FIELD OF THE INVENTION

This invention relates to compounds which are particularly useful in radioimaging and radio- chemotherapy as it particularly relates to the treatment and location of malignant melanomas and other pigmenting disorders. Novel acyl derivatives of phenolic thioether amines and their use in compositions for blocking melanin synthesis in human or animal melanocyte cells is also described. More particularly, the compositions involving the novel derivatives are useful in treating pigmentation problems due to a variety of skin disorders, including skin cancer in the form of melanoma. BACKGROUND OF THE INVENTION

Phenylthioalkylamines is a broad class of compounds having a variety of uses which include therapeutics. U.S. patents 4,134,996 and 4,183,927 disclose certain phenylthioalkylamine compounds which are useful as platelet aggregation inhibitors. Other phenylthioalkyl¬ amine compounds of applicant\'s published International application 091/16302 are useful as depigmenting agents in treating a variety of pigmentary diseases. Such diseases are often characterized in the elevated levels of the enzyme tyrosinase in melanocytes; i.e., human and animal cells which synthesize the pigment melanin. There are a variety of pigmentary diseases, such as melas a, melanoma, moles and the like. In particular, moles are susceptible to becoming melanoma after exposure to sunlight which precipitates increased synthesis of tyrosinase.

Usually commercial forms of depigmenting compositions are based on the use of hydroguinone.

However, the hydroquinone preparations are very unstable and cause skin irritation. Hydroguinone compositions can also cause permanent whitening of the skin if used for a prolonged period and at a high concentration. As to

treatment of melanoma, this is.presently attended to by surgical procedures, since any type of known non-surgical treatment of melanoma is unsatisfactory.

Research work has been conducted in the field of phenolic and diphenolic compounds to serve as a basis for chemotherapeutic treatment of melanoma and skin depigmentation. In particular, 4-S-cysteinylphenol (4-S- CP) and 4-S-cysteaminylphenol (4-S-CAP) have been synthesized and evaluated for cytotoxicity to normal epidermal melanocytes to determine their effectiveness as depigmenting agents and antimelanoma agents. Miura et al, "Synthesis of Cysteinylphenol, Cysteaminylphenol, and Related Compounds, and In Vivo Evaluation of Antimelanoma Effect", Arch. Dermatol Res. (1987) 279:219-225, disclose the effect of 4-S-CAP and 4-S-CP in depigmentation of black hair follicles as manifested by loss of functioning melanocytes. It was established that 4-S-CAP was a potent agent in prolonging the lifespan of melanoma- bearing mice and hence exhibited inhibition of melanoma growth. The 4-S-CP and the methyl ester of 4-S-CP also exhibited some inhibition of melanoma growth, although not as active as the 4-S-CAP.

The same compounds, 4-S-CP and 4-S-CAP, were also investigated for properties of depigmentation of black guinea pig skin by topical application. The results of this work is reported by Ito et al, "Depigmentation of Black Guinea Pig Skin by Topical Application of Cysteaminylphenol, Cysteinylphenol, and Related Compoundε", The Journal of Investigative Dermatology, Vol. 88 No. 1, January 1987. Although 4-S-CAP demonstrated depigmenting properties, inflammatory changes of the skin of the guinea pigs was prominent. The 4-S-CAP was capable, however, of:

1. decreasing the number of functioning melanocytes;

2. decreasing the amount of epidermal melanin pigments; and

3. degenerating and destroying melanocytes.

4-S-CP and 4-S-CAP were also investigated for their selective cytotoxicity on follicular melanocytes. This was reported by Ito et al, "Selective Cytotoxicity of 4- S-Cysteaminylphenol on Follicular Melanocyte of the Black Mouse: Rational Basis for its Application to Melanoma Chemotherapy", Cancer Research, June 15, 1987 47:3278- 3284. It was reported that 4-S-CAP demonstrated cytotoxicity in the depigmentation of black hair follicles, whereas it had no effect on the albino follicles. Hence 4-S-CAP is actively engaged in the melanin synthesis of the melanocytes.

The 4-S-CAP compound, however, has several limitations from the standpoint of practical clinical use. These limitations include: a. hypotensive effect; and b. high toxicity due to the 4-S-CAP being a substrate for monoamineoxidase (MAO) which in the plasma converts 4-S-CAP into an aldehyde form which produces a non-specific cytotoxicity. Homologs of the 4-S-CAP compound have been investigated for antimelanoma and depigmenting properties. As reported in Alena et al, Melanocytotoxicity and Antimelanoma Effects of Phenolic Amine Compounds in Mice In Vivo, Cancer Research 50:3743- 3747, June 15, 1990, such homologs demonstrate depigmentation properties and N-acetyl-4-S-CAP demonstrates potent antimelanoma properties. The benefits and uses of these homologs and related compounds for use as depigmenting agents and antimelanoma agents are described in applicant\'s published International application WO 91/16302. Although the compounds disclosed in that application are effective depigmenting agents as well as antimelanoma agents, there are stability and toxicity problems associated with some of the compounds. In particular the very effective N-

acetyl-4-S-CAP compound tends to be unstable and can cause topical irritations on administration.

As noted, melanin synthesis is a biological property unique to the pigment cell, melanocyte and its neoplastic counterpart, malignant melanoma. In order to develop a targeted chemotherapy of malignant melanoma, phenolic and catecholic melanin precursors have been synthesized and their melanocytotoxicity and antimelanoma effects have been examined (Ito, Y. et al, 1987; Miura, S. et al, 1987; Alena, F. et al, 1990; Jimbow, K. et al, 1992).

Among these, S-substituted phenolic amines, N-acetyl-4-S- cysteaminylphenol (N-Ac-4-S-CAP) was found to be a superior substrate of melanin synthesizing enzyme, tyrosinase (Pankovich, J. et al, 1990; Miura, T. et al, 1990) , and to possess the most effective melanocytotoxic activity and antimelanoma effect (Miura, T. et al, 1990; Alena, F. et al, 1990; Wong, M. et al, 1991). 4-S-CAP was, however, as noted, above, found to have some general cytotoxicity (Alena, F. et al, 1990) , because it is metabolically transformed a cytotoxic aldehyde metabolite by plasm monoamine oxidase (Pankovich, J. et al; Inoue, S. et al, 1990) . On the other hand, N-Ac-4-S-CAP has shown an absence of such cytotoxicity (Alena, F. et al, 1990) , indicating that N-Ac-4-S-CAP may be a compound suitable for the development of melanoma chemotherapy.

