mol) in dioxane (250 ml). flask

- 27 044823 degassed with nitrogen, sealed and heated to 100°C overnight. The reaction was then complete (as determined by TLC using 10% ethyl acetate/hexanes). The solvent was evaporated under reduced pressure. The reaction mixture was diluted with ethyl acetate (400 ml), washed with water, saturated NaCl solution and dried over Na ₂ SO ₄ . The solvent was filtered and concentrated in vacuo to give a brown oil. The crude compound was purified by column chromatography (10% ethyl acetate/hexane). Compound 5 was obtained as a light brown solid (6.5 g, 47%).

Synthesis of compound 6.

Potassium carbonate (7.4 g, 0.054 mol) was added to a solution of compound 5 (6.5 g, 0.018 mol) in a mixture of methanol (150 ml) and water (50 ml) at ambient temperature. The resulting suspension was heated to reflux overnight. The reaction was then complete (as determined by TLC using 20% ethyl acetate/hexanes). The reaction mixture was cooled to ambient temperature and concentrated with the solvent under vacuum. The residue was dissolved in ethyl acetate (200 ml) and washed with water and brine, then dried (sodium sulfate), filtered, concentrated with the solvent in vacuo to give a brown solid. The crude compound was purified by column chromatography (20% ethyl acetate/hexane). Compound 6 was obtained as an orange solid (3.3 g, 72%).

Synthesis of compound I (CV-8686).

Dimethylformamide (20 mL) was added to a dry 100 mL two-neck round bottom flask under argon at 0°C. POCl ₃ (0.6 g, 0.0038 mol) was added slowly while maintaining the internal temperature below 5°C for 10 minutes. After 30 min of stirring at 0°C, a solution of compound 6 (1.0 g, 0.0038 mol) in dimethylformamide (5 ml) was slowly added, maintaining the internal temperature below 5°C for 10 min. The resulting mixture was stirred at ambient temperature overnight. The reaction was then complete (as determined by TLC using 20% ethyl acetate/hexanes). The reaction mixture was poured into saturated aqueous sodium bicarbonate (150 ml) and stirred for 1 hour. The resulting mixture was extracted with ethyl acetate (2x100 ml). The organic layers were combined and washed with water, saturated NaCl solution and dried over Na ₂ SO ₄ . The solvent was filtered and concentrated in vacuo to obtain a brown solid. The crude product was purified by column chromatography (0-20% ethyl acetate/hexanes). Compound I (CV-8686) was obtained as a light pink solid (0.84 g, 75%).

HPLC purification: 97.01% (area%). ¹ H-NMR, ¹³ C spectrum corresponding to the structure. IERMS: Calc. for C ₁₉ H ₁₇ N ₂ O (M+H)+: 289, obtained value: 289.1.

Synthesis of compound IV (CV-8687).

Concentrated HCl (0.3 ml) was added to a solution of compound I (1.0 g) in tetrahydrofuran (125 ml) at ambient temperature. The resulting mixture was heated to reflux overnight. The reaction was then complete (as determined by TLC using 40% ethyl acetate/hexanes). The reaction mixture was cooled to ambient temperature and stirred for 1 hour. The solid was filtered, washed with tetrahydrofuran (20 ml) and dried to give compound IV (CV-8687) as a light yellow solid (0.84 g, 89% ).

HPLC purification: 98.4% (area%). ¹ H-NMR, ¹³ C spectrum corresponding to the structure. IER-MS: Calc. for C ₁₉ H ₁₅ N ₂ (M+H)+: 271, obtained value: 271.3.

Compound II (CV-8688) was synthesized according to the scheme shown in FIG. 2C.

Synthesis of compound 7.

Copper iodide (0.53 g, 10 mol percent) and PdCl ₂ (PPh ₃ ) ₄ (1/0 g, 5 mol percent) were added to the degassed solution of compound 1 (9.0 g, 0.03 mol), 3- methoxy-1-butyne (2.8 g, 0.035 mol) in triethylamine (150 ml) at ambient temperature. The mixture was then stirred at ambient temperature for 2 hours. The reaction was complete (TLC using 10% ethyl acetate/hexanes). The reaction mixture was diluted with ethyl acetate (300 ml), washed with water, saturated NaCl solution and dried over Na2SO4. The solvent was filtered and concentrated in vacuo to give a brown oil. The crude product was purified by column chromatography (10% ethyl acetate/hexane). Compound 7 was obtained as a light yellow liquid (7.0 g, 90%).

Synthesis of compound 8.

PtCl ₂ (0.68 g, 0.0025 mol), Na ₂ CO ₃ (4.0 g, 0.038 mol), indole (6.0 g, 0.05 mol) and compound 7 were added to the oven-dried flask (10.0 g, 0.025 mol) in dioxane (250 ml). The flask was degassed with nitrogen, sealed and heated to 100°C overnight. The reaction was then complete (as determined by TLC using 10% ethyl acetate/hexanes). The solvent was evaporated under reduced pressure. The reaction mixture was diluted with ethyl acetate (400 ml), washed with water, saturated NaCl solution and dried over Na2SO4. The solvent was filtered and concentrated in vacuo to give a brown oil. The crude compound was purified by column chromatography (10% ethyl acetate/hexane). Compound 8 was obtained as a light brown solid (3.5 g, 77%).

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Synthesis of compound 9.

