Field of Invention

The present invention relates to the field of skin whitening compositions. Specifically, the invention relates to: compounds that act as inhibitors of tyrosinase and/or melanogenesis; compositions comprising the compounds; and methods/uses using the compounds and compositions.

Background

There is a large market worldwide for skin whitening agents, especially in Asia. It is estimated that approximately 15% of the world’s population use skin whitening agents (Pillaiyar et ai, J Enzyme Inhib Med Chem. 2017; 32(1): 403-425). As explained in Pillaiyar, melanin is primarily responsible for the pigmentation of human skin, and melanin is produced from epidermis melanocytes by melanogenesis. A precursor for the production of melanin is L-tyrosine, and L-tyrosine is used to produce melanin by the action of tyrosinase ( via the Raper Mason pathway). Since tyrosinase is produced only in melanocytes, inhibition of tyrosinase is a promising target for skin whitening, and most commercially available cosmetic skin lightening agents are tyrosinase inhibitors.

However, currently available synthetic tyrosinase inhibitors result in undesired side effects, such as skin irritation and cytotoxicity. In addition, known naturally occurring tyrosinase inhibitors are expensive to isolate and generally have poor stability. For example, Arndt, Kenneth A., and Thomas B. Fitzpatrick, Jama 1949 (1965): 965-967 reported that 5 % hydroquinone creams used to treat hyperpigmentation caused skin irritation as a side effect. Kojic acid has been used in the cosmetic industry as a skin whitening agent but is reported to have carcinogenic effects and stability issues during storage (Fujimoto, Nariaki, et al. Carcinogenesis 20.8 (1999): 1567-1572). 20% solutions of azelaic acid have been used to treat melisma but side effects of itching, burning, and scaling were reported (Balina, Luis M., and Klaus Graupe, International journal of dermatology 30.12 (1991): 893-895). Tranexamic acid is another compound used to treat melisma, and causes the side effects nausea/diarrhoea and, orthostatic reactions (Tse, Tsz Wah, and Edith Hui, Journal of cosmetic dermatology 12.1 (2013): 57-66). There is a need for an easy to manufacture tyrosinase inhibitor that does not produce significant undesired side effects. There is also a need for an inhibitor which can be manufactured and/or isolated in an environmentally friendly way.

Although previous literature studies have suggested that extracts of Wrightia antidysenterica do not have significant activity against tyrosinase (Ito et ai, Data Brief. 2018 Oct; 20: 573- 576), the inventors have surprisingly found that certain compounds extracted from Wrightia antidysenterica are highly active against tyrosinase.

Summary of Invention

Specifically, the inventors have surprisingly found that extracts from the plant Wrightia antidysenterica, particularly extracts from the leaves of Wrightia antidysenterica, contain compounds having potent tyrosinase inhibition activity. As such, these compounds can act as inhibitors of melanogenesis and therefore produce a skin whitening effect.

Extracts from the leaves of Wrightia antidysenterica have been used as a traditional medicine in communities in Malaysia, Indonesia and Sri Lanka to treat skin disorders such as psoriasis and nonspecific dermatitis (but not to provide a skin whitening effect). These extracts have proved to be well tolerated by the skin and to have few side effects. It can therefore be concluded that the compounds within Wrightia antidysenterica have low cytotoxicity. The low cytotoxicity of certain compounds isolated from Wrightia antidysenterica has also been confirmed by the present inventors in the below Examples.

Due to the surprising tyrosinase inhibition activity identified by the present inventors, combined with the tolerability and low cytotoxicity shown both by traditional uses and the present inventors, compounds extracted from Wrightia antidysenterica can be used to provide skin whitening without causing significant undesired side effects.

Accordingly, the present invention provides the following.

1 Use of a compound of formula (I):

wherein Ai , A ₂ , A ₃ , A ₄ and A ₅ are each independently hydrogen or -O-Z, where each Z is independently hydrogen, or a mono-, di- or tri-saccharide connected to the oxygen atom via a glycosidic bond,

or a physiologically acceptable salt thereof,

in the manufacture of an agent for whitening a subject’s skin.

2. The use according to Clause 1 , wherein each glycosidic bond is independently an Clglycosidic bond or a C-glycosidic bond.

3. The use according to Clause 1 or 2, wherein when Ai , A ₂ and/or A ₄ are a mono-, di- or tri-saccharide, they are connected to the remainder of the molecule via an O-glycosidic bond.

4. The use according to any one of the preceding clauses, wherein A ₃ and As are hydrogen.

5. The use according to any one of the preceding clauses, wherein A ₂ and A ₄ are each a hydroxyl group.

6. The use according to any one of the preceding clauses, wherein Ai is a trisaccharide. 7. The use according to any one of the preceding clauses, wherein: A ₃ and A ₅ are hydrogen;

A ₂ and A ₄ are each a hydroxyl group; and

Ai is a tri-saccharide. 8. The use according to Clause 1 , 2 or 5, wherein when A ₃ and/or As are a mono-, di- or tri-saccharide, they are connected to the remainder of the molecule via a C-glycosidic bond.

9. The use according to any one of Clauses 1 , 2, 5 or 8, wherein A ₃ and As are a mono saccharide.

10. The use according to any one of Clauses 1 , 2, 5, 8 or 9, wherein Ai is hydrogen.

11 . The use according to any one of Clauses 1 , 2, 5, 8, 9 or 10, wherein:

A ₃ and A ₅ are a mono-saccharide;

A ₂ and A ₄ are each a hydroxyl group; and

Ai is hydrogen.

12. The use according to any one of the preceding clauses, wherein each mono-, di- or tri-saccharide respectively is composed of one, two or three glucose moieties.