In the treatment of various pigmentary disorders, it would also be beneficial to determine the specificity of the compounds used in the treatment and in particular, compounds such as N-Ac-4-S-CAP. In this regard, we have also discovered that certain anti-melanoma and depigmenting agents can be labelled with radioactive molecules to achieve not only radioimaging but as well, radio-chemotherapy particularly in the treatment of malignant melanoma. In accordance with thiε invention, we have also discovered esterified compounds which exhibit excellent stability and very low general toxicity, but have

selective toxicity for melanocytes.

gTOfflft-RY OF THE INVENTION

In accordance with one aspect of the invention, there is provided a compound for use in radioimaging and radiochemotherapy in the diagnosis and/or treatment of melanoma and other pigmentary disorders, said compound being selected from the group of compounds represented by the formula:

wherein

Ri is H or Cj-Cg alkyl; R ₂ is H or Cj-Cg alkyl; R ₃ is H, Cj-Cg alkyl or Cj-Cg alkanoyl; ₄ is H or Cj-Cg alkanoyl; Ra is a radioactive material useful in radioimaging and/or radiochemotherapy where the dotted line ( ) indicates:

1) Ra is covalently bound to the structure of formula (I) ; 2) Ra is ionically associated with the structure of formula (I) ; or

3) Ra is radioactive carbon which is part of the structure of formula (I) ; and x is 1 to 5, with the provisos that when x is 1, one of R-, R_ or R ₃ is other than H, and that the sulphur containing group

and the Ra group are in the 2, 4 or 6 positions of the phenyl ring.

In accordance with a further aspect of the invention, there is provided a- process for radioimaging melanoma colonies in vivo in a subject comprising: i) administering to the subject\'ε circulatory εyεtem one or more radioimaging compoundε εelected from the group of compoundε represented by the formula:

wherein

Ri is H or C.-C ₈ alkyl;

R ₂ iε H pr Cj-Cgalkyl;

R ₃ iε H, C,-Cg alkyl or C _j -C ₈ alkanoyl;

R* is H or C,-C ₈ alkanoyl;

Ra iε a radioactive material useful in radioimaging and/or radiochemotherapy where the dotted line ( ) indicates:

1) Ra is covalently bound to the structure of formula (I) ;

2) Ra iε ionically associated with the structure of formula (I) ; or

3) Ra is radioactive carbon which is part of the structure of formula (I) ; and x is 1 to 5, with the provisos that when x iε 1, one of R„ 1* ₂ or R ₃ is other than H, and that the sulphur containing group

and the Ra group are in the 2, 4 or 6 positions of the phenyl ring; and ii) detecting the presence of emitted radiation from an accumulation of said selected radioimaging compound as εaid adminiεtered compound binds solely to any melanoma tiεsue present in said subject. In accordance with another aspect of the invention, there iε provided a proceεε for radiochemotherapy treatment of melanoma cells in a subject comprising: i) administering to the subject\'s circulatory syεtem one or more of the radiochemotherapy compoundε εelected from the group of compoundε represented by the formula:

wherein

R, is H or Cj-Cg alkyl;

R ₂ is H pr C^Cgalkyl ;

R ₃ is H, Ci-Cg alkyl or C _j -Cg alkanoyl;

R, is H or C,-Cg alkanoyl;

Ra is a radioactive material useful in radioimaging and/or radiochemotherapy where the dotted line ( ) indicates:

1) Ra is covalently bound to the structure of formula (I) ;

2) Ra is ionically aεεociated with the structure of formula (I) ; or

3) Ra is radioactive carbon which iε part of the structure of formula (I) ; and x is 1 to 5, with the provisoε that when x is 1, one of R„ _? or R ₃ is other than H, and that the εulphur containing group and the Ra group are in the 2, 4 or 6 positions of the phenyl ring.

In accordance with another aspect of the invention, the preferred compounds of formula (I) are when X is 1 and , is alkanoyl.

In accordance with another aspect of the invention, a compound of the formula (II) :

wherein Ri is H or Ci - C ₈ alkyl;

-ft, iε H, Ci - c ₈ alkyl or Cj - Cg alkanoyl; R ₃ is H or C, - C _g alkyl; R ₄ is Cj - Cg alkanoyl; and x is 1 to 5; with the proviso that when x is 1 one of R,, R ₂ or R ₃ is other than H, and that the sulphur containing group is in the 2, 4 or 6 poεition of the phenyl ring.

In accordance with another aspect of the invention, the preferred compoundε of formula (II) are when x iε 1 and R-, iε alkanoyl.

In accordance with a further aspect of the invention, a pharmaceutical composition compriseε a

compound of formula (II) and a.pharmacologically or biologically acceptable carrier.

According to a preferred aspect of the invention, this compoεition iε particularly uεeful aε a depigmenting compoεition in sunscreening lotions. The composition is also useful as an antimelanoma agent and useful for the treatment of melasma.

In accordance with another aspect of this invention, a composition uεeful for blocking melanin synthesis in human or animal melanocyte cells, compriseε: i) a biologically effective amount of an active compound εelected from the group represented by formula (II) ; and ii) a suitable biologically compatible carrier for the selected active compound.

According to another aspect of the invention, a method for blocking melanin εynthesiε in human or animal melanocyte cellε compriεeε treating human or animal skin by use of a composition compriεing a biologically effective amount of an active compound εelected from the group repreεented by formula (II) .

Such methods of treatment are preferably applied in the depigmenting of εkin due to UV exposure, melasma and is alεo uεeful in the treatment of melanoma. According to another aεpect of the invention, there iε provided a method of formulating a composition useful for blocking melanin synthesis in human or animal melanocyte cells of skin compriseε mixing: i) a biologically effective amount of an active compound εelected from the group repreεented by formula (II) ; and ii) a suitable biologically compatible carrier.

BRIEF DESCRIPTION OF THE DRAWINGS: Figure 1 is a whole body autoradiogram of the sectional mouεe of Figure IA; and

Figure IA is a sectioned mouse.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The radiolabelled compoundε of thiε invention sure capable of binding to melanoma tiεsue such that when the tissue is subjected to radioimaging the existence of such melanoma colonies or cells appears in the image.