Potassium carbonate (3.8 g, 0.027 mol) was added to a solution of compound 8 (3.3 g, 0.0091 mol) in a mixture of methanol (75 ml) and water (25 ml) at ambient temperature. The resulting suspension was heated to reflux overnight. The reaction was then complete (as determined by TLC using 20% ethyl acetate/hexanes). The reaction mixture was cooled to ambient temperature and concentrated with the solvent under vacuum. The residue was dissolved in ethyl acetate (200 ml) and washed with water and brine, then dried (sodium sulfate), filtered, concentrated with the solvent in vacuo to give a brown solid. The crude compound was purified by column chromatography (20% ethyl acetate/hexane). Compound 9 was obtained as an orange solid (2.1 g, 88%).

Synthesis of compound II (CV-8688).

Dimethylformamide (20 mL) was added to a dry 100 mL two-neck round bottom flask under argon at 0°C. POCl ₃ (0.76 g, 0.005 mol) was added slowly while maintaining the internal temperature below 5°C for 10 minutes. After 30 min of stirring at 0°C, a solution of compound 9 (1.3 g, 0.005 mol) in dimethylformamide (5 ml) was slowly added, maintaining the internal temperature below 5°C for 10 min. The resulting mixture was stirred at ambient temperature overnight. The reaction was then complete (as determined by TLC using 20% ethyl acetate/hexanes). The reaction mixture was poured into saturated aqueous sodium bicarbonate (150 ml) and stirred for 1 hour. The resulting mixture was extracted with ethyl acetate (2x100 ml). The organic layers were combined and washed with water, saturated NaCl solution and dried over Na ₂ SO ₄ . The solvent was filtered and concentrated in vacuo to obtain a brown solid. The crude compound was crystallized in chloroform (25 ml). Compound II (CV-8688) was obtained as a light pink solid (0.81 g, 53%).

HPLC purification: 98.94% (area%). ¹ H-NMR, ¹³ C spectrum corresponding to the structure. IERMS: Calc. for C ₁₉ H ₁₇ N ₂ O (M+H)+: 289, obtained value: 289.0.

Example 6.

Cell morphology.

Typical cell morphology after different treatments is shown in Fig. 5A-5K, 6A-6K, 7A7K, 8A-8K, 9A-9K, 10A-10K, 11A-11K and 12A-12K. The morphology of both cell lines was significantly affected by treatment with 100 μM CV-8684 and CV-8688, as well as by treatment with staurosporine after 6 h. CV-8685 had an effect only on WM115 at 100 μM.

Example 7.

Apoptosis-inducing activity of malassesin and malassesin derivatives - preliminary analysis with annexin V.

Materials and reagents.

Annexin V-FITC assay kit was purchased from Beyotime Biotechnology, RPMI 1640 medium and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco, fetal bovine serum (FBS) was purchased from Invitrogen, stabilized antifungal solution with antibiotic (100x) was purchased from Sigma, and 0.25% trypsin-EDTA (1X) phenol red were purchased from Invitrogen.

Cell culture.

MeWo (ATCC® HTB-65™) and WM115 (ATCC® CRL-1675) cells were purchased from ATCC (Manassas, VA) and maintained under the following conditions: for MeWo: DMEM supplemented with 10% FBS; for WM115: RPMI 1640, supplemented with 10% FBS (10% FBS, 1% stabilized antifungal solution with antibiotic).

Review of the study.

During intermediate stages of apoptosis, phosphatidylserine (PS) moves from the inner to the outer layer of the cell membrane, exposing PS to an extracellular environment in which it can be detected. Highly fluorescent Annexin V conjugates provide rapid and reliable detection methods for PS externalization studies.

During the first set of studies, MeWo and WM115 cells were treated with test compounds in 10 doses, starting at 100 μM with a 3-fold dilution. Staurosporine was used as a positive control. After 6 hours of treatment, cell apoptosis was assessed using the Annexin V assay. The test compounds assessed were CV-8684, CV-8685, CV-8686, CV8687 and CV-8688.

Analytical procedures.

For cell seeding, cells were harvested and cell numbers were determined using a Countess® cytometer. Then the cells were diluted with culture medium to the required density. 40 μl of cell suspension per well was added to the required number of wells in a 384-well plate (Corning 3712 - clear bottom plate). The final cell density was 6000 cells/well. After seeding, the plates were incubated at 37°C and 5% CO2 overnight.

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To prepare a compound source plate, each test compound was dissolved in DMSO to make a 10 mM stock solution. A 3-fold serial dilution was performed using an EVO200™ Fluid Handler (TECAN) to create ten concentrations of the test compound. 0.1% DMSO was used as a control (negative) vehicle. The compound source plate was then centrifuged at room temperature at 1000 rpm for 1 min and shaken using a plate shaker for 2 min.

For compound treatment, 40 nL of compound was transferred from the compound source plate to a 384-well culture plate using an Echo550 fluid handler (LabCyte Inc.). After 6 hours of incubation, the plates were removed from the incubator for detection.

For the Annexin V pre-assay, plates were removed from the incubator and allowed to equilibrate at room temperature for 15 min. Then the culture medium was removed. 20 μl of a premixed working solution of annexin V-FITC and Hoechst33342 dye was added to each well. The cells were then incubated at room temperature for 20 min. The plates were sealed and centrifuged for 1 min at 1000 rpm to remove bubbles. The plates were then read using an Acumen eX3 plate reader. Relative activity was calculated using the following formula: Activity (%)=100%x( _{Annexin v} number/ _B c _e cell number), and EC ₅₀ was calculated using GraphPad Prism (v. 5.01).

Results.

In the preliminary screening assay described above, CV-8688 markedly increased annexin V staining of MeWo cells with an EC _{of 50,908.57} nM. Staurosporine, a positive control, strongly increased annexin V staining in both cell lines. (Figures 3A-3M).

Example 8.