13. Use of a compound of Formula (la) or (lb):

(la) (lb) or a glycoside or physiologically acceptable salt thereof, in the manufacture of an agent for whitening a subject’s skin.

14. The use according to Clause 13, wherein the compound has the formula (la) or (lb):

(la) (lb)

or a physiologically acceptable salt thereof.

15. The use according to Clause 13, wherein the compound has the formula (la):

(la),

or a glycoside or physiologically acceptable salt thereof.

16. Use of a compound of formula (III)

or a glycoside or physiologically acceptable salt thereof,

in the manufacture of an agent for whitening a subject’s skin.

17. The use according to Clause 16, wherein the compound is of formula (III):

or a physiologically acceptable salt thereof.

18. A compound of formula (II): or a physiologically acceptable salt thereof, wherein:

each Ri is independently selected from a Ci~e alkyl group and a phenyl group, each of which may be unsubstituted or substituted with one, two or three groups selected from the group consisting of F, Cl, Br, methyl, ethyl, hydroxyl and amino,

R ₂ is a Ci alkyl group, which may be unsubstituted or substituted by one, two or three groups selected from the group consisting of F, Cl, Br, methyl, ethyl, hydroxyl and amino,

R ₃ is hydrogen, Ci-e alkyl or -O-Ci- ₆ alkyl;

n is an integer of from 1 to 3, and

m is an integer of from 0 to 4.

19. The compound according to Clause 18, or physiologically acceptable salt thereof, wherein at most one Ri is phenyl.

20. The compound according to Clause 18 or 19, or physiologically acceptable salt thereof, wherein m is an integer of from 0 to 2.

21. The compound according to any one of Clauses 18 to 20, or physiologically acceptable salt thereof, wherein m is 0.

22. The compound according to any one of Clauses 18 to 21 , or physiologically acceptable salt thereof, wherein n is i .

23. The compound according to any one of Clauses 18 to 22, or physiologically acceptable salt thereof, wherein R ₂ is hydrogen, methyl or ethyl, optionally wherein R ₂ is methyl.

24. The compound according to any one of Clauses 18 to 23, or physiologically acceptable salt thereof, wherein R ₃ is hydrogen. 25. The compound according to any one of Clauses 18 to 23, or physiologically acceptable salt thereof, wherein:

Ri is -Ci- ₆ alkyl or phenyl;

R _{2 IS} -C1-4 alkyl;

R ₃ is hydrogen;

n is an integer of from 1 to 3; and

m is an integer of from 0 to 4.

26. The compound according to any one of Clauses 18 to 25, wherein the compound of formula (II) has the formula:

or a physiologically acceptable salt thereof. 27. A compound of formula (IV):

or a physiologically acceptable salt thereof, wherein R is hydrogen, Ci-e alkyl, -C(=0)-Ci- ₆ alkyl, or a mono-, di- or tri-saccharide connected to the oxygen atom via a glycosidic bond.

28. A compound according to Clause 27, wherein R is hydrogen, -C1 -4 alkyl, -C(=0)-Ci- ₄ alkyl or a mono-, di- or tri-saccharide connected to the oxygen atom via a glycosidic bond, optionally wherein R is hydrogen.

29. A composition comprising a compound or physiologically acceptable salt or glycoside as described in any one of Clauses 18 to 28 and at least one physiologically acceptable excipient.

30. The composition according to Clause 29, wherein the composition is a cosmetic composition, a food or drink composition or a supplement composition.

31. A skin whitening composition comprising a compound or physiologically acceptable salt or glycoside as described in any one of Clauses 1 to 28.

32. The composition according to any one of Clauses 29 to 31 , comprising two or more compounds or physiologically acceptable salts or glycosides as described in any one of Clauses 1 to 27, and at least physiologically acceptable excipient.

33. Use of a compound as defined in any one of Clauses 18 to 28 in the manufacture of an agent for whitening a subject’s skin.

34. The use according to any one of Clauses 1 to 17 and 33, wherein the agent is for topical or oral administration.

35. A method of inhibiting tyrosinase in a subject, said method comprising a step of administering to said subject an effective amount of a compound or physiologically acceptable salt or glycoside as described in any one of Clauses 1 to 28, or a composition according to any one of Clauses 29 to 32.

36. A method of inhibiting melanogenesis in a subject, said method comprising a step of administering to said subject an effective amount of a compound or physiologically acceptable salt or glycoside as described in any one of Clauses 1 to 28, or a composition according to any one of Clauses 29 to 32. 37. A method of whitening a subject’s skin, said method comprising a step of administering to said subject an effective amount of a compound or physiologically acceptable salt or glycoside as described in any one of Clauses 1 to 28, or a composition according to any one of Clauses 29 to 32.

38. The method according to any one of Clauses 35 to 37, wherein the compound, physiologically acceptable salt, glycoside or composition is administered topically or orally.

39. The method according to any one of Clauses 35 to 38, which is a non-therapeutic and/or cosmetic method.

40. Use of a compound as described in any one of Clauses 1 to 28 as a skin whitening agent.

41. Use of a compound as described in any one of Clauses 1 to 28, in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of a hyperpigmentation disorder, lentigo, vitiligo, skin cancer and a neurodegenerative disorder.

42. The use according to Clause 41 , wherein the skin cancer is a melanoma.

Brief Description of Drawings

Fig. 1 Depicts the HPLC chromatogram of 17 fractions from the Wrightia antidysenterica extract (first batch), with optimal eluent gradient and detection at 254 nm.

Fig. 2 Depicts the tyrosinase inhibition activity of each fraction from the Wrightia antidysenterica extract (first batch).