Further, by virtue of the binding of these compounds to melanoma sites, the radioactive material may be used in radiochemotherapy treatment of the melanoma cells to either kill such cells or effect their remission. The preferred compounds of this invention exhibit considerably leεs toxicity than known forms of phenolic amine compounds such as the aforementioned 4-S-CP and 4- S-CAP. In particular, when 4-S-CAP is used in radioimaging or radiochemotherapy, the compound collects in the eye as well as in the melanoma tiεεue. However, the compoundε of formula (I) do not have such drawbacks and are therefore particularly suited to this new use. When the compoundε of the formula (I) are used in radiochemotherapy they may be combined with suitable pharmaceutical excipientε and carriers wherein the radioactive selected compounds are preεent in a biologically effective amount in the carrierε. Suitable biologically, compatible carrierε may be εolutionε when injected by either the IP or SC route. Suitable biologically, compatible carrierε for injection include physiologically normal εaline and other types of readily injectable εolutionε which are well understood by those skilled in the art and readily available. The preferred injectable carrier is a neutral buffer having a pH of 7 to 7.4 and does not in any way effect the activity of the radiolabelled compoundε. For purposes of injection, the concentration of the radiolabelled compound in the injectable εolution is normally in the range of 200 to 1200 mg per kg of body weight where the preferred dosage is in the range of 300 mg to 500 mg per kg of body weight. In the following examples, further information is provided as to the amount of compounds injected for

purpoεeε of radioimaging.

Although one skilled in the art appreciates that there are a variety of techniques for making the compounds of formula (I) before they are radiolabelled the proposed method for manufacturing the compounds iε via the Weirmeiεter reaction as described in WO 91/16302.

As will be demonstrated in the following examples, radiolabelled compounds of this invention bind specifically to melanoma tissues and the radiolabelled compound or radiolabelled derivatives thereof remain in the melanoma tissue for a sufficient period of time for either therapeutic treatment or for subεequent radioimaging. The radiolabelling of the compound occurs normally in the phenyl ring in accordance with the following reaction schemes.

HO— ([ — SH

Ra wherein I is a radioactive form of iodine ,

Exemplary iodine labelling iε as follows:

N-acetyl-3-iodo-4-S-cysteaminy1-phenol;

N-acetyl-2-S-cysteaminyl-3-iodo-phenol;

N-acetyl-2-S-cysteaminyl-5-iodo-phenol; N-{2-{(2\'-iodo-4\'-acetoxy-phenyl)thio}ethyl}- acetamide;

N-{2-{(2\'-acetoxy-5\'-iodo-pheny1)thio>ethy1}- acetamide; and

N-{2-{(2\'-acetoxy-3\'-iodo-pheny1)thio>ethy1}- acetamide.

Variouε radiolabelling agents may be used such as carbon 14, U-14-C, iodine (such aε ¹²³ I, ¹²⁵ I and ^I31 I) , technetium (εuch aε "Tc) , flourine (εuch aε ¹⁸ F) , indium (such aε ^ιn In) and 35 _s for example. The method of introduction of the radiolabelling agent to the compound will depend on the choice of radiolabelling agent, aε one skilled in the art would appreciate. The radiolabelling agent will be asεociated with the εtructure of the compound through one of the several ways: (1) through a covalent bond with the compound structure; (2) through ionic association with the compound structure; or (3) aε part of the εtructure of the compound.

Novel acylated compoundε of thiε invention are represented by the compoundε of formula (II) . It haε been found that thoεe compoundε are particularly uεeful in the treatment of variouε pigmentation diεorderε and, when formulated into suitable pharmaceutical compositionε, exhibit excellent stability and very low general toxicity, but selective toxicity with respect to melanocytes. Hence, in accordance with another aεpect of thiε invention, the pharmaceutical compositions are very useful in the treatment of pigmentary diseases such aε treatmentε involving one or more of the following: blocking melanin synthesis in human or animal melanocyte cells, inhibiting the metabolic pathway involving tyrosinase in human or animal melanocyte cells, and

treatment of melanoma, treatment of hyperpig- mentation and treatment of cutaneouε hyperpigmentation.

In formulae (I) and (II) , preferred lower alkyl and lower alkanoyl groups are C- to C ₆ . The most preferred lower alkyl and lower alkanoyl groups are C- to C ₄ .

Preferred compounds of formula (I) include N-acetyl- 4-S-cysteaminylphenol, N,N-dimethyl-4-S-cysteaminyl- phenol, 4-S-homo-cysteaminylphenol, α-methyl-4-S- cyεteaminylphenol, N-{2-{( \'-acetoxypheny1)-thio)ethyl)- acetamide, N-{l-methyl {2—[(4\'-acetoxypheny1)thio}ethyl}- acetamide, N,N-dimethy1 (2-{(4\'-acetoxypheny1)- thio}ethylamine or N-propionyl-4-S-cysteaminylphenol.

Preferred specific compounds of the invention of Formula (II) are:

Compound R. R _: R ₃ R ₄

1. N-(l-Methyl-{2-{(4\'- acetoxyphenyl)th_o}- ethyljacetamide CH ₃ H COCH ₃ COCH ₃

2. N,N,-Dimethyl {2-{(4- acetoxyphenyl)thio}- ethylamine H CH ₃ CH ₃ COCH ₃

3. N-{2-{(4\'-acetoxy- phenyl)thio}ethyl}- acetamide H H COCH ₃ C0CH ₃

4. N-{2-{(4-acetoxyphenyl)- tfaio}e hyl}propionamide H H COC_H ₅ COCH ₃

The compounds of formula (II) may be manufactured by a variety of synthesis techniques. Generally, they may be prepared by acylation of the corresponding cysteaminylphenol compounds.

The cysteaminylphenol compounds may also be prepared in accordance with the technique discloεed by Miura et al, "Syntheεiε of Cysteinylphenol, Cysteaminylphenol, and

Related Compoundε, and in vivo Evaluation of Antimelanoma Effect", Arch. Dermatol. Reε. (1987) 279:219-225. Thiε entailε the reaction of a phenol with a cyεteamine to yield the 4-S-cysteaminylphenol or the 2-S- cysteaminylphenol. The 4-S-cysteaminylphenol may then be isolated from the reaction product by Silica gel column chromatography or crystallization with changing pH of εolution. It iε understood that the various derivatives of this invention may be made by various radical subεtitutionε to the εulphur chain, or substituting a thiol with the desired chain.

Details of procesεeε for making εome of the preferred compoundε of the invention are set out in the accompanying Examples. The intermediate cysteaminylphenol compounds may be acylated by reaction with the appropriate alkanoyl; for example, acetyl chloride may be reacted with N-{2-((4- hydroxyphenyl)thio)ethyl}acetamide, to give the preferred compound of the invention N-{2-((4\'-acetoxypheny1)- thio)ethyl}acetamide (NAP-TEA).