Apoptosis-inducing activity of Malassesin and Malassesin derivatives—further assessed by Annexin V assay.

Review of the study.

To further examine the effect of the test compounds on apoptosis, multiple data acquisitions covering different stages of apoptosis were performed for MeWo and WM115 cells. Both cell types were treated with test compounds in 3 doses (100 μM, 10 μM and 1 μM). Staurosporine was used as a positive control. After the required treatment period (6, 24, 48 or 72 h), apoptosis was assessed by measuring the percentage of cells exhibiting annexin V binding after exposure to the test compounds. The test compounds evaluated were CV-8684, CV-8685 and CV-8688.

Analytical procedures.

Cell seeding was performed as described above with the following exceptions: the final cell density was 4000 cells/well for 6-hour and 24-hour detection, whereas 2000 cells/well was used for 48-hour and 72-hour detection. For each time point, clear-bottom 384-well plates (Corning 3712) and white-bottom hard plates (Corning 3570) were prepared. The plates were incubated as described above.

To prepare a compound source plate, each test compound was dissolved in DMSO to make a 10 mM stock solution. Two additional concentrations were obtained by 10-fold dilution to 1 mM and 0.1 mM. Staurosporine was used as a positive control and 1% DMSO was used as a baseline (negative) control. The source plate of the compound was centrifuged at room temperature at 1000 rpm for 1 min and shaken using a plate shaker for 2 min.

400 nL of test compound was transferred from the compound source plate to 384-well culture plates using an Echo550 liquid manipulator. After 6, 24, 48, and 72 h, the plates were removed from the incubator for detection.

For the Annexin V assay, plates were removed from the incubator and allowed to equilibrate at room temperature for 15 min. The culture medium was removed and the cells were washed twice with PBS. 20 μL of premixed Annexin VFITC working solution was added to each well. Cells were incubated at room temperature for 20 min. The plates were read using Acumen eX3 to count the number of FITC-positive cells. Relative activity was calculated using the following formula:

Relative activity (%) = 100% x (number _{of sample} / number _{of filler} ).

Results.

CV-8684 induced apoptosis at the highest concentration tested after 6 h of treatment in both MeWo and WM115 cells. CV-8685 after 24 h of treatment showed an induction effect on WM115, while 48 h of treatment induced apoptosis in both cell types. Finally, CV-8688, after 6 h of treatment, demonstrated an induction effect in both cell types in a dose-dependent manner (Fig. 4A-4L).

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Example 9.

Cell viability after exposure to malassesin and malassesin derivatives - CellTiter-Glo® assay.

Analytical procedures.

CellTiter-Glo ® 2.0 Assay Kit was purchased from Promega. Cell seeding, preparation of the compound source plate, and exposure of the cells to the test compounds were carried out as described in example 8.

For the CellTiter-Glo® assay, plates were removed from the incubator and equilibrated at room temperature for 15 min. CellTiter-Glo® reagents were thawed and brought to room temperature before experimentation. 40 µl of CellTiterGlo® detection reagent was then added to each well (1:1 ratio with culture medium). The plates were then incubated at room temperature for 30 min and read using an EnSpire plate reader (PerkinElmer). Residual activity was calculated using the following formula:

Residual activity (%)=100%x (Lum _sample -Lumf _he )/(Lum _filler -Lumf _he ).

Results.

CV-8684 exhibited dose-dependent inhibition of cell viability in both cell lines tested, although the inhibitory effect was stronger in MeWo cells. CV-8685 exhibited an inhibitory effect on WM115 cell viability in a dose-dependent manner only after 24-h treatment. CV-8688 inhibited the viability of both cell types in a dose-dependent manner. Staurosporine, a positive control, showed 100% inhibition of cell viability in both cell lines after 24-hour treatment. (Figs. 13A-13K).

Example 10.

Cytotoxicity of Malassesin and Malassesin derivatives lactade dehydrogenase release assay.

Review of the study.

The LDH assay provides a quantitative measurement of lactate dehydrogenase (LDH) released into the media from damaged cells as a biomarker of cytotoxicity and cytolysis.

Analytical procedures.

The CytoTox-ONE™ Homogeneous Membrane Integrity Assay Kit was purchased from Promega. Cell seeding, preparation of the compound source plate, and exposure of the cells to the test compounds were carried out as described in example 8.

For LDH release assay, plates were removed from the incubator and equilibrated at room temperature for 15 min. The plates were then centrifuged at 1000 rpm for 1 min. 20 μl of cell culture medium was transferred to a new 384-well black hard plate. 20 µl of CytoTOX-ONE™ was then added to each well and incubated at room temperature for 10 min. After this, 10 μl of stop solution was added to each well and the plates were shaken at 500 rpm for 1 min. The plates were read using an excitation wavelength of 560 nm and an emission wavelength of 590 nm on an EnSpire. Relative activity was calculated using the following formula:

Relative activity (%)=100%x (Lum _sample -Lumf _he )/(Lum _filler -Lumf _he ).

Results.

CV-8684 did not induce significant release in any of the cell lines after 72 hours of incubation. CV-8685 showed a dose-dependent induction effect on LDH release from WM115, but not MeWo, cells after 24-h treatment. CV-8688 induced LDH release at the highest concentration tested. (Fig. 14A-14L).

Example 11.

Efficiency of aryl-hydrocarbon receptor activation for malassesin and malassesin derivatives.

Analytical procedures.

HepG2-AhR-Luc cells were purchased from Pharmaron, OneGlo luciferase assay system was purchased from Promega, DMEM was purchased from Hyclone, and penicillin/streptomycin was purchased from Solabio.