Fig. 3 Depicts the UV spectra of: (a) fraction 1 1 ; (b) fraction 14; and (c) fraction 15 from the Wrightia antidysenterica extract (first batch).

Fig. 4 Depicts the mass spectra (MS) and proposed structure of the active compound in fraction 11 from the Wrightia antidysenterica extract (first batch): (a) ESI-MS spectrum in negative ion mode; (b) MS-MS spectrum in negative ionization mode; and (c) proposed chemical structure of the active compound in fraction 11. Fig. 5 Depicts the ESI-MS spectra of fraction 14 from the Wrightia antidysenterica extract (first batch) in negative ion (top) and positive ion mode (bottom).

Fig. 6 Depicts: (a) high-resolution ESI-MS spectrum in negative ion mode; (b) MS-MS spectrum in negative mode; and (c) proposed fragmentation pathway of the active compound (a kaempferol-3-O-glycoside derivative) in fraction 14 from the Wrightia antidysenterica extract (first batch).

Fig. 7 Depicts: (a) ¹ H NMR spectrum of fraction 14 from the Wrightia antidysenterica extract (first batch); and (b) proposed isomeric structures of the kaempferol-3-O-glycoside derivative from fraction 14.

Fig.8 Depicts: (a) ESI-MS spectrum; and (b) high-resolution ESI-MS spectrum of fraction 15 from the Wrightia antidysenterica extract (first batch) in positive ion mode.

Fig. 9 Depicts ESI-MS-MS spectra of fraction 15 from the Wrightia antidysenterica extract (first batch) in positive ion mode.

Fig. 10 Depicts: (a) ESI-MS spectrum of fraction 15 from the Wrightia antidysenterica extract (first batch) in negative ion mode; and (b) the proposed chemical structure of the active compound in fraction 15.

Fig. 11 Depicts the ESI-MS-MS spectra of fraction 15 from the Wrightia antidysenterica extract (first batch) in negative ion mode.

Fig. 12 Depicts the ¹ H NMR spectrum of fraction 15 from the Wrightia antidysenterica extract (first batch).

Fig. 13 Depicts the ¹³ C NMR spectrum of fraction 15 from the Wrightia antidysenterica extract (first batch).

Fig. 14 Depicts: (a) HPLC chromatogram from the first round of semi-preparative HPLC of the Wrightia antidysenterica extract (second batch); and (b) tyrosinase inhibition activity of the respective 9 fractions in (a). Fig. 15 Depicts: (a) HPLC chromatogram from the second round of semi-preparative HPLC of fraction 7 (from the first round of HPLC separation) of the Wrightia antidysenterica extract (second batch); and (b) tyrosinase inhibition activity of the respective 8 fractions in (a).

Fig. 16 Depicts: (a) the LC-MS spectrum; and (b) ESI-MS spectrum of fraction 4 from the second round of HPLC purification of the Wrightia antidysenterica extract (second batch) in negative ion mode.

Fig. 17 Depicts: (a) the high-resolution ESI-MS spectrum; and (b) ESI-MS-MS spectrum of fraction 4 from the second round of HPLC purification of the Wrightia antidysenterica extract (second batch) in negative ion mode.

Fig. 18 Depicts the ¹ H NMR spectrum of fraction 4 from the second round of HPLC purification of the Wrightia antidysenterica extract (second batch) in negative ion mode.

Fig. 19 Depicts the proposed chemical structure of the active compound in fraction 4 from the second round of HPLC purification of the Wrightia antidysenterica extract (second batch).

Fig. 20 Depicts the IC ₅ o of isolated compounds in comparison with that of crude dried leave extracts of a in inhibiting tyrosinase.

Fig. 21 Depicts data used to calculate the IC50 of the known tyrosinase inhibitor Kojic acid.

Fig. 22 Depicts cytotoxicity assay results for three compounds isolated from Wrightia antidysenterica.

Description

The invention provides methods and uses involving compounds of formula (I), (la), (lb), (II), (III), and (IV), and physiologically acceptable salts thereof, and physiologically acceptable glycosides of compounds of formula (la), (lb) and (III). The invention also provides compounds of formula (II), and (IV), and physiologically acceptable salts thereof, as well as compositions comprising the compounds. Specifically, the invention provides methods of using the compounds and uses of the compounds to inhibit tyrosinase, inhibit melanogenesis and provide a skin whitening effect, and use of the compounds as skin whitening agents. A skilled person will understand that skin whitening refers to the lightening the colour of a subject’s skin, and does not require that the skin becomes white.

wherein each Ri is independently selected from a Ci~ ₆ alkyl group and a phenyl group, each of which may be unsubstituted or substituted with one, two or three groups selected from the group consisting of F, Cl, Br, methyl, ethyl, hydroxyl and amino, R2 is a C1-4 alkyl group, which may be unsubstituted or substituted by one, two or three groups selected from the group consisting of F, Cl, Br, methyl, ethyl, hydroxyl and amino,

R3 is hydrogen, C1-6 alkyl or -O-Ci-6 alkyl;

n is an integer of from 1 to 3, and

m is an integer of from 0 to 4.

wherein R is hydrogen, C1-6 alkyl, -C(=0)-Ci- ₆ alkyl, or a mono-, di- or tri-saccharide connected to the oxygen atom via a glycosidic bond.

In particular, the invention provides the following methods and uses of the compounds of formula (I), (la), (lb), (II), (I II), and (IV), 1. Use of a compound, physiologically acceptable salt or glycoside described herein in the manufacture of an agent for whitening a subject’s skin.

2. A compound, physiologically acceptable salt or glycoside described herein for use in a method of whitening a subject’s skin.

3. A method of whitening a subject’s skin, comprising a step of administering to said subject an effective amount of a compound, physiologically acceptable salt or glycoside described herein.