The compositions of this invention having active agents of formula (II) provide a mechanism for treating a variety of pigmentary diseaεeε, εuch aε melaεma and other hyperpigmentation diseases and melanoma and other skin cancerε and for preventing melaεma and skin cancers which are normally induced by exposure to UV radiation. This activity of the composition iε achieved by the εelected active compound of the composition becoming toxic in the melanocytes in the preεence of tyrosinase without being a substrate for plasma MAO. The compositionε upon adminiεtration, according to thiε invention, have significant melanoma cytotoxicity and antimelanoma effects. This is recognized in the significant depigmentation of hair on black experimental animals, as well as significantly inhibiting the experimental growth of lung metastases of B16 F10 melanoma cells. The compositionε of thiε invention have alεo demonstrated

15 selective toxicity to other neural crest tumors, such as pheochromocytoma and neuroblastoma cells.

Experimental tests on animals indicate per single dose toxicity levels in the range of 1,400 mg/kg of body weight for the preferred compound of formula (II) , NAP- TEA.

In the pharmaceutical compositions of the invention, the term "a biologically effective amount of the active compound" means that a sufficient amount of the compound in the composition is provided such that upon administration to the human or animal by, for example, i.p., s.c. or topical route provides εufficient active agent on each application to give the desired result in treating various pigmentary diseases, including melanoma, melasma, or acting as UV blocker and/or depigmenting agent in sunscreen lotions. However, the biologically effective amount of the active compound is at a level that it is not toxic to the human or animal during the term of treatment. The term "a suitable biologically compatible carrier" includes a solvent for the compound when the compound is injected by either i.p. or s.c. route. Suitable biologically compatible carrierε for injection include physiologically normal saline and other types of readily injectable solutions which are well understood by those skilled in the art. The preferred injectable carrier is a neutral buffer having a pH of 7.0 to 7.4. When the compound is topically applied, the carrier may be any type of suitable excipient or carrier in the form of coεmetic compositions, pharmaceutical adjuvants, sunscreen lotions and the like.

Suitable carrierε for topical application include conventional εkin treatment compositions, εuch as cosmetic compositionε and pharmaceutical preparations. Examples of such ingredients for topical application are oils such as liquid paraffin, vaseline, methyl- polyεiloxane, castor oil, squalane, and coconut oil;

anti-oxidantε εuch aε butylated hydroxyaniεole, butylhydroxytoluene, ethyl gallate, and tocopherol; surfactants such as sodium laureate, laurylpyridinium chloride, polyoxyethylene sorbitan monooleate, glyceryl monoarachate, sodium N-stearyl-N,N-dimethyl glycine, oleyl hydrolysed animal protein, and polyoxypropylene glyceryl ether phosphate; humectantε εuch aε glycerol, sodium 2-pyrrolidone-5-carboxylate, and sodium lactate; -thickeners such as tragacanth gum, quince seed gum, zanthan gum, carboxyvinyl polymer and bentonite; preservatives such aε benzoic acid, allyl p- hydroxybenzoates, dehydroacetic acid and trichlorocarbonilide; coloring agents and pigments such as Acid Red Rhodamine B, Violamin R, Orange SS, Naphthol, Yellow-S, Tartrazine, Alizarin, Cyanine Green F,

Brilliant Blue CFC, Acid Violet, Carthamine, ^-carotene. Red, Blue & Yellow Oxide of Iron, titanium dioxide. Yellow Iron Oxide, Cobalt Blue, Ultramarine Blue, Rose & Violet, tri-iron tetroxide, and carbon black; waxes εuch aε Beeε wax, Japan wax, Carnauba wax, Candelilla wax, and lanolin; film-forming agentε εuch as nitrocellulose and polyvinyl alcohol; εolventε or dispersing media such a water and alcohols (e.g. ethanol) ; powderε εuch aε aluminum powder, talc, kaolin, zinc oxide, titanium dioxide, mica, calcium, carbonate, and treated powderε; plaεticizerε such as acetyl tributyl citrate, and dibutylphthalate; pharmaceutically active agents such aε retinol, palmitate; ₇ -orizanol, pyridoxine dipalmitate, ascrobyl dipalmitate, ergocalciferol, di-α-tocopherol acetate, biotin, ethinylestradoil eεtrone, hydro- crotiεone, calcium pantothenate, ammonium glycyrrhizinate, allantoid, quaiasulene, and hinokitiol; and perfumes εuch aε musk, civet, amber, jasmine absolute, and rose oil. The skin treatment compositions, according to thiε invention, can be prepared in any conventional form, for example, solubilized forms εuch aε cosmetic lotions, and

emulsified forms such as liquid creams, creams, ointments and disperεions.

The pharmaceutical compositions of this invention are particularly uεeful for depigmenting εkin where the compoεition iε applied topically or injected by s.c. or i.p. route to the afflicted area to achieve desired degree of depigmentation of the skin. Hence as another aεpect of the invention, the compoεition iε uεeful in the method of treating εkin for purposes of depigmentation. As well, the invention provides for formulating a composition which is useful for depigmenting skin.

The compositionε of the invention are alεo especially useful for treating melanoma. The composition is adminiεtered either topically or by injection through i.p. or s.c. route. The compoεition is used in treating melanoma to either clear it up or place it in remission. Furthermore, a method of the invention iε in the formulating of the composition useful for treating melanoma. The compoundε of formula (II) may be used in sunscreening lotions as depigmenting agents. It has been found that the active agentε of formula (II) :

1. prevent UV radiation activating melanin synthesis in melanocytes; 2. prevent moles from becoming melanoma after exposure to sunlight; and 3. are substrateε for the enzyme tyrosinase where it is thought that enzymatic conversion of the substrateε produce components which act aε depigmenting agentε and poεεibly aε UV blockers in the sunscreen lotion.

Quite surprisingly, we have found that, upon administration of the acylated derivatives of formula (II) , the compoundε are deacylated to become potent melanocytotoxic and depigmenting agentε. Such in vivo conversion of the compounds is extremely beneficial from the standpoint of product stability and reduced general toxicity. Once the compounds of formula (II) are

deacylated upon administration, they become excellent tyrosinase substrateε. According to a preferred embodiment of the invention, NAP-TEA, once incorporated into the body, iε deacylated to become N-acetyl-4-S-CAP which iε, aε already demonεtrated in the aforementioned International Application WO91/16302, a εtrong tyroεinaεe substrate that is present only in pigment cells. This enzyme activity becomeε highly expreεsed in malignant melanoma and melasma cells where the specificity of the N-acetyl-4-S-CAP is important in avoiding general toxicity to the patient. The therapeutic approach in the use of these compounds for pigmentary diseases such as melasma and skin cancer, such as malignant melanoma, have become very important aε well aε preventive medicine for photoaging and εkin cancer developed after expoεure to εunlight. Melanoma repreεentε 2% of all cancer by instance and iε generally increaεing in the population at a rate exceeded only by lung cancer.