Culture medium for stably transfected HepG2 cells was prepared by supplementing DMEM with large amounts of glucose and L-glutamine, as well as 10% FBS.

HepG2-AhR-Luc cells were cultured in T-75 flasks at 37°C, 5% CO ₂ and 95% relative humidity. Cells were allowed to reach 80–90% confluence before detachment and separation.

The cultured cells were washed with 5 ml of PBS. The PBS was aspirated back, 1.5 ml of trypsin was added to the flask, and the cells were incubated at 37°C for approximately 5 min or until the cells detached and floated. Trypsin was activated by adding excess serum-containing medium.

The cell suspension was transferred into a conical tube and centrifuged at 120 g for

- 31 044823 min for cell sedimentation. Cells were resuspended in seeding medium at the required density.

µl of cells were transferred into a 384-well culture plate (5x103 cells/well). The plates were placed in an incubator at 37°C for 24 h.

After this, stock solutions of the test compounds and omeprazole were prepared as a positive control. 40 nL of compound solutions were transferred to an assay plate using an Echo550. The plate was then placed back into the incubator for compound treatment.

Later, after 24 h of treatment, the plate was removed from the incubator and allowed to cool to ambient temperature. 30 μl of One-Glo reagent equivalent to that in the culture medium was added to each well. Cells were allowed to undergo lysis for at least 3 minutes and then measured on a luminometer.

Responses were plotted against dose using nonlinear regression analysis in XLfit and EC ₅₀ values were also calculated.

Results.

The results of the AhR-luciferase assay are shown in FIG. 15A-15F.

Example 12.

MelanoDerm™ analysis.

Review of the study.

The purpose of this study is to evaluate the potential skin irritation caused by the study drug in the MelanoDerm™ skin model after repeated exposure, to guide dose selection for a subsequent study. Toxicity will be determined by measuring the relative conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in tissues treated with the study drug compared to tissues treated with a negative control/control solution.

This study will use the MelanoDerm™ skin model supplied by MatTek Corporation (Ashland, MA). MelanoDerm™ tissue is composed of human-derived normal epidermal keratinocytes (NEKCs) and melanocytes (NMPs) that have been cultured to form a multilayered, highly differentiated human epidermal model. NMPs in co-cultures undergo spontaneous melanogenesis, leading to the formation of tissues with different levels of pigmentation. Cultures are grown on cell culture inserts at the air-liquid interface, which allows for topical application of skin modulators. The MelanoDerm™ model exhibits in vivo-like morphological and ultrastructural characteristics. NMPs localized in the basal cell layer of MelanoDerm™ tissues are dendritic and spontaneously produce melanin granules that progressively populate the tissue layers. Thus, the research system can be used to screen for materials that may inhibit or stimulate melanin production compared to a negative control.

The experimental design of this study consists of determining the pH of pure study drugs, if possible (and/or dosing solution as appropriate), and a determination assay to determine the relative viability of tissues after repeated exposure. The MelanoDerm™ skin model will be exposed to the study drugs for only 7 days. The study drug will be applied topically to a skin model of MelanoDerm™ every 48 hours (within a time frame of 48±2 hours from the previous treatment). Study drug toxicity will be determined by NAD(P)H-dependent microsomal enzymatic reduction of MTT (and, to a lesser extent, succinate dehydrogenase-induced reduction of MTT) in control and test drug-treated tissues. (Berridge et al., 1996). Data will be presented in the form of relative survival (MTT converted versus negative control).

Materials.

MelanoDerm™ maintenance medium (EPI-100-LLMM) and MelanoDerm™ skin model (MEL-300A) were supplied by MatTek Corporation. 1% kojic acid (prepared in sterile deionized water) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were supplied by Sigma. Dulbecco's modified Eagle's medium (DMEM) containing 2 mM L-glutamine (MTT supplement medium) was supplied by Quality Biological. Isopropanol was supplied by Aldrich. Sterile Ca++ and Mg~ free Dulbecco's phosphate buffered saline (CMF-DPBS) was supplied by Invitrogen or a similar supplier. Sterile deionized water was supplied by Quality Biological or a similar supplier. DMSO was supplied by CiVenti Chem.

Analytical procedures.

Investigational drugs will generally be tested alone or as directed by the Sponsor (see Protocol Appendix 1). Ten microliters (10 µl) or 25 µl of each investigational drug will be applied directly to the tissue to cover the top surface. Depending on the nature of the drug being studied (liquids, gels, creams, foams, etc.), the use of a dosing device, sieve, or other auxiliary device may be necessary.

- 32 044823 to ensure uniform distribution of the test drug over the tissue surface.

On application days, each study drug will be diluted at least 200-fold using the appropriate volume of EPI-100-LLMM (or an alternative diluent determined during the solubility study). For each dosing, a fresh dilution will be prepared in EPI-100-LLMM. The final dilution to be made for the dosing solution preparation will be determined from the solubility assessment mentioned above and recorded in the study workbook.

DMSO diluted to 0.5% (v/v) in EPI-100-LLMM will be used as vehicle control and applied to tissues (at doses of 10 μL and 25 μL) based on the same procedure as for study drugs and analytical control.

Study drugs will be applied topically to MelanoDerm™ tissue every 48 hours (within a time frame of 48 ± 2 hours from the previous treatment) during the 7-day study. Ten and 25 microliters, respectively, of each study drug will be applied to each tissue. Twenty-five microliters of positive and negative controls, respectively, will be applied to each tissue.