In embodiments of the invention, the above methods and uses are non-therapeutic and/or cosmetic methods or uses.

In some embodiments of the invention, the compound of formula (II) has the formula:

or a physiologically acceptable salt or glycoside thereof.

In some embodiments of the invention, the compound of formula (II) does not have the formula:

or a physiologically acceptable salt or glycoside thereof.

In some embodiments of the invention, the compound of formula (IV) has the formula: or a physiologically acceptable salt or glycoside thereof.

In some embodiments of the invention, the compound of formula (IV) does not have the formula:

or a physiologically acceptable salt or glycoside thereof.

In embodiments herein, the word“comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word“comprising” may be replaced by the phrases“consists of or “consists essentially of). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word“comprising” and synonyms thereof may be replaced by the phrase“consisting of” or the phrase“consists essentially of or synonyms thereof and vice versa.

“Saccharide” as used herein refers to a moiety composed of one or more sugars. A sugar molecule generally has an empirical formula CnF nOn. A skilled person will understand that the saccharide substituents mentioned herein are formed from the removal of a hydrogen atom from a saccharide molecule, and will generally have the empirical formula C _n Fb _n -iO _n . For example, when a moiety mentioned herein is said to be a mono-, di- or tri-saccharide, it is a moiety formed from removing a hydrogen atom from a mono-, di- or tri-saccharide molecule.

A mono-saccharide is a moiety formed from a single sugar, and is also known as a simple sugar. Examples of mono-saccharides include pentose and hexose sugars. An example of a pentose sugar that may be mentioned herein is ribose. Examples of hexose sugars that may be mentioned herein include glucose, galactose, mannose and fructose. A skilled person will understand that these are merely examples of mono-saccharides/sugars and that other sugar moieties may be included in the compounds of formula (I), (la), (lb), (II), (III), and (IV).

A di-saccharide is a moiety formed from two mono-saccharides, i.e. from two simple sugars, where the two mono-saccharides are bonded together by a glycosidic bond. Any two mono saccharides may be combined to form a di-saccharide. Examples of common di-saccharides include sucrose (formed from glucose and fructose), lactose (formed from glucose and galactose) and maltose (formed from two glucose moieties).

A tri-saccharide is a moiety formed from three mono-saccharides, i.e. from three simple sugars, where the three mono-saccharides are bonded together by a glycosidic bond.

The point of attachment between two sugars may vary, and different di- and tri-saccharides may be formed from the same constituent mono-saccharides, depending on the location of the bond between the mono-saccharides. A skilled person will understand that when compounds of formula (I), (la), (lb), (II), (III), or (IV) comprise a di- or tri-saccharide, they may be formed from mono-saccharides linked any possible position. An example of this phenomenon is seen in the compounds of formula (la) and (lb) disclosed herein, where both formulae comprise a tri-saccharide moiety composed of three glucose moieties, but which glucose moieties are linked via different atoms in each formula.

A glycosidic bond is a bond formed between a sugar moiety and another moiety (which may be a sugar or non-sugar). A glycosidic bond may be via an oxygen atom (O-glycosidic bond), carbon atom (C-glycosidic bond), or when present, via a S atom or N atom. In an O- glycosidic bond, the bond is typically formed between the hemiacetal or hemiketal group carbon of the sugar moiety and an oxygen atom of the other moiety, and is a C-0 bond. In a C-glycosidic bond, the bond is formed between a carbon atom of one moiety and a carbon atom in the other moiety, i.e. is a C-C bond.

"Alkyl" refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, /so-propyl, n-butyl, /so-butyl, n-hexyl, and the like. As used herein, C1 -C5 alkyl refers to an alkyl group having 1 to 5 carbon atoms.

References herein (in any aspect or embodiment of the invention) to compounds of formula formula (I), (la), (lb), (II), (III), or (IV) includes references to such compounds per se, to tautomers of such compounds, as well as to physiologically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.

Physiologically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula formula (I), (la), (lb), (II), (III) or (IV) with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula (I), (la), (lb), (II), (III) or (IV) in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

Examples of physiologically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium. Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulfonic acids (e.g. benzenesulfonic, naphthalene-2- sulfonic, naphthalene-1 , 5-disulfonic and p-toluenesulfonic), ascorbic (e.g. L-ascorbic), L- aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+)-camphoric, camphor-sulfonic, (+)-(1 S)- camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1 , 2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), ooxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L- malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, tartaric (e.g.(+)-L- tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulfonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

As mentioned above, also encompassed by formula (I), (la), (lb), (II), (III) or (IV) are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of formula (I), (la), (lb), (II), (III) or (IV), of molecules of a non-toxic physiologically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulfoxide. Solvates can be prepared by recrystallising the compounds of formula (I), (la), (lb), (II), (III) or (IV) with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates. For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

“Pharmaceutically functional derivatives” of compounds of formula (I), (la), (lb), (II), (III) or (IV) as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of formula (I), (la), (lb), (II), (III) or (IV). Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula (I), (la), (lb), (II), (III) or (IV).

The term“prodrug” of a relevant compound of formula (I), (la), (lb), (II), (III) or (IV) includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).

Prodrugs of compounds of formula (I), (la), (lb), (II), (III) or (IV) may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formula (I), (la), (lb), (II), (III) or (IV) wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula (I), (la), (lb), (II), (III) or (IV) is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. I-92, Elsevier, New York-Oxford (1985).

Compounds of formula (I), (la), (lb), (II), (III) or (IV), as well as physiologically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds of formula (I)” or “compounds of formula (II)”.