For purposes of treatment, it is understood that depending upon the route of administration the composition of this invention may have variouε concentrationε of active agent in the biologically compatible carrier. For purposes of injection, the concentration of the active agent in the injectable εolution uεually iε in the range of 200 to 1200 mg/kg of body weight with a preferred doεage in the range of 300 mg to 500 mg/kg of body weight. For purposes of topical application, the concentration of the active agent in the cosmetic cream or the like usually is in the range of 4% to 10% by weight in a cream base with a preferred compoεition in the range of 4% to 6% by weight in a cream baεe.

It iε appreciated that various complementary agents may be included with the active agentε of this invention. For example, the composition may be adminiεtered in combination with L-dopa and/or antidecarboxylase. Such combinations may have εelected uεe in the treatment of

various melanomas.

The following Examples demonstrate various aspects of the invention in providing the diagnoεiε and treatment of pigmentation diseases, depigmenting properties and antimelanoma propertieε. The variouε compoundε of formula (I) may be radiolabelled in accordance with a preferred procedure outlined in the following Examples. The procedures have been described in particular reference to the compound N-acetyl-4-S-CAP; however, it is underεtood that the procedure may be εimilarly applied to the other compoundε of formula (I) . Furthermore, the Examples alεo demonεtrate effectiveness in blocking melanin synthesis for only one of the compounds of formula (II) . However, it is understood that the compoundε of formula (II) all exhibit in a predictable way εimilar propertieε.

EXAMPLE 1 Synthesis of N-Acβt l-4-S-cysteaminyl (ϋ- ^u C) henol (U- ^I C)phenol (9.26 MBq, 333.3 MBq/mmol) in toluene (0.25 mL) waε added to aqueous HBr (47% w/v ; 0.5 mL solution containing unlabelled phenol (1.7 mg; 0.018 mmol) and cystamine*2HCl (45 mg; 0.2 mmol). The mixture was heated for 30 min at 130°C in a εcrew-capped reaction vial. After cooling, the solvent was removed under vacuum and the residue was disεolved in aqueous methanol (3% v/v) . The solution was applied to a Sep-Pak C _ig cartridge. The cartridge waε washed with aqueous methanol (10% v/v ; 0.6 mL for removal of exceεε cyεtamine and ( ^l C) labelled 4-S-CAP and 2-S-CAP were eluted with aqueous methanol (20% v/v ; 3.0 mL) . The eluent waε concentrated under vacuum. The major compoundε were εeparated on preparative TLC uεing the mobile phaεe I. The productε were extracted from TLC with MeOH in CHC1 ₃ (20% v/v) . The two iεomerε were acetylated with acetic anhydride in dry pyridine at 25 ^β C for 24 hours. The εolvent waε removed under vacuum. The

residue was dissolved in NH ₃ (2.0 M) and stirred at 25°C for 30 min for O-deacetylation. After reaction, the methanol solvent was removed under vacuum and the residue was purified cn preparative TLC using mobile phase II. ( ¹⁴ C)N-Ac-4-S-CAP (9.5μCi, 351.9 kBq) and ( ¹⁴ C)N-Ac-2-S- CAP(5.7 μCi, 211.1 kBq) in 3.8% and 2.3% radiochemical yield respectively. The radiochemical purity of the two isomers was more than 99%.

This Example demonstrates that one is able to synthesize ( ¹⁴ C)N-Ac-4-S-CAP with conventional acetyacety- 21ation and O-deacylation after electrophilic substitution on phenol of the NH ₂ CH ₂ CH ₂ S ⁺ cation in HBr solution, as per the following reaction scheme.

^HO ~£___) ^{+ S} 2 ^(CH 2 ^CH 2 ^N 2 ⁾ 2- ^2HC1 in\'lr (47%) ► ( ¹ C) Phenol ( ¹

In the above reaction process, ( ¹⁴ C)-acetyl-2-S- cysteaminylphenol (( ¹⁴ C)-Ac-2-S-CAP was also obtained as a minor isomer. The εyntheεiε of ( ¹⁴ C)4-S-CAP using the above reaction was as described previously (Somayaji et al, 1989) . It was difficult to remove cysta ine by methanol precipitation or to separate the 2 and 4 isomers of CAP by TLC system. However, it was possible to remove cystamine and accomplish preliminary purification using a Sep-pak C ¹⁸ cartridge. The chromatographic separation of the two isomerε waε accomplished with mobile phase I. This basic mobile phase allowed a complete separation of

the two iso eric CAP compounds.of the reaction mixture (4-S-CAP : Rf ■ 0.66; 2-S-CAP : Rf = 0.53).

Unlabelled cryεtralline phenol ( 2 mg) reacted smoothly with cystamine in HBr solution in a small scale reaction. An overall yield was approximately 76% as a mixture of the two isomers, the ratio of 4-S-CAP to 2-S- CAP being 2.5. Acetylation of the O-position and the N- position on isomers was carried out with acetic anhydride in dry pyridine. O-Deacetylation for the synthesis of N- Ac-4-S-CAP was performed with NH ₃ in methanol (2.o M) under mild conditions without N-deacetylation. A mixture of 4-S-CAP (a major product) and 2-S-CAP (a minor product) containing the ¹ C-radiolabel were obtained by using a two phase reaction system (toluene : HBr) under reflux, and with the addition of unlabelled phenol this gave low yield of products. If a source of (U" ¹ C) phenol were available as a solid material or as a more concentrated solution, the yield and the specific activity can be greatly increaεed. ( ¹⁴ C)N-Ac-4-S-CAP εyntheεized from ( ¹ C)4-S-CAP gave a high radiochemical yield (68%). Although the specific activity of ( ^l4 C)N-Ac- 4-S-CAP and ( ¹ C)N-Ac-2-S-CAP was low (450 kBq/μmol) , the productε would be εufficient to carry out experimental studies. The use of solid (U" ¹⁴ C)phenol with a high specific activity for the synthesiε of ( ¹ C)4-S-CAP and

( ¹ C)2-S-CAP would increaεe both the specific activity and the yield of the final products.