If possible, the pH of the pure liquid study drug (and/or dosing solution as appropriate) will be determined. The pH will be determined using a pH test paper (eg, with a pH range of 0-14 for estimation and/or a pH range of 5-10 for more precise determination). Typical pH increment values on narrower range pH indicator paper are approximately 0.3 to 0.5 pH units. The maximum increment on pH indicator paper is 1.0 pH units.

The determination assay will include a negative control and a positive control. MelanoDerm™ tissues designated as an analytical negative control will be treated with 25 µl of sterile deionized water. Twenty-five microliters of 1% kojic acid (prepared in sterile deionized water and filtered during preparation) will be used to apply to tissues designated as analytical positive controls. 1% kojic acid will be stored in a tube covered with aluminum foil until use within 2 hours of preparation time. The exposure times of the positive and negative controls will be identical to those used for the study drugs.

The ability of each investigational drug to directly reduce MTT should be assessed. Prepare a 1.0 mg/mL solution of MTT in MTT supplement medium as described below. Approximately 25 µl of the test compound will be added to 1 ml of MTT solution and incubate the mixture in the dark at 37 ± 1°C for 1 to 3 hours. A negative control of 25 µl of sterile deionized water will be tested at the same time. If the color of the MTT solution turns blue/purple, the test drug is believed to reduce MTT. Water-insoluble test materials may exhibit direct reduction (darkening) only at the interface between the test drug and the medium.

Determination of direct MTT reduction for the test compound(s) may have been previously performed in an independent study. In such cases, the results of the direct MTT reduction study can be used in this particular study with reference to the original study.

Tissue Exposure: At least 16 hours after culture initiation, photographs of two MelanoDerm™ tissues (considered untreated at day 0) will be taken using a digital camera to assist with visual assessment of the degree of pigmentation of the tissues at time zero of analysis. The exact procedures used to obtain tissue images will be described in the workbook and study report. MelanoDerm™ tissues will be washed with CMF-DPBS, dry blotted onto sterile absorbent paper and cleared of excess fluid. MelanoDerm™ tissues will be transferred to appropriate MTT-containing wells after washing and processed in the MTT assay as described in the MTT assay section.

At least 16 hours after culture initiation, tissues will be transferred to a new 6-well plate containing 0.9 ml of fresh, prewarmed EPI-100-LLMM. The study will be conducted over a 7-day period. Two tissues will be topically treated on the first day and every 48 hours (within a time frame of 48 ± 2 hours from the previous treatment) with 10 and 25 microliters, respectively, of each study drug. The medium will be refreshed daily (within a time frame of 24±2 hours from the previous treatment); tissues will be transferred to a new 6-well plate containing 0.9 ml of fresh, prewarmed EPI-100-LLMM.

Two tissues will be topically treated on the first day and every 48 hours (within a time frame of 48 ± 2 hours from the previous treatment) with 25 microliters of positive and negative control, respectively. The medium will be refreshed daily (within a time frame of 24±2 hours from the previous treatment); tissues will be transferred to a new 6-well plate containing 0.9 ml of fresh, prewarmed EPI-100-LLMM. Tissues will be incubated at 37±1°C in a humidified atmosphere

- 33 044823

5±1% CO2 in air (standard culture conditions) for appropriate exposure time.

On application days, MelanoDerm™ tissue will first be gently washed three times with ~500 μL CMF-DPBS to remove any residual test drug. Tissues will then be transferred to a new 6-well plate containing 0.9 ml of fresh, pre-warmed EPI-100LLMM and treated with the appropriate test drug, negative or positive control. Tissues will be incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) for the appropriate exposure time. The exact flushing procedure will be indicated in the work log.

At the end of the 7-day study, MelanoDerm™ tissues treated with negative or positive control and each study drug will be photographed using a digital camera to assist with visual assessment of the degree of tissue pigmentation at the end of the assay (Day 7). The exact procedures used to obtain tissue images will be described in the workbook and study report. Tissue viability will then be determined by MTT recovery as outlined below.

MTT Assay: A 10X stock solution of MTT prepared in PBS (filtered during batch preparation) will be thawed and diluted in warm MTT supplement medium to obtain a 1.0 mg/mL solution, no more than 2 hours prior to use. 300 µl of MTT solution will be added to each designated well of a pre-labeled 24-well plate.

At the end of the exposure time, each MelanoDerm™ tissue intended for MTT analysis will be washed with CMF-DPBS, dry blotted onto sterile absorbent paper, and cleared of excess fluid. MelanoDerm™ tissues will be transferred to the appropriate MTT-containing wells after washing. 24-well plates will be incubated under standard conditions for 3±0.1 hours.

After 3±0.1 hours, MelanoDerm™ tissues will be blotted onto sterile absorbent paper, cleared of excess liquid, and transferred to a pre-labeled 24-well plate containing 2.0 ml of isopropanol in each designated well. The plates will be covered with parafilm and stored in a freezer (2-8°C) until samples are collected for the final exposure time. If necessary, plates can be stored overnight (or up to 24 h after sample collection for the last exposure time) in the freezer before MTT extraction. The plates will then be shaken for at least 2 hours at room temperature. At the end of the extraction period, the liquid in the cell culture inserts will be decanted into the well from which the cell culture insert was taken. The extract solution will be mixed and 200 µL will be transferred to the appropriate well of a 96-well plate. I will add two hundred microliters of isopropanol to the wells designated as blank control. The absorbance at 550 nm (OD550) for each well will be measured using a Molecular Devices Vmax plate reader (with AUTOMIX enabled).

In cases where a study drug is shown to restore MTT, the only problem is those study drugs that remain bound to the tissue after washing, resulting in a false signal of MTT recovery. To demonstrate that the effect of a possible residual test drug does not directly result in MTT restoration, a functional test was performed in a determination assay to show that the test material does not bind to tissue and does not lead to a spurious MTT restoration signal.