Compounds of formula (I), (la), (lb), (II), (III) or (IV) may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention. Compounds of formula (I), (la), (lb), (II), (III) or (IV) may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ‘chiral pool’ method), by reaction of the appropriate starting material with a‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

In particular, when a compound of formula (I), (la), (lb), (II), (III) or (IV) comprises a mono-, di- or tri-saccharide moiety, the compound of formula (I), (la), (lb), (II), (III) or (IV) will comprise multiple asymmetric carbon atoms. Typically, the mono-, di- or tri-saccharide will be composed of naturally occurring sugar moieties, i.e. the D-form of said natural sugar.

Further embodiments of the invention that may be mentioned include those in which the compound of formula (I), (la), (lb), (II), (III) or (IV) is isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compound of formula (I), (la), (lb), (II), (III) or (IV) is not isotopically labelled.

The term "isotopically labelled", when used herein includes references to compounds of formula (I), (la), (lb), (II), (III) or (IV) in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound" will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula (I), (la), (lb), (II), (III) or (IV). Thus, the term "isotopically labelled" includes references to compounds of formula (I), (la), (lb), (II), (III) or (IV) that are isotopically enriched at one or more positions in the compound.

The isotopic labelling or enrichment of the compound of formula (I), (la), (lb), (II), (III) or (IV) may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and/or iodine. Particular isotopes that may be mentioned in this respect include ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ 0, ¹⁷ 0, ¹⁸ 0, ³⁵ S, ¹⁸ F, ³⁷ CI , ⁷⁷ Br, ⁸² Br and ¹²⁵ l).

When the compound of formula (I), (la), (lb), (II), (I II) or (IV) is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula (I), (la), (lb), (II), (III) or (IV) that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or non-radioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or nonradioactive isotope.

The term“effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

For the avoidance of doubt, in cases in which the identity of two or more substituents in a compound of formula (I), (la), (lb), (I I), (I I I) or (IV) may be the same, the actual identities of the respective substituents are not in any way interdependent.

Compounds of formula (I), (la), (lb), (II), (I II) or (IV) may be administered by any suitable route, but may particularly be administered orally or topically, for example as an ointment, cream or gel.

Compounds of formula (I), (la), (lb), (I I), (I II) or (IV) may be synthesised using routine synthetic routes and methods, or extracted from parts (e.g. leaves) of the plant Wrightia antidysenterica. Derivatives of compounds naturally occurring in Wrightia antidysenterica may be prepared from the isolated naturally occurring compounds using routine synthetic routes and methods.

Compounds of formula (I), (la), (lb), (I I), (I I I) or (IV) are potent inhibitors of tyrosinase, and are well tolerated by the human body, having minimal side effects and cytotoxicity. As such, they may be used in the treatment of diseases in which inhibition of tyrosinase is indicated, such as a hyperpigmentation disorder, lentigo, vitiligo and skin cancer (Deri, B., Kanteev, M. , Goldfeder, M. et al. The unravelling of the complex pattern of tyrosinase inhibition. Sci Rep 6, 34993 (2016)). Tyrosinase inhibitors are also proposed as agents for treating Parkinson’s disease and Huntingdon’s disease (Pillaiyar T, Manickam M, Namasivayam V. Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors. J Enzyme Inhib Med Chem. 2017;32(1):403-425). A skilled person will understand that the appropriate claim language for such uses of compounds described herein varies depending on the territory in question. Accordingly, the present invention provides:

1. Use of a compound described herein in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of a hyperpigmentation disorder, lentigo, vitiligo, skin cancer and a neurodegenerative disorder.

2. A compound described herein for use in the treatment of a disease or disorder selected from the group consisting of a hyperpigmentation disorder, lentigo, vitiligo, skin cancer and a neurodegenerative disorder.

3. A method of treating a disease or disorder selected from the group consisting of a hyperpigmentation disorder, lentigo, vitiligo, skin cancer and a neurodegenerative disorder, said method comprising administering to a subject an effective amount of a compound described herein.

Where any aspect or feature of the invention is described in relation to a use, compound for use or method as described above, it is explicitly contemplated that said aspect or feature should be understood as being disclosed in the context of all of the uses, compounds for use and methods. Similarly, a reference herein to a use, compound for use or method as described above should be interpreted as a reference to all of the use, compound for use and method.

In some embodiments of the invention, the disease or disorder may be selected from the group consisting of a hyperpigmentation disorder, lentigo, vitiligo, and skin cancer. In some embodiments of the invention, the skin cancer may be a melanoma. In some embodiments of the invention the neurodegenerative disorder may be Parkinson’s disease or Huntingdon’s disease.

The below examples illustrate the invention and are not to be construed as limitative.

Examples

Materials

328 tropical plant samples used in this work were obtained from the Singapore Botanic Garden. A first batch of Wrightia antidysenterica was provided by/purchased from Pioneer Landscape Nursery (Singapore), and a second batch was provided by/ purchased from Yi Li Nursery Sdn Bhd, Muar, Johor, Malaysia. L-tyrosine and tyrosinase from mushroom (T3824) were purchased from Sigma Aldrich. Acetone, ethanol, butylene glycol, dipotassium phosphate and monopotassium phosphate (phosphate buffer, PBS) of analytical grade were obtained and used without further purification.

General methods

ESI-MS (Thermo Scientific LCQ), LC-MS (Bruker AmaZonX LCMS), High resolution MS (Bruker MicroTOF-QII) and ¹ H NMR (AVNEO500 NMR) techniques have been used to characterize the structure of the compounds. The flow rate of LC-MS is 1 mL/min, the column used is 250 mm ^c 4.6 mm i.d. 5pm phenomenex C-18 prepChrom HPLC column. The solvent gradient is the same with semi-preparative HPLC.