All chemicals used were of reagent grade quality. (U- ¹ C)Phenol in toluene was purchased from Sigma Chemical Company (Saint Louis, Miεεouri, USA) . Preliminary purification waε performed on Sep-Pak C _lg Cartridges (Waterε Chromatography Division, Millipore Corporation, Milford, USA) . Whatman PLK5F preparative silica gel 150A plates were used for separation of 4-S-cysteaminyl (U- ^, C)phenol, ( ¹ C) 4-S-CAP and 2-cysteaminyl (U- ¹⁴ C) phenol (( ^, C)2-S-CAP) using mobile phase I (CHC1 ₃ MeOH : NH ₄ OH = 24 :12 :1. 1 v/v/v) and using mobile phaεe II

(CHCI ₃ : MeOH - 85 15 v/v) for the purification of the N-acetyl derivativeε of the two iεomerε. ^I C- Radioactivity determination of productε waε performed in Aquaεal*-2 (Biotechnology Systems NEN* Research Product, Boston, MA, USA) with a Beckman LS 3801 liquid scintillation spectrometer. HPLC analyses were carried out with a Waters system consiεting of a Model 600E εyεtem controller. Model U6K injector and Model 441 detector at 254 nm, using μBonapak C ₁₈ reverse phase compression (lOμ) column (Waters Millipore Canada Ltd., Mississauga, Ontario) and MeOH : 0.1% acetic acid in H ₂ 0 (1 : 1 v/v) as eluent with a flow rate of 1 mL/min. The final productε, N-acetyl-4-S-cyεteaminyl (U- ¹ C) phenol (( ¹⁴ C) N-Ac-4-S-CAP) and N-acetyl-2-S-cyεteaminyl (U- ¹⁴ C)phenol (( ¹⁴ C) N-Ac-2-S-CAP) were confirmed by comparing their retention times to those of authentic unlabelled compounds on HPLC. Radiochemical purity after TLC was determined by collect eluent fractionε (0.5 mL) from an HPLC analyεiε and counting by a liquid scintillation spectrometer.

EXAMPLE 2A Synthesis of N-{2-((4-Acetoxyphenyl)- thio)ethyl} cetamide (NAP-TEA) Procedure Acetyl chloride method

To solution of N-{2-((4-Hydroxyphenyl)thio)ethyl} acetamide (200 mg, 0.95 mmol) in dry pyridine was added εlowly acetyl chloride at -15°C with εtirring. The reεulting εolution waε εtirred at 25 ^β C for 1.5 hour. The reεidue obtained after removal of solvent was chromatographed on a low preεεure εilica gel column (CANAG, D-0, fine grain ; Mobile phaεe : 5% MeOH/CHCL _j ) . The fractionε containing product were collected and then the white powder obtained after removal of εolvent waε recrystallized to gain the white crystals from H ₂ 0-MeOH. (Chemical yield : 217 mg, 0.85 mmol 90.3% ) mp= 87-88°

ςharac-fcerjgatipn

*H NMR (Acetone-dβ) ; δ 7.52 (bro, ε, IH, NH) , 7.41 (d, fine structure, 2H, J=8.5 Hz , aromatic C (2 ) -H, C (6) -H) , 7.04 (d, fine structure 2H, J=8.5 Hz , aromatic C(3) -H, C (5) -H) , 3.33 (m, 2H, J (CH ₂ -CH ₂ ) =6

Hz , J(CH ₂ -NH) =7.5 HZ , CH _j C-H _j NH) , 3.05 (dd, 2H, J (CH ₂ . _CH 2 ) =8 Hz , SC-H _j ) , 2.22 (S, 3H, C&COO) , 1.83 (s, 3H, CH ₃ CONH) .

"C NMR (Acetone-ό^) ppm; 169.95 (IC, CHaCONH) 169.29

(IC, CH _j COO) , 150.12 (IC, aromatic, C(4) ) , 133 .61 (IC, aromatic, C(l) ) , 130.76 (2C, aromatic, £H) 122.98 (2C, aromatic, £H) , 39.35 (IC, £H ₂ NH) , 33.48 (IC, SCH _j ) , 22.52 (IC, £H ₃ CONH) , 20.61 (IC, CH ₃ COO) .

Exact mass calc. for C„H ₁₅ N0 ₃ S : 253.07725; found (HRMS) , 253.0773 ; intensity = 18.18% .

EXAMPLE 2B Synthesis of N-{2-{(4-Aceotxyphβnyl)thio}- ethyl}propionamide (NAP-TEP)

A mixture of 2-ethyl-2-oxazoline (786 mg, 7.94 mmol) and 4-hydroxythiophenol (1 g, 7.94 mmol) was heated at 125° for 2 h under Ar gaε. After cooling of the reaction mixture, the mixture waε evaporated under vacuum. The reεidue waε purified with a low preεεure normal phaεe silica gel column chromatography (mobile phase: 10% MeOF in CHClj) . N-propionyl-4-S-cystaminylphenol (N-Pro-4-S- CAP) was obtained as a white powder on evaporation of the solvent (Chemical yield: 53.4%, 953 mg) . A mixture of N-propionyl-4-S-cysteaminylphenol, dry pyridine, and acetic anhydride is then stirred at 25° for 24 hours. The reaction mixture is evaporated under vacuum and the reεidue iε purified with a low preεεure normal phase silica gel column chromatography (mobile phase : 8% MeOH in CHC1 ₃ ) . N-{2-{(4-aceotxyphenyl)thio}- ethyl}-propionamide (NAP-TEP) is obtained as a white powder upon evaporation of the solvent.

EXAMPLE 3

In vivo covalent binding of ( ^, C)-N-acetyl-4-S-CAP waε investigated in mice. Female C57BL/6J mice received a εingle intraperitoneal injection of 2.0 mmol/kg ( ¹⁴ C)-N- acetyl-4-S-CAP compound. The animalε were killed 48 hourε later. The tissues were removed and the covalent binding was determined as follows.

Five different tissues were examined for covalent binding of radioactively labelled N-acetyl-4-S-CAP intermediate(s) . The maximum binding of N-acetyl-4-S-CAP was detected in the s.c. melanoma tumor and melanoma colony-bearing lung tiεεueε. In contraεt, the normal organs, e.g., the lung without melanoma colonies, kidney and liver, which are actively involved in drug metabolism of N-acetyl-4-S-CAP, did not reveal a significant accumulation of radioactive material. The results of such εtudy are εet out in the following Table 1.