To determine whether the effects of residual test drug resulted in MTT restoration, freeze-killed control tissue was used. Freeze-killed tissue is prepared at IIVS by placing untreated MelanoDerm™/EpiDerm™ (Melanoderm™ without melanocytes) tissue in a freezer at -20°C at least overnight, thawing to room temperature, and then refrozen. Once killed, the tissue can be stored indefinitely in the freezer. Freeze-killed tissue can be obtained ready-made from MatTek Corporation and stored in a freezer at -20°C until use. To examine the residual reduction in the amount of the test drug, killed tissues are treated with the test drug in the usual manner. Analytical procedures will be carried out in the same manner as for viable tissue. At least one killed control treated with sterile deionized water (negative killed control) will be examined in parallel, as some recovery of MTT from residual NADH and associated enzymes in the killed tissue is expected.

If little or no MTT recovery is observed in the test drug-treated killed control, the observed MTT recovery in the test drug-treated viable tissue can be attributed to viable cells. If significant MTT recovery is observed in study drug-treated killed controls (compared to treated viable tissue), additional steps must be taken to account for chemical recovery of the study drug, or the study drug may be considered non-investigational within the system. OD550 values for killed controls will be analyzed as described below.

Raw absorption data will be recorded and saved as a print file and imported into an Excel spreadsheet. The average OD550 value for empty wells will be calculated. The adjusted mean OD550 for negative controls will be determined by subtracting the mean OD550 for the blank wells from their mean OD550 values. Adjusted OD550 values for individual test drug exposure and positive control exposure will be determined by subtracting the average OD550 value for the blank wells from each. All calculations will be carried out using a table in Excel format. Although the algorithms discussed are used to calculate endpoint analysis at the treatment group level, the same calculations can be applied to individual runs.

Accor. OD550 for exposure to the study drug = OD550 for exposure to the study drug - Average OD550 for empty wells.

If a killed control (QC) is used, the following additional calculations will be performed to adjust for the amount of MTT recovered directly from the study drug residues. The raw OD550 value for the negative control-treated killed control will be subtracted from the raw OD550 values for each of the test-treated killed controls to determine the net OD550 values for the test-treated killed controls.

Net OD550 for each UC treated with the study drug = Initial OD550 treated with the study drug UC - Initial OD550 treated with the negative control UC.

Net OD550 values represent the amount of MTT reduced due to direct reduction by test drug residues at specific exposure times. In general, if the net OD550 value is greater than 0.150, the net MTT recovery value will be subtracted from the adjusted OD550 values of the viable treated tissues to obtain the final adjusted OD550 value. These final adjusted OD550 values will then be used to determine % viability of control samples.

Final adjusted OD550=Adjusted OD550 for test compound (viable samples) - Net OD550 for test compound (UC).

Finally, the following % of control calculations will be carried out:

% viability=[(Final adjusted OD550 for test drug or positive control)/(Adjusted mean OD550 for negative control)] x 100

Results.

MelanoDerm™ analysis results are shown in FIG. 16A-16K. Tissues treated with Malassesin, Compound I and Compound II showed a decrease in pigmentation on day 7 of the experiment. In fig. 17A-17K show 15X magnification images of MelanoDerm™ samples subjected to the indicated treatments.

Example 13.

Zebrafish assay.

Analytical procedures.

Compounds: Compounds will be provided by the Study Sponsor as stock solution (BS) at the highest soluble concentration in water/PBS or DMSO.

Standard Embryo Retrieval Procedures: Phylonix AB zebrafish will be produced through natural mating or using a mass embryo production system (MEPS, Aquatic Habitats). There will be approximately 50 newborn zebrafish per female zebrafish. The zebrafish will be kept in fish water at 28°C. The zebrafish will be cleaned (dead zebrafish removed) and sorted by developmental stage. Since zebrafish receive their nutrition from the attached yolk sac, no nutrition is required until 6 days after fertilization (dpo).

Solubility of compounds: The main stock solution (SS) (using the highest concentration) will be diluted in pure DMSO to obtain additional stock solutions (DS), i.e.: 10, 50, 100, 200, 300 mM, etc. Fish water [200 mg sea salt from Instant Ocean (Aquarium Systems) per liter of deionized water; pH 6.6-7.0, maintained with 2.5 mg/L neutral regulator (Seachem Laboratories Inc.); conductivity 850-950 µS] supplied by Phylonix will be dispensed into a test bottle, 4 ml/bottle.

To create a test compound (TS) solution, 4 μL of each TS will be added directly to the fish water. Example: 4 µl of 10 mM DR added to fish water will produce 10 µM IR; the final concentration of DMSO will be 0.1%. Alternatively, to obtain the same final concentrations of TS and DMSO, 10 µl of TS can be added to 10 ml/container of fish water. For assays where up to 1% DMSO may be acceptable, 40 µL of IR can be used to obtain 100 µM IR. When using 10 ml of fish water, the volume of TS should be proportionally increased to obtain the same final concentrations of TS and DMSO. The solution will be incubated at 28°C for the length of time specified for each assay and

- 35 044823 visually inspect daily for the presence of sediment.

Maximum Tolerated Concentration (MTC): The MIC (LC10) will be used as the standard criterion for lethality of a compound, determined by 10 concentrations of the compound. After determining the highest soluble concentration of a compound, the Study Sponsor will select 10 concentrations.

Thirty ~2 dpo chorionic wild-type AB zebrafish from Phylonix will be dispensed into wells of 6-well microplates containing 4 ml/well water and DMSO at a concentration ranging from 0.1-1% depending on the solubility of the compound.