Extraction of plant materials

Plant materials were pulverized and used as received. The plant materials were extracted with a mixture containing acetone, ethanol and water (AEW, 2:2:1 by volume), or 50% butylene glycol (BG) in water. Briefly, the plant materials (0.1 g) were mixed with extraction solvent (2.0 ml.) under gentle agitation. For AEW extraction, the plant materials were extracted at 25°C for 2 hours. For BG extraction, the plant materials were extracted at 25°C for 96 hours (~4 days), or at 70°C for 6 hours. After extraction, the extract was centrifuged for 15 minutes and the supernatant was collected and stored in the freezer (-4°C) for the tyrosinase inhibition assay.

Tyrosinase inhibition assay

The tyrosinase inhibitory activity of the plant extracts was determined using a previously described method (Uchida, Ryuji, Seiko Ishikawa, and Hiroshi Tomoda, Acta Pharmaceutica Sinica B 4.2 (2014): 141-145, and Lo, Yuan-Hsin, et ai, Journal of

Ethnopharmacology 124.3 (2009): 625-629.) with minor modifications. Prior to the assay, the plant extracts were diluted to two different concentrations, 0.5 and 1.0 mg/mL, using 50 mM PBS (pH 6.8). The tyrosinase inhibition assay was conducted using a 96 well microplate with 140 pL L-tyrosine (0.6 g/L), 20 pL PBS and 20 pL diluted sample in each well. The mixture was pre-incubated at 25°C for 10 minutes before adding 20 pL of tyrosinase (500 U/mL). The absorbance changes were monitored using a microplate absorbance reader (Bio-Tek Synergy HT, Bio-Tek Instruments, Inc. , Winooski, VT) at 475 nm for 30 minutes. The percentage tyrosinase inhibition was calculated as:

% tyrosinase inhibition = (l - ) ^c 100

where S is the slope of a plot of absorbance of the sample versus time, and B is the slope of a plot of absorbance of the blank versus time.

The blank contained the only extraction solvents and the same chemicals used in the assay, without any plant materials or extracts.

Example 1. Screening of plant extracts and Kojic acid for tyrosinase inhibitory activity

A total of 328 plant extracts were screened for their tyrosinase inhibitory activity using mushroom tyrosinase (500 U/mL), at relative plant concentrations of 0.5 and 1.0 mg/ml_ (see general method above). It was observed that 46 samples exhibited some tyrosinase inhibition effect and they were selected for further investigation.

AEW extracts and BG extracts (at 25 or 70°C) of the selected 46 plant samples were further screened for tyrosinase inhibition activity at four different concentrations of 0.2, 0.4, 0.8 and 1.0 mg/mL. Kojic acid was also tested using the same protocol as a positive control. The results of tyrosinase inhibition of the selected plant extracts are shown in Table 1 below. From the above preliminary screening, it was observed that the extract from the leaves of Wrightia antidysenterica (entry 12) demonstrated the highest tyrosinase inhibition, and was therefore used for further studies and characterization.

Table 1. Tyrosinase inhibition (%) activity of selected tropical plant extracts.

The known tyrosinase inhibitor Kojic acid was used as a positive control, and had an IC50 of 1 1 .80 ± 1 .18 pg/ml (data shown in Figure 21). This is more than twice as high as the IC50 of crude extract of Wrightia antidysenterica, which was 4.80±0.46 pg/ml.

Example 2. Extraction and separation of first batch of Wrightia antidysenterica extract and structural identification of key fractions

To identify the active compounds in the Wrightia antidysenterica extract, HPLC separation and structural identification of the key fractions in the first batch of the extract were carried out as discussed below.

Experimental Fresh leaves were removed from the plants, and freeze-dried for one week to remove moisture. The dried leaves (30 g) were then blended into powder using high force blender and the resulting powder were transferred into a 500 ml. blue cap bottle. To the bottle, 300 ml. solvent AEW (2:2: 1 by volume) was added. The blue cap bottle was put into a shaker and incubated for 12 hours with 2000 rpm at room temperature. After extraction, the sample was passed through a Buchner funnel with filter paper to remove the solid residues. The supernatant (300 mL) was then transferred to a 500 mL round bottle flask, and the solvent was removed using a rotary evaporator (BUCHI system) using a 55°C water bath and under reduced pressure (20 mbar). The AEW crude extract of the freeze-dried leaves was weighed to determine the extraction yield.

The AEW crude extract was diluted with deionized water to give a final concentration of 30 mg/ml_, and was filtered through a 0.45 pm PTFE membrane filter. 200 pL of the filtrate was injected into HPLC column (250 mm ^c 10 mm i.d. 10pm BUCHI C-18 prepChrom) and eluted with mobile phase A (deionsied water) and phase B (acetonitrile). The flow rate was 2.0 mL/min and the column temperature was 30 °C. The sample was eluted using a gradient elution as follows: 0-5min, 97-80% A (3-20% B); 5-10 min, 80-75% A (20-25% B); 10-12 min, 75-70% A (25-30% B); 12-15 min, 70-68% A (30-32% B); 15-20 min, 68-60% A (32-40% B); and 20-25min, 55-60% A (40-45% B).

A total of 17 fractions were identified from the HPLC separation using gradient elution. Each fraction was collected and their tyrosinase inhibition activites were determined (Fig. 2). Fractions 11 , 14, and 15 were selected for further structure characterization as they were found to be more active in inhibiting tyrosinase as compared to the other fractions (Fig. 2). Given this, fractions 11 , 14 and 15 were re-injected into HPLC to verify their purity, with their respective UV spectra identified (Fig. 3a-c, respectively).