TABLE 1

In Vivo Covalent Binding of (14Q-NACAP to Lung with and without B16F10

Melanoma Colonies, B16F10 Melanoma s.c. Tumor Tissue, Liver and Kidney

Tissue* Covalent Binding ¹ \'

(nmol covalently bound/mg of protein

Lungs with B16F10 0.330 + 0.118 melanoma colonies

Lungs without B16F10 0.010 + 0.001 melanoma colonies

B16F10 melanoma 0.521 + 0.076 s.c. tumor Liver 0.013 + 0.003 Kidney 0.008 + 0.001

\'Female C57BL/6J mice received a single intraperitoneal injection of 2.0 mmol/kg (" -NACAP. The animals were killed 48 hr later, the tissues were removed, and the covalent binding was determined as described under above.

The data are presented as the means +S.D. (n=3).

In vivo biodiεtribution by whole-body autoradiography was determined in use of the compounds of this invention and in particular, ( ¹⁴ C)N-acetyl-4-S-CAP at 48 hours after i.p. injection of ( ¹⁴ C)N-acetyl-4-S-CAP in the mouse, radioactive material was cleared from the body and was not detectable in any normal organs except the lumen of the large intestine. This is shown in the attached Figure 1. A whole-body autoradiogram of the mouse with both an s.c. B16F10 melanoma tumor and a B16F10 lung melanoma colonies after single i.p. injection of ( ^M C)N-acetyl-4-S-CAP (5.0 μCi) . 48 hours after injection the animal was killed, frozen and prepared for sectioning. The 20-μm sections were freeze-dried and exposed to x-ray film for whole-body autoradiography. A) A whole-body section without exposure to x-ray film. E, eye; B, brain; Lu, lung with B16F10 melanoma colonies; H, heart; Li, liver; Me, s.c. melanoma tumor; I, intestines. B) after exposure to x-ray film. Positive εignal of accumulated radioactivity iε clearly detectable in the melanoma colony-bearing lung and s.c. melanoma tissues, as well in the lumen of the distal partes of inteεtine. Bar, 1 cm.

Thiε result aε shown in Figure 1 demonstrates the significant detoxication of N-acetyl-4-S-CAP in the liver followed by the excretion of N-acetyl-4-S-CAP metabolites into the bile. The s.c. melanoma tumor and the lung with melanoma colonies were the only organs diεplaying a significant accumulation of the radioactivity.

The radiolabelled compounds of thiε invention and in particular, radiolabelled N-acetyl-4-S-CAP and radiolabelled NAP-TEA are particularly uεeful aε radioimaging agentε without causing toxic εide effectε in the hoεt. Alεo, in appropriate doεageε the compoundε may be uεed effect radio-chemotherapy at specific melanoma sites within the host.

EXAMPLE 4 - EXPERIMENTS FOR O-DEACYLATION OF NAP-TEA IN MOUSE PLASMA NAP-TEA waε introduced to mouεe plasma to determine by in vitro experimentation the extent to which NAP-TEA is deacetylated to form N-Acetyl-4-S-CAP. The procedure was as follows. Into 100 μl of mouse plasma, 10 μg of NAP-TEA was introduced. The mixture was shook for one- half of a minute and then pasεed through a Sep-Pak C cartridge in either 7 ml of 5% methanol/water to form eluent 1 or in 4 ml of 50% methanol/water to form eluent 2. Both eluentε 1 and eluentε 2 were collected. Aqueous me-thanol waε removed from eluent 2 to provide a reεidue which was subjected to HPLC under the following conditions. The HPLC had a column of μ Bonapak C ₁₈ under reverse phase radical compresεion. The reεidue waε mixed with methanol:water at 50:50 with a flow rate through the radiograph at 1 ml/min. The detector waε a Water Associates Model 441 with detection by ultraviolet at 250 nm. All that was detected in eluent 2 as passed through the chromatograph was N-Acetyl-4-S-CAP which indicateε that NAP-TEA waε readily deacylated by the mouεe plasma to give the desired active agent.

EXAMPLE 5 - DETERMINATION OF NAP-TEA, N-ACETYL-4-S-CAP AND METABOLITES IN IN VIVO EXPERIMENTATION

A C57BL/6J Black Mouse was injected at 300 mg/kg body weight by i.p. adminiεtration of NAP-TEA. 100 μl of plaεma waε extracted from the mouεe after 5 minutes of injection and after 20 minutes of injection. The plasma samples were treated in accordance with the procedure of Example 2. The plasma sampleε were paεεed through a Sep-Pak C _I8 cartridge in either 5% methanol:water to produce eluent 1 or 50% methanol/water to produce eluent 2. Eluent 2 had the aqueous methanol removed therefrom to provide a reεidue. The reεidue waε rediεεolved in methanol/0.1% acetic acid and water in a ratio of 50:50 and subjected to HPLC for both the 5 minute and 20 minute

plasma samples. Their was no evidence of NAP-TEA but heir was evidence of N-Acetyl-4-S-CAP. The concentration of N-Acetyl-4-S-CAP in the 5 minute extracted sample being considerably greater than in the 20 minute extracted sample to indicate that in vivo NAP- TEA is deacylated to N-Acetyl-4-S-CAP which in turn iε then taken up by the mouse melanocytes.

EXAMPLE 6 - IN VITRO CYTOTOXICITY OF NAP-TEA MTT aεsav in accordance with the standard procedure

MTT solution (Sigma catalog No. M2128) was disεolved in PBS at l mg/ml and filtered to sterilize the solution. The solution was stored in the dark at 4°C until used. The growing cellε were harvested, counted and inoculated into 96 well microtiter plates (1-2 x 10 ³ cellε/well) .

After 24 h, drugs were applied to culture wells, and the cultures were incubated for 5 days at 37 ^β C. At this time, 50 μl of MTT solution was added to microculture wells. After 4 h incubation at 37°C, εupernatant waε removed from each well. 150 μl of DMSO waε added to solubilized the MTT-formazan product. After mixing gently, absorbance at 540 run waε measured with ELISA plate spectrophotometer.

Results

NAP-TEA showed lower in vitro cytotoxicity than that of N-Acetyl-4-S-CAP on 3 cell lines.