Initially, 10 concentrations will be tested (i.e. 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 and 500 µM (or up to the concentration that the solubility of the compound allows) If necessary, higher (up to 2000 µM) or lower (up to 0.001 µM) concentrations will be investigated.

Zebrafish will be incubated with each concentration of test compound in the dark at 28°C for 3 days. Untreated and 0.1-1% DMSO treated zebrafish will be used as analytical and vehicle controls. To calculate % mortality after treatment, the number of dead zebrafish will be counted and removed. At 5 dpo, dead animals will be counted to calculate % lethality (= total number of dead zebrafish/30). It should be noted that in case of decomposition of dead zebrafish, the number of dead zebrafish will be calculated by counting the number of live zebrafish.

To estimate MICs, lethality curves will be generated by plotting % lethality versus concentration using EXCEL software. To obtain the average value and CO for the MIC, experiments will be carried out 3 times.

Visual assessment of the effect of the compound on zebrafish skin pigmentation: Zebrafish skin pigment cells, including xanthophores, iridophores and melanophores (melanocytes), are derived from neural crest cells. In zebrafish, differentiated skin pigment progenitor cells express pigment at ~24 dpo. This study focuses on melanocytes, which express melanin, the black pigment on the surface of the skin. Melanocytes first appear as small black areas on the back of the head. As zebrafish develop, the number of such regions increases and they merge to form stripes that extend toward the tail region. In contrast, albino mutant zebrafish exhibit scattered skin pigmentation. Compounds will be administered at 2 dpo to evaluate whether the compounds can arrest the ongoing process of embryonic pigmentation that is completed by 5 dpo. For each compound, three concentrations will be tested - MIC, 50% MIC and 25% MIC.

Thirty 2 dpo self-hatched AB wild-type zebrafish from Phylonix will be treated with a concentration of each compound for 3 days. Untreated and 0.1% DMSO treated zebrafish will be used as controls. Positive control: phenylthiourea (PTM, 0.03%).

The zebrafish will be visually examined daily using a dissecting microscope; Compound- and FTM-treated zebrafish will be compared with untreated and vehicle-treated control zebrafish. The number of zebrafish showing decreased pigmentation will be counted daily and expressed as a % of the animals studied; will provide representative images. To determine the optimal compound concentration and treatment time to reduce pigmentation, a kinetic curve will be constructed by plotting the % of zebrafish exhibiting decreased skin pigmentation versus time (dpo). Fisher's exact test (P<0.05) will be used to determine whether the effect of a compound is significant.

Additional visual assessment of the effect of the compound on zebrafish skin pigmentation will be carried out after treatment: 0.1, 1 and 3 μM. 32 dpo self-hatched AB wild-type zebrafish from Phylonix will be treated with a concentration of each compound for 3 days. Untreated and 0.1% DMSO treated zebrafish will be used as controls. Positive control: phenylthiourea (PTM, 0.003%). The zebrafish will be visually examined daily using a dissecting microscope; Compound- and FTM-treated zebrafish will be compared with untreated and vehicle-treated control zebrafish.

At 5 dpo, the number of zebrafish showing decreased pigmentation will be counted and expressed as a % of the animals studied; will provide representative images. To determine the optimal compound concentration and treatment time to reduce pigmentation, a kinetic curve will be constructed by plotting the % of zebrafish exhibiting reduced skin pigmentation versus concentration. Fisher's exact test (P<0.05) will be used to determine whether the effect of a compound is significant.

Quantitative effect of the compound on zebrafish skin pigmentation. Based on the visual assessment results, we will use optimal conditions (concentration, compound treatment time) to quantify the effect of the compound on zebrafish skin pigmentation.

Twenty wild-type AB zebrafish from Phylonix at the optimal stage, as determined by visual assessment, will be treated with the optimal concentration of compound. Untreated and 0.1% DMSO treated zebrafish will be used as controls. Positive control:

- 36 044823 phenylthiourea (PTM, 0.03%).

Full posterior images of the zebrafish will be obtained using a SPOT camera at 2X magnification. The posterior region of the head and torso will be defined as a region of interest (ROI) using the selection function in Adobe Photoshop. The black pigmentation on the skin in the ROI will be highlighted using the selection function in Photoshop. The total pigment signal (TS) in pixels will be determined using the histogram function in Photoshop.

If the connection affects the growth of the zebrafish, the length (L) of the body and the width (W) of the trunk will be smaller, which will affect the RO area and the final PS value. Therefore, we will normalize the final signal (CS) measurement using CS=FS/LxN.

Untreated and vehicle-treated zebrafish are expected to exhibit similar CS to indicate that the vehicle has no effect. FTM-treated zebrafish are expected to exhibit low CS to validate the assay. Compound-treated zebrafish will be compared to vehicle-treated control zebrafish.

To determine whether the effect of a compound is significant (P<0.05), the mean CS of compound-treated zebrafish will be compared with the mean CS of vehicle-treated zebrafish using a Student's t test.

Additional quantitative assessment of the effect of the compound on zebrafish skin pigmentation will be carried out after treatment with concentrations of compounds of 0.5 and 1.5 μM.

dpo wild-type AB zebrafish from Phylonix will be treated with 0.5 and 1.5 µM concentrations of compounds. Untreated and 0.1% DMSO treated zebrafish will be used as controls. Positive control: phenylthiourea (PTM, 0.003%).