Further, fractions 11 , 14 and 15 from the first batch were collected from semi-preparative HPLC and the solvents were removed by rotary evaporator. The active compounds from these fractions were characterized by ESI-MS, LC-MS, high-resolution MS and ¹ H NMR spectroscopies to identify the chemical structures of the compounds. While the crude fractions 14 and 15 were less active in inhibiting tyrosinase than fraction 11 , the isolated active agents in these compounds demonstrated high activity as discussed below.

The flow rate of LC-MS is 1 mL/min, the column used is 250 mm ^c 4.6 mm i.d. 5pm phenomenex C-18 prepChrom HPLC column. The solvent gradient is the same with semi preparative HPLC. From the result of LC-MS, the fragment pattern of active compound was identified and further analyzed. High resolution ESI-MS have also been performed to identify the exact fomular of the active compounds. ¹ H NMR (D2O 500Hz) for fraction 14, ¹ H NMR (CD3OD 500Hz) and ¹³ C NMR for fraction 15 have also been carried out to further determine the structure of the active compound.

Fraction 11 The UV-vis spectrum of fraction 11 is as shown in Fig. 3a. ESI-MS spectrum of fraction 1 1 in Fig 4a shows an m/z of 592.87, which suggests the presence of [M-H] ^· ion of apigenin 6,8-di- C-glucoside (also known as vicenin-2, the structure of which is shown in Fig. 4c and below). Further, the MS-MS spectrum of ion m/z [M-H]- of 592.87 shows fragmentation pattern with major ion fragments that are similar to those of vicenin-2 (Fig. 4b).

Apigenin 6,8-di-C-glucoside (Vicenin-2)

Fraction 14

The UV-vis spectrum of fraction 14 is as shown in Fig. 3b. The ESI-MS spectrum (negative mode) of fraction 14 shows m/z values of 740.1 and 739.2 (Fig. 5a), and was further confirmed by the m/z value of 739.2090 in the high-resolution MS spectrum, which corresponds to a [M-H] ^· ion of a kaempferol-3-O-glycoside derivative, with a formula C33H39O19 (Fig. 6a). The secondary MS fragmentation patterns are shown in Fig. 6b, which suggests the proposed major fragments of the kaempferol-3-O-glycoside derivative shown in Fig. 6c.

The chemical structure of the active compound in fraction 14 was further confirmed by the ¹ H NMR spectra shown in Fig. 7a. It was observed from the ¹ H NMR spectrum that there are four methyl groups (all doublets) at 0.90, 0.95, 1 .12 and 1.14 ppm, which suggests the possibility of two position isomers of kampferol-3-O-glycoside derivative due to different linkage of the two rhamnose (at 1 ,2 and 1 ,3 positions) as shown in Fig. 7b. Proposed active kampferol-3-O-glycoside compounds of Fraction 14

Fraction 15

The UV-vis spectrum of fraction 15 is as shown in Fig. 3c. The ESI-MS spectrum (positive mode) of fraction 15 shows m/z values of 228.0 and 432.7, which correspond to [M+Na] ⁺ and [2M+Na] ⁺ , respectively, of a compound (M) with formulae C _{I I} H _H N0 ₃ (Fig. 8a and 10b). This was further confirmed by the m/z value of 228.0627 in the high-resolution MS spectrum (positive mode), which corresponds to a compound with formulae CnHiiN0 ₃ Na (Fig. 8b). The MS-MS spectrum (positive mode) in Fig. 9 shows the fragmentation pattern similar to that of 2-methyl-3-hydroxyl-indole-3-acetic acid (Fig. 10b).

Further, the ESI-MS spectrum (negative mode) of fraction 15 shows an m/z value of 204, which corresponds to [M-H] (Fig. 10a), and the fragmentation pattern (negative mode) further confirms the identity of the proposed compound 2-methyl-3-hydroxyl-indole-3-acetic acid (Fig. 11). Lastly, the ¹ H NMR and ¹³ C NMR spectra confirm the identity of the compound (Fig. 12 and 13). The Ή NMR shows four hydrogens A, B, C and D at the chemical shift of 6.90, 7.02, 7.25, 7.33, that can be assigned to the benzene ring. There are three hydrogens G at the chemical shift of 2.09, that can be assigned to the methyl group on the pyrrole ring. The two hydrogens E and F at the chemical shift of 3.18 and 3.39 can be assigned to the acetic acid. Example 3. Extraction and separation of second batch of Wrightia antidysenterica extract and structural identification of key fraction

Similarly, for the second batch of Wrightia antidysenterica extract, HPLC separation and structural identification were carried out to identify the active compounds in the extracts. Experimental

The leaves (2.5kg) of Wrightia antidysenterica from Malaysia were mixed with ethanol (12.5 L) at room temperature and homogenized to form a slurry. After extraction, filter bags were used to filter the solid residue of the sample, with the filtrate collected. The filtrate was then transferred into a 50 L round bottle flask and the solvent was removed using a rotary evaporator (BUCHI system) using a 55°C water bath and under reduced pressure (100 mbar). After rotary evaporation, the extraction (500 ml.) was freeze-dried for 5 days to give the crude solid extract (100 g), which was used for C18 column chromatography. The freeze-dried sample was dissolved in water to give a concentration of 1 g/mL. Thereafter, 2 mL of the as-prepared solution was loaded into the semi-preparative C18 column and eluted with mobile phase A (water) and phase B (methanol) at a flow rate of 5.0 mL/min on a C18 column. A gradient elution was performed starting as follows: 0-40 min, 100% A (0% B); 40-80 min, 80% A (20% B); 80-120 min, 60% A (40% B); 120-160 min, 40% A