In vitro cytotoxicity(IC*,) of NAP-TEA and N-Acetyl-4-S-CAP with MTT assay

Cell line Compound

NAP-TEA N-Acetyl-4-S-CAP

IP* HeLa 77μg/ml 75μg/ml

SK-MEL-23 31μg/ml 25μg/ml

UT amelanotic melanoma I4μg/ml 12μg/ml

EXAMPLE 7 - UTILIZATION OF NAP-TEA WITH MUSHROOM TYROSINASE

Procedure

A reaction mixture consisting of NAP-TEA (500μmol) and of mushroom tyrosinase (11 units) in 1.0 ml of 0.05 M sodium phosphate buffer (pH 6.8) was incubated at 37°C in a water bath. The reaction waε εtopped periodically by cooling with ice. Aliquots removed at set time intervals were rapidly cooled to 0°C in an ice bath and the remaining NAP-TEA waε meaεured uεing HPLC.

HPLC instrument and conditions Water 600 E liquid chromatograph εyεtem Conditions: μBondapak C, ₈ , Radial-Pak Cartridge (Waters) Mobile phase; 70% MeOH/H ₂ 0, 1 ml/min

Detector; Waters Model 441 UV absorbance detector

Detection; Wavelength 254 nm

Results

NAP-TEA was not a substrate of mushroom tyroεinase, due to the blocking effect of acetoxy group on the 4- poεition of the benzene ring becauεe itε concentration remained conεtant in the reaction mixture. However, the concentration of N-Acetyl-4-S-CAP in the reaction mixture dropped off rapidly over 25 minutes to almoεt zero amount after 50 minuteε in the reaction mixture.

EXAMPLE 8 - MELANOCYTOTOXICITY IN VIVO OF NAP-TEA (DEPIGMENTATION OF BLACK HAIR)

Hairs were plucked manually from the back of 6-8 week old C57BL/6J black mice (4 mice) . Starting on day 2, daily for 14 days NAP-TEA was injected i.p. The doεe was 300 mg/kg body weight.

Resultε

After completion of the i.p. injections of NAP-TEA,

mice were depigmented with the. new replacement hair being almost pure white.

EXAMPLE 9 - DEPIGMENTING EFFECT OF NAP-TEA ON NEWBORN BLACK MICE

Six newborn (3 day old) C57BL/6J black mice were injected i.p. with a single dose of 0.1 mL of NAP-TEA solution (300 mg/kg body weight) .

Results

The newborn mice were depigmented in all hair follicles with the growing hair being light grey after a single i.p. injection of NAP-TEA.

EXAMPLE 10 - IN VIVO MELANOCYTOTOXICITY OF NAP-TEA IN PIG Solutions of 15% NAP-TEA in DMSO(w/v) and of 15% NAP-TEA in 60% EtOH(w/v) were prepared for topical application to a Yucatan pig. NAP-TEA was applied to the back skin of the pig twice a day for 8 weeks. After 8 weeks, punch biopεy waε performed from the application area and from normal skin. Specimens obtained were kept in 2N NaBr solution for 3 hours followed by εeparation of the epidermiε from dermis. Specimens of epidermis were incubated for 3 hours in phosphate buffer (pH 7.4) containing L-Dopa. The specimens were fixed in 10% neutral formalin solution and then washed twice in PBS. Finally, the specimens were mounted in glycerol gel. The numbers of Dopa-positive melanocytes in 9 adjacent 1 mm ² fieldε from each εample were counted.

Results

The fieldε from εkin samples treated with NAP-TEA revealed a significant reduction in the number of functioning melanocytes;

Number of functioning melanocytes (Average of 9 fields)

Normal epidermis 166 ± 74/mm ² NAP-TEA in DMSO 32 ± 14/mm ²

NAP-TEA in 60% EtOH 74 ± 25/mm ²

DMSO only 114 ± 56/mm ²

EtOH only 117 ± 38/mm ²

From the above specific examples, it iε apparent that one of the preferred compoundε, according to this invention, is converted in vivo to N-Acetyl-4-S-CAP which functionε, aε already discussed in applicant\'s co-pending International Application W091/16302 as an excellent -therapeutic agent in the treatment of pigmentary diseases. It is therefore understood that the acylated forms of the compoundε of thiε invention are altered in vivo to yield compounds which already have been established as excellent therapeutic agents in the field of treating pigmentary disorders.

Throughout the specification we have referenced several Journal articles which relate to standard procedures and techniques. Those articleε are listed as follows.

1. Alena F. , Jimbow K. and Ito S. (1990)

Melanocytotoxicity and Antimelanoma effects of Phenolic Amine Compounds in Mice in vivo. Cancer Res. 50, 3743.

2. Ito Y. and Jimbow K. (1987) Selective Cytotoxicity of 4-S-cyεteaminylphenol on Follicular Melanocytes of the Black Mouεe - Rational Basis for its Application to Melanoma Chemotherapy. Cancer Res. 47, 3278.

3. Inoue S., Ito S., Wakamatεu K. , Jimbow K. and

Fujita K. (1990) Mechanism of Growth Inhibition of Melanoma Cells by 4-S-cysteaminylphenol and its Analogues. Biochem. Pharmacol. 39, 1077.

4. Jimbow K., Iwashina T. , Alena F. , Yamada K. ,

Pankovich J. and Umemura T. (1992) Exploitation of Pigment Biosynthesis Pathway as a Selective Chemotherapeutic Approach for Malignant Melanoma. J. Invest. Derm ol. (in press) .

5. Miura S., Ueda T. , Jimbow K. , Ito S. and Fujita K. (1987) Synthesis of Cysteinylphenol, Cystesuαinylphenol and Related Compounds, and in vivo Evaluation of Antimelanoma Effect. Arch. Derm tol. Res. 279, 219.

6. Miura T., Jimbow K. and Ito S. (1990) The in vivo Antimelanoma Effect of 4-S- cyεteaminylphenol and itε N-acetyl derivative. int. J. Cancer 46, 931.

7. Pankovich, J. Jimbow K. and Ito S. (1990) 4-S- cysteaminylphenol and its Analogues aε Substrates for Tyrosinaεe and Monoamine Oxidase. Pigment Cells Res. 3, 146.

8. Somayaji V.V., Wiebe, L.I. and Jimbow K. (1989) Experimental Antimelanoma Agents: The Synthesis of 4-S-cysteaminylphenol (U- ¹⁴ C)phenol. Nuc. compact 20, 158.

9. Wong M. and Jimbow K. (1991) Selective Cytotoxicity of N-acetyl-4-S-cyεteaminylphenol on Follicular Melanocyteε of Black Mice. Br. J. Dβrmatol 124, 56.

Although preferred embodimentε of the invention are deεcribed herein in detail, it will be underεtood by thoεe skilled in the art that variationε may be made thereto without departing from.the εpirit of the invention or the scope of the appended claims.