Full posterior images of the zebrafish will be obtained using a SPOT camera at 2X magnification. The back of the head will be defined as a region of interest (ROI) using the selection function in Adobe Photoshop. The black pigmentation on the skin in the ROI will be highlighted using the selection function in Photoshop. The total pigment signal (TS) in pixels will be determined using the histogram function in Photoshop.

If the connection affects the growth of zebrafish, the length (L) of the body will be shorter and the width (W) of the body will be smaller, which will affect the RO area and the final PS value. Therefore, we will normalize the final signal (KS) using KS=FS/LxN.

Untreated and vehicle-treated zebrafish are expected to exhibit similar CS to confirm the lack of effect of the vehicle. FTM-treated zebrafish are expected to exhibit low CS, allowing assay validation. Compound-treated zebrafish will be compared to vehicle-treated control zebrafish.

To determine whether the effect of a compound is significant (P<0.05), the mean CS of compound-treated zebrafish will be compared with the mean CS of vehicle-treated zebrafish using a Student's t test.

Results.

The results of visual assessment of zebrafish exposed to Compound II are shown in FIG. 18A-18F and FIGS. 19A-19F. A table with summarized results for the visual assessment part of the study is shown in Fig. 20.

Quantification of the ROIs and results for zebrafish exposed to Compound II are shown in FIG. 21A-21E and FIGS. 22A-22B.

Example 14.

Stability of malassezin and malassezin derivatives in DMSO and cell culture medium.

The test compounds were prepared at a concentration of 100 μM in DMSO and culture medium. The solutions were incubated at room temperature for 2 h and analyzed by LC-MS. Peak area was used to estimate the compound remaining in the solvent.

Results.

The LC-MS results are shown in FIG. 23A-23J. These results indicate that the compounds are stable in the culture medium after two hours of incubation.

- 37 044823

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Claims (14)

Winston-McPherson, et al. Synthesis and Biological Evaluation of 2,3'-diindolylmethanes as Agonists of Aryl Hydrocarbon Receptor. Bioorganic & Medicinal Chemistry Letters 2014; 24:4023–4025. Whyte, et al. Ethoxyresorufin-O-deethylase (EROD) Activity in Fish As a Biomarker of Chemical Exposure. Critical Reviews in Toxicology 2000; 30(4):347-570. Yamaguchi, et al. Melanocytes and Their Diseases. Cold Spring Harb Perspect Med 2014; 4:a017046. Zonios, et al. Skin Melanin, Hemoglobin, and Light Scattering Properties can be Quantitatively Assessed In Vivo Using Diffuse Reflectance Spectroscopy. J Invest Dermatol. 2001; 117: 1452-1457. All documents cited in this invention are incorporated herein by reference as if they were cited in their entirety. Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to these described embodiments, and various other changes or modifications may be made by those skilled in the art without departing from the scope and spirit of the invention. CLAIM 1. A cosmetic composition for skin lightening containing a compound having the structure of formula (II) where Rp R2, _R3 , R4, R5, _R6 , _R7 , R8, _R9 , _R10 and Rn are independently selected from the group consisting of hydrogen and methyl; or a cosmetically acceptable salt, and a cosmetically acceptable excipient, diluent or carrier, and further comprising one or more ingredients selected from the group consisting of fragrance, estrogen, vitamin A, vitamin C, vitamin E, pyruvic acid, lactic acid, glycolic acid, lanolin, petrolatum, aloe vera, methylparaben, propylparaben and pigments. 2. The composition according to claim 1, characterized in that it is a composition for topical or transdermal administration, selected from the group consisting of powders, sprays, ointments, pastes, creams, lotions, gels, patches, emulsions, suspensions, aerosols and inhalers . 3. Cosmetic composition according to claim 2, characterized in that the compound is selected from the group consisting of: - 40 044823 or a cosmetically acceptable salt thereof. 4. The composition according to claim 3, characterized in that the compound is or a cosmetically acceptable salt thereof. 5. A method of lightening the skin of a human subject, comprising applying to the human subject a compound selected from the group consisting of: or a cosmetically or pharmaceutically acceptable salt thereof. 6. A method for lightening the skin of a human subject, comprising applying to the human subject a composition according to claim 1. 7. A method for lightening the skin of a human subject, comprising applying to the human subject a composition according to claim 2. 8. The method according to claim 7, characterized in that the compound is selected from the group consisting of: and a cosmetically or pharmaceutically acceptable salt thereof. 9. The method according to claim 5, characterized in that the connection is: - 41 044823 or a cosmetically or pharmaceutically acceptable salt thereof. 10. The method according to claim 8, characterized in that the connection is or a cosmetically or pharmaceutically acceptable salt thereof. 11. Pharmaceutical composition for skin lightening containing a compound having the structure of formula (II) r ₄ (P) where R _b R2, R ₃ , R ₄ , R5, R ₆ , R ₇ , R8, R9, R ₁₀ and Rn are independently selected from the group consisting of hydrogen and methyl; or a pharmaceutically acceptable salt, and a pharmaceutically acceptable excipient, diluent or carrier, and further containing one or more ingredients selected from the group consisting of fragrances, estrogen, vitamin A, vitamin C, vitamin E, pyruvic acid, lactic acid, glycolic acid, lanolin, petrolatum, aloe vera, methylparaben, propylparaben and pigments. 12. The pharmaceutical composition according to claim 11, characterized in that it is a composition for topical or transdermal administration selected from the group consisting of powders, sprays, ointments, pastes, creams, lotions, gels, patches, emulsions, suspensions, aerosols and inhalers. 13. Pharmaceutical composition according to claim 12, characterized in that the compound is selected from the group consisting of: or a pharmaceutically acceptable salt thereof. 14. The composition according to claim 13, characterized in that the compound is -