(60% B); 160-200 min, 20% A (80% B); and 200-230min, 0% A (100% B). The fractions from the first two gradients were collected in separate 100 mL in blue cap bottles, while fractions from the remaining gradients were collected in 50 mL centrafuge tubes. A total of 19 fractions were collected with their inhibition activity tested. It was observed that the fraction with 80% methanol showed the highest inhibition activity and was therefore, collected and concentrated to 200 mg/mL. This fraction was concentrated and purified by semi-preparative HPLC to provide pure active compounds. The fraction was filtered through a 0.45 pm PTFE membrane filter, and the filtrate (800 pL) was injected into a semi-Prep HPLC system (Waters, 2996 Photodiode Array Detector) with a HPLC column (250 mm ^c 10 mm i.d. 5 pm BUCHI C-18 prepChrom), eluting with mobile phase A (water with 0.1% formic acid) and phase B (acetonitrile with 0.1 % formic acid). The flow rate was 5.0 mL/min and the column temperature was 25°C.

Results and discussion

In the first round of semi-preparative HPLC separation, an isocratic elution was used with 72% A (28% B) for 40 min, and 9 fractions were collected based on the peaks eluted from the HPLC (Fig. 14a), with each fraction tested for tyrosinase inhibition activity. As shown in Fig. 14b, fraction 7 was found to have the highest inhibition activity. As such, fraction 7 was further purified by a second round semi-preparative HPLC under the similar conditions mentioned above, but with the change of isocratic elution to 78% A (22% B) for 60 min. Eight fractions were collected (Fig. 15a), and fraction 4 was observed to show the highest tyrosinase inhibition activity (Fig. 15b). Further, the HPLC chromatogram shows a single peak for fraction 4, which suggests that fraction 4 is likely to be a pure compound suitable for structural determination.

It was observed that the mass spectra of fraction 4 showed ionization only in the negative mode (not in positive mode), suggesting that fraction 4 is an acidic compound. The anionic LC-MS spectrum shows a m/z of 139, which corresponds to [M-H]- of the proposed structure of p-hydroxyphenyl nitrite (Fig. 19a). This was further confirmed by the m/z shown in the ESI-MS spectrum (Fig. 16b), and high-resolution MS spectrum (Fig. 17a), which was assigned to a species with molecular formula C6H4NO3. The MS(n) fragmentation pattern is shown in Fig. 17b, which give rise to two fragments at m/z 108 [O-C6H4-O] ^· and 46 [NO2] .The ¹ H NMR spectrum of the compound (in Fig. 18) shows two groups of aryl protons, each being a doublet at 6.91 and 8.15 ppm, respectively. Taken together, the structure of this compound was deduced to be p-hydroxyphenyl nitrite (Fig. 19).

Example 4. Comparison of the tyrosinase inhibition activity of Wrightia antidysenterica crude leaves extract with the isolated compounds from the first batch To understanding the correlation between the efficacies of the isolated compounds with the crude leaves extract of Whghtia antidysenterica, the tyrosinase inhibition activities of each isolated compounds were determined and compared with that of the crude leave extract.

As shown in Fig. 20, the crude extract of Wrightia antidysenterica has an IC ₅₀ value of 19.01 ±1.39 pg/mL (based on fresh leaves as sample weight). The IC ₅₀ value of the kampferol-3-O-glycoside derivative, (MW of 740) is 36.9 ± 2 mM, which is equivalent to 27.3 ± 1.5 pg/mL. The IC ₅₀ value of vicenin-2 (MW of 594) is 7.84 ± 0.78 pM which is equivalent to 4.65 ± 0.46 pg/mL. The IC ₅₀ value of the 2-methyl-3-hydroxy-indole-3-acetic acid (MW of 205) is 80.8 ± 2.3 pM, which is equivalent to 16.6 ± 0.5 pg/mL.

The low IC ₅₀ of the crude extract appears to suggest that the presence of synergistic effects among the various constituents of the active compounds in the crude extracts (Fig. 20).

Different compounds were isolated from the first and second batches of Wrightia antidysenterica. This is believed to be a result of the different extraction techniques and isolation processes used on each batch.

Example 5. Cell cytotoxicity assay for compounds isolated from Wrightia antidysentrica

The cytotoxicity of the below three compounds isolated from Wrightia antidysentrica was determined as set out below. The isolation of the compounds is set out above.

MW740: kaempferol-3-glucoside (Fraction 14)

MW594: vicenin-2 (Fraction 11)

MW139: 4-hydroxyphenyl nitrite or catecholic ester of nitrate (exact structure not determined) (Fraction 4)

MW139 was dissolved in 100% DMSO, while MW740 and MW594 were dissolved in sterile distilled water.

B16-F10 melanoma cells were prepared using culture medium, DMEM medium containing 10% FBS and Penicillin/Streptomycin (Gibco) in T-175 flasks. After that, cells were seeded in 24-well plates at 2.5x10 ⁴ cells/well in 500 ml_ of assay medium. After 24 h, assay medium was refreshed to allow the cells to be incubated in the presence or absence (negative control) of various concentrations of samples and 20 ng/mL a-MSH. After 3 days, the cell viability assay of B16-F10 cells was performed using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies Inc.). The medium was removed, and 500 mI_ of growth medium containing 10% CCK-8 solution was added and incubated for 30 - 60 min at 37°C. The amount of formazan salt was quantified by measuring the absorbance at 450 nm with a microplate reader. Number of viable cells were determined against a standard of B16-F10 cells (0 - 20x10 ⁵ cells/well) exposed to 20 ng/mL a-MSH for 72 h.

No cytotoxicity was observed for any of the tested compounds. Results are shown in Figure 